The Interactive Fly
The centrosome is the main microtubule-organizing center in animal cells. It comprises of two centrioles and the surrounding pericentriolar material. Protein organization at the outer layer of the centriole and outward has been studied extensively; however, an overall picture of the protein architecture at the centriole core has been missing. This paper reports a direct view of Drosophila centriolar proteins at ~50-nm resolution. This reveals a Sas6 ring at the C-terminus, where it overlaps with the C-terminus of Cep135. The ninefold symmetrical pattern of Cep135 is further conveyed through Ana1-Asterless axes that extend past the microtubule wall from between the blades. Ana3 and Rcd4, whose termini are close to Cep135, are arranged in ninefold symmetry that does not match the above axes. During centriole biogenesis, Ana3 and Rcd4 are sequentially loaded on the newly formed centriole and are required for centriole-to-centrosome conversion through recruiting the Cep135-Ana1-Asterless complex. Together, these results provide a spatiotemporal map of the centriole core and implications of how the structure might be built (Tian, 2021).
The centrosome has multiple crucial functions, including the assembly of the mitotic spindle and establishing the axis of cell division. It comprises two principal components: a pair of orthogonally arranged centrioles and the surrounding pericentriolar material (PCM). Centrioles are stable cylindrical structures comprising nine microtubule blades arranged at the end of nine spokes that radiate from a central hub. During each cell cycle, the centriole pair disengages at the mitotic exit, allowing the new centrioles (or daughter centrioles) to gradually assemble next to each preexisting centriole (the mother centriole). A mother centriole serves as a recruitment and assembly scaffold for the PCM proteins to form spindle poles in mitosis; in many cell types, it also provides a template for cilium or flagellum assembly during cell quiescence, forming a crucial organelle for chemical sensation, signal transduction, locomotion, and so forth. Centrosome defects have been related to a wide range of human diseases, including cancer, microcephaly, and a group of disorders collectively known as the 'ciliopathies' (Tian, 2021).
Understanding how the centrosome functions requires knowledge of its protein composition and organization. The centrosome is composed of >100 different proteins. Their architectural arrangement has begun to be systematically examined since the application of superresolution microscopy. Using 3D structured illumination microscopy (3D-SIM), distinct concentric domains within a centrosome have been documented (e.g., zones I-V of the Drosophila centrosome) and that the PCM has a conserved, ordered structure. Protein organization at several compartments of the centrosome, such as the distal and subdistal appendages, the transition zone, the centrosome linker, and the longitudinal axis of the centriole, has also been studied via 3D-SIM, stimulated emission depletion (STED) microscopy, or stochastic optical reconstruction microscopy. Meanwhile, proteins at the core of the centriole remain largely unresolved. This cartwheel region, revealed as zone I by 3D-SIM, contains the central hub of ~22-nm diameter and the nine spokes that determine the ninefold symmetrical feature of the centriole (Tian, 2021).
Drosophila cultured cells present a consistent model for the study of the centriole core because, contrary to the vertebrate centrosome, the cartwheel persists in the mature centriole. The centriole is composed of doublet microtubules arranged in a ninefold symmetrical cylinder, which is ∼200 nm wide and long and has a cartwheel formation along the entire length. This study first determined which proteins known to be required for Drosophila centriole duplication are the components of the centriole core. A direct view of these proteins is presented at ~50-nm resolution, and a timing order of their assembly is presented using several superresolution techniques. These revealed a ninefold radial scaffold comprising Spindle assembly abnormal 6 (Sas6), Centrosomal protein 135kDa (Cep135), Anastral spindle 1 (Ana1), and Asterless (Asl), as well as concentric toroids formed by Anastral spindle 3 (Ana3) and Reduction in Cnn dots 4 (Rcd4), two novel core centriolar components that are also organized in ninefold symmetry. During centriole biogenesis, Ana3 is recruited to the newly formed daughter centriole later than Sas6 but before Rcd4 and Cep135. These findings thus provide a spatiotemporal map of the centriole core and a model of how the proteins might interact to build the structure (Tian, 2021).
These data reveal the spatiotemporal organization of the proteins at the core region of the Drosophila centriole (see Schematics depicting the lateral organization of centriole core). By superimposing the current measurements to the electron cryotomography data of the Trichonympha, Chlamydomonas, and Drosophila centrioles, this study found that Cep135 overlaps with the C-terminus of Sas6 on the spokes via its C-terminus and extends to the pinheads via the N-terminus. Ana1 localizes from the pinheads to the outer edge of the doublet microtubules. Asl slightly overlaps with the doublet microtubules and extends into PCM in a ninefold manner. It is proposed that the core region of the centriole is composed of two dimensions. One is the ninefold radial dimension that is established by elongated molecules overlapping through their adjacent termini: Sas6, Cep135, Ana1, and Asl. They likely constitute the spoke-pinhead axes and further transmit the ninefold symmetrical geometry to the microtubule wall and into the core PCM. The other is a circular dimension established by a group of compact proteins that are also arranged in ninefold symmetry: Ana3, Rcd4, and possibly Ana2. They likely decorate the radial axes and provide the physical support for the ninefold configuration (Tian, 2021).
Previous work has shown that Cep135, Ana1, and Asl form a complex that is responsible for the centriole-to-centrosome conversion), the final stage in the assembly of the daughter centriole that converts it into a mother centriole able to duplicate. With improved spatial resolution, this study shows that the three proteins are each organized in ninefold manner, reinforcing the idea they are the bona fide components of the spoke-pinhead scaffold. The ninefold radial axes then extend past the centriole microtubule wall via the C-terminus of Ana1, which is positioned between the microtubule blades. Recently, an electron cryotomography study showed that, between adjacent microtubule blades, there are ninefold amorphous brushlike structures in the Drosophila S2 centriole. This study suggests that it could contain Ana1 and Asl, both of which exhibit ninefold symmetry at this region (Tian, 2021).
These findings allocate a role to Drosophila Ana3 and Rcd4, previously known from genome-wide RNAi screens to be required for centriole duplication. Ana3 was later reported to be responsible for the structural integrity of centrioles and basal bodies and for centriole cohesion in the Drosophila testes. This study now provides evidence that both Ana3 and Rcd4 are core centriolar components, localizing to the region where Cep135 is. The N-terminus of Ana3 localizes closest to the center of the centriole, followed by the C-termini of Ana3 and Rcd4 and the N-terminus of Rcd4. Both Ana3 and Rcd4 are organized in ninefold symmetry but seem to be positioned in axes that are not in line with the Cep135-Ana1-Asl complex. Spatial overlapping of Ana3 and Rcd4 indicates these two proteins might interact, which has recently been reported (Panda, 2020) and is conserved to their human counterparts, RTTN and PPP1R35. Depletion of either Ana3 or Rcd4 leads to failure in loading the Cep135-Ana1-Asl complex during centriole biogenesis and thus causes defects in centriole-to-centrosome conversion and the reduction of the centrosome number. This pathway is also conserved in human cells, where PPP1R35 was reported to promote centriole-to-centrosome conversion upstream of Cep295 (human homologue of Ana1) and RTTN and PPP1R35 serve as upstream effectors of Cep295 in mediating centriole elongation (Tian, 2021).
Taken together, these data provide an overall picture of the protein architecture at the centriole core and implications of how the ninefold symmetrical structure might be built. Knowing the spatiotemporal restraints of individual centriolar components will guide the immediate study of the molecular interaction partners and understanding of their functions. Meanwhile, it would also provide information for a higher-resolution approach, including cryo-EM, to eventually obtain a 3D map of the centriole (Tian, 2021).
Asymmetric inheritance of mother versus daughter centrosome in stem cell division
Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. Developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation (Yamashita, 2007).
Adult stem cells maintain populations of highly differentiated but short-lived cells throughout the life of the organism. To maintain the critical balance between stem cell and differentiating cell populations, stem cells have a potential to divide asymmetrically, producing one stem and one differentiating cell. The asymmetric outcome of stem cell divisions can be specified by regulated spindle orientation, such that the two daughter cells are placed in different microenvironments that either specify stem cell identity (stem cell niche) or allow differentiation (Yamashita, 2007).
Drosophila male germline stem cells (GSCs) are maintained through attachment to somatic hub cells, which constitute the stem cell niche. Hub cells secrete the signaling ligand Unpaired, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the neighboring germ cells to specify stem cell identity. Drosophila male GSCs normally divide asymmetrically, producing one stem cell, which remains attached to the hub, and one gonialblast, which initiates differentiation. This stereotyped asymmetric outcome is controlled by the orientation of the mitotic spindle in GSCs: The spindle lies perpendicular to the hub so that one daughter cell inherits the attachment to the hub, whereas the other is displaced away (Yamashita, 2007).
The stereotyped orientation of the mitotic spindle is set up by the positioning of centrosomes during interphase. GSCs remain oriented toward the niche throughout the cell cycle. In G1 phase, the single centrosome is located near the interface with the hub. When the duplicated centrosomes separate in G2 phase, one stays next to the hub, whereas the other migrates to the opposite side of the cell. Centrosomes in the GSCs separate unusually early in interphase, rather than at the G2-prophase transition, so it is common to see GSCs with fully separated centrosomes without a spindle (Yamashita, 2007).
Differences between the mother and daughter centrosomes underlie the stereotyped behavior of the centrosomes in Drosophila male GSCs. The mother centrosome normally remains anchored to the hub-GSC interface and is inherited by the GSC, whereas the daughter centrosome moves away from the hub and is inherited by the cell that commits to differentiation. Mother and daughter centrosomes were differentially labeled by transient expression of green fluorescent protein-pericentrin/AKAP450 C-terminus (GFP-PACT) from the Drosophila pericentrin-like protein under heat shock-Gal4 control. The PACT domain, which is necessary and sufficient for centriolar localization, is incorporated into centrioles only during centrosome duplication and does not exchange with the cytoplasmic pool. Both the mother and daughter centrosomes are labeled by GFP-PACT in the first cell cycle after heat shock. In the second cell cycle, the daughter centrosome retains GFP-PACT, whereas the mother centrosome is not labeled, thus distinguishing the mother and daughter centrosomes. After a short burst of GFP-PACT expression induced by a 2.5-hour heat shock, 20% to 30% of the GSCs had GFP-labeled centrosomes, indicating the duplication of centrosomes during the window of GFP-PACT expression. By 12 hours after heat shock, >90% of the labeled GSCs had two GFP-positive centrosomes, indicating that they had progressed to the G2 phase of the first cell cycle after GFP-PACT incorporation (Yamashita, 2007).
By 18 to 24 hours after heat shock, the number of GSCs with two GFP-positive centrosomes had decreased, whereas the number of GSCs with one GFP-positive and one GFP-negative centrosome had increased, suggesting progression into the second cell cycle. Generally, the centrosome distal to the hub was labeled, whereas the centrosome proximal to the hub was GFP-negative, indicating that the daughter centrosomes migrate away from the hub-GSC interface during asymmetric GSC divisions (Yamashita, 2007).
Labeling the mother rather than the daughter centrosomes confirmed that the male GSCs in the niche preferentially retain mother centrosomes over time. Centrioles assembled during early embryogenesis were labeled using the NGT40 Gal4 driver to drive the expression of GFP-PACT in blastoderm-stage embryos, shutting off after germband extension. In the first cell cycle after the depletion of the cytoplasmic pool of GFP-PACT in the GSCs, both the mother and daughter centrosomes should be labeled. In subsequent cell cycles, only the mother centrosomes should be labeled (Yamashita, 2007).
In most GSCs in the second or later cell cycle after the depletion of cytoplasmic GFP-PACT, the labeled centrosome was positioned next to the hub-GSC interface, and the unlabeled centrosome had moved away from the hub. The frequency of GSCs that had the proximal, but not distal, centrosome labeled remained constant over time for 10 days (L3 larvae to day-3 adults), suggesting that the mother centrosomes are reliably retained by the GSCs, even through multiple rounds of GSC divisions. Some GSCs maintained cytoplasmic GFP-PACT, especially in L3 larvae, suggesting that the GFP-PACT had not yet been diluted out. Some GSCs were also observed with two labeled centrosomes, suggesting that they are in the first cell cycle after the depletion of cytoplasmic GFP-PACT (Yamashita, 2007).
The mother centrosomes in GSCs appeared to maintain robust interphase microtubule arrays. Ultrastructural analysis of the GSCs revealed that the centrosome proximal to the hub was commonly associated with many microtubules throughout the cell cycle. Nineteen centrosomes in GSCs were scored in serial sections of the apical tips of five wild-type testes. Eleven centrosomes were localized close to the adherens junctions between the hub and the GSCs. Nine of these proximal centrosomes appeared to be in interphase cells, based on nuclear morphology and microtubule arrangement. Typically, these interphase centrosomes proximal to the hub were associated with numerous microtubules. In some samples, microtubules appeared to extend from the centrosome toward the adherens junctions. The other two proximal centrosomes appeared to be in cells in mitotic prophase, based on their robust microtubule arrays containing bundled microtubules running parallel to or piercing the nuclear surface (Yamashita, 2007).
In contrast, of the five distal centrosomes in the apparently interphase cells that were scored, four had few associated microtubules. The remaining three distal centrosomes appeared to be in cells in mitotic prophase, based on microtubule arrays containing bundled microtubules. Thus, the mother centrosomes may maintain interphase microtubule arrays that anchor them to the hub-GSC interface, whereas the daughter centrosomes may initially have few associated microtubules and be free to move, establishing a robust microtubule array only later in the cell cycle (Yamashita, 2007).
Consistent with the idea that astral microtubules anchor the mother centrosomes to the hub-GSC interface, mother versus daughter-centrosome positioning was randomized in GSCs that were homozygous mutant for centrosomin (cnn), an integral centrosomal protein required to anchor astral microtubules to centrosomes. Analysis of mother and daughter centrosomes after transient expression of GFP-PACT revealed that, for cnn homozygous mutant GSCs where one of the two centrosomes was positioned next to the hub, it was essentially random whether the mother or the daughter centrosome stayed next to the hub. In addition, in >25% of total labeled GSCs, neither of the two centrosomes was next to the hub (Yamashita, 2007).
These results indicate that the two centrosomes in Drosophila male GSCs have different characters and fates. The mother centrosome stays next to the junction with the niche and is inherited by the cell that self-renews stem cell fate. Thus, GSCs can maintain an old centriole assembled many cell generations earlier. In contrast, the daughter centrosome migrates away from the niche and is inherited by the cell that will initiate differentiation. It is postulated that the mother centrosomes in male GSCs may remain anchored to the GSC-niche interface throughout the cell cycle by attachment to astral microtubules connected to the adherens junction, whereas the daughter centrosomes may initially have few associated microtubules and thus can move away from the niche. Microtubule-dependent differential segregation of mother and daughter spindle-pole bodies (equivalent to centrosomes in higher organisms) is observed in budding yeast (Pereira, 2001). In cultured vertebrate cells, the centrioles mature slowly over the cell cycle, and the mother centrosomes (containing a mature centriole) attach astral microtubules more effectively and are more stationary than daughter centrosomes in interphase (Piel, 2000). The unusually early separation of centrosomes in interphase male GSCs may provide a way to move the daughter centrosome out of range of the stabilizing influence of the adherens junction complex before it becomes competent to hold a robust microtubule array (Yamashita, 2007).
Developmentally programmed anchoring of the mother centrosome may provide a key mechanism to ensure the stereotyped orientation of the mitotic spindle and thus the reliably asymmetric outcome of the male GSC divisions. Although it is tempting to speculate that determinants associated with the mother or daughter centrosome may play a role in specifying stem cell or differentiating-cell fates, such determinants are yet to be identified. Rather, the asymmetric inheritance of mother and daughter centrosomes in male GSCs may be a consequence of the cytoskeletal mechanisms that are imposed as part of the stem cell program to anchor one centrosome next to the niche throughout the interphase, ensuring a properly oriented spindle (Yamashita, 2007).
Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles, were examined. Early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function (Rebollo, 2007).
Immediately after cytokinesis, the single dot revealed by both PCM and centriole reporters splits in two, strongly suggesting that centrosome duplication has taken place. The YFP-Asl marker, like other centriolar markers in Drosophila, does not allow for resolution of individual centrioles within a centrosome in larval NBs. Therefore, timing of centriole duplication in these cells remains uncertain. The two resulting centrosomes migrate together within the single major aster of the cell to the apical cortex. Later on, one of the centrosomes loses PCM and starts to migrate. At this early time point in the cell cycle, unequal centrosome fate is already established: one, apical, will remain in the stem cell; the other will go into the differentiating daughter. Migration of the downregulated centrosome (revealed by the centriolar marker), initially within the apical side of the cell and more basally later on, occupies most of the cell cycle and is the most variable stage, its duration being dependent on cell-cycle length. The apical centrosome organizes the only aster found in the NB for most of the cell cycle. As mitosis onset approaches, the moving downregulated centrosome becomes stabilized at the basal side and starts to accumulate PCM and organize the second aster. As a direct consequence, the spindle is assembled already in alignment with the polarity axis of the cell. In Drosophila male germline stem cells (Yamashita, 2007), one of the centrosomes is also consistently located adjacent to the hub from early interphase onward. Only this centrosome maintains a robust aster through the cell cycle. The other, associated with only a few microtubules, moves away from the hub and is inherited by the gonialblast. In these cells, the oldest centriole is always in the centrosome that is proximal to the hub and is therefore retained by the stem cell (Yamashita, 2007). It has not yet been possible to determine which of the two centrosomes contains the oldest centriole in larval neural stem cells. In pins NBs, unequal centrosome fate and function are established, but, eventually, the stable, aster-forming apical centrosome is downregulated and starts to behave like the other, migrating across the cell. Like the other too, it organizes an aster only shortly before NEB. The place of assembly of the two asters in pins mutant NBs is not fixed and consequently spindle orientation is randomized, and so is the size difference between the two daughter cells (Rebollo, 2007).
It is still unclear how NB polarity is maintained from one cycle to the next; a distinct Baz apical crescent is only assembled at prophase. The permanent positioning of the NB centrosome in the apical side of the cell, through the cell cycle, suggests that it could be contributing to specifying the apical cortex after mitosis (Rebollo, 2007).
In summary, four main conclusions can be derived from these observations: (1) the two centrosomes of asymmetrically dividing Drosophila larval NBs become unequal early in the cell cycle in terms of mobility, MTOC activity, and fate; (2) such elaborated unequal centrosome regulation provides a means to position the asters, thus ensuring spindle alignment along the polarity axis of the cell; (3) Pins contributes to spindle orientation in NBs by preventing the downregulation of the MTOC capability of the apical centrosome, thus maintaining the apical aster in place, and (4) spindle orientation is predetermined and can be accurately predicted as soon as the aster reaches the apical cortex during the initial stages of the cell cycle. Altogether, these observations reveal that asymmetry in Drosophila neural stem cells goes beyond the polarized localization of a number of protein complexes during mitosis and may affect entire organelles such as the centrosome, which exerts a major effect on cell architecture and function throughout the cell cycle (Rebollo, 2007).
Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. It has been shown that their mitotic spindle orientation is established before spindle formation. This study investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a 'dominant centrosome.' This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. A two-step mechanism is proposed to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment (Rusan, 2007).
By prophase, NBs contain two MTOCs that are almost fully separated and aligned along the NB/GMC axis, but analysis of fixed NBs revealed a single MTOC positioned opposite the GMCs before mitotic entry. Thus MTOC behavior was examined throughout the cell cycle as an initial approach to test the hypothesis that fixing the position of one MTOC through successive divisions helps ensure persistent spindle orientation. Live NBs expressing GFP-G147, an MT-associated protein were examined, revealing a striking temporal difference in MTOC activity. During interphase, a single detectable MTOC persists opposite the previous division site; this is referred to as the dominant MTOC. As NBs approach mitosis, this MTOC increases activity (matures; empirically judged by size and MT fluorescence intensity), forming an MT basket around the nucleus. This is referred to as preprophase; this stage is also seen in fixed samples stained for tubulin. Soon after, sometime before the dominant MTOC fully matures, something striking happens: a second MTOC appears distant from the first. This is the second MTOC and this stage is referred to as prophase onset. The second MTOC increases activity, maturing ~10 min before nuclear envelope breakdown (NEB). Using 4D imaging, the possibility was excluded that the second MTOC was present earlier in another focal plane. To further assess this, forming spindles were imaged end on. It is clear that the second MTOC did not emerge from the dominant MTOC or travel around the nucleus. Instead, the second MTOC appeared roughly opposite the dominant MTOC. MTOC separation began immediately, and by NEB, they were 146 ± 20° apart. Thus, NBs form two distinct MTOCs: an MTOC persisting from the previous division and another only activated at mitotic entry (Rusan, 2007).
This distant activation of the second MTOC raised questions about the centrosome cycle. One possibility is that NBs have two MT nucleating centrosomes, but only one can retain MTs and act as an MTOC during interphase, whereas the second acquires MT retention ability during mitotic entry, explaining the second MTOC's sudden appearance. There is precedent for this: mouse L929 cells have two γtub-bearing centrosomes that can nucleate MTs, but only one contains Ninein and can retain MTs to form an MTOC (Rusan, 2007).
To test this hypothesis in NBs, EB1-GFP was used. This binds growing MT plus ends and reliably identifies MT nucleation sites. Only one nucleation site was present in interphase and preprophase, and a new nucleation site appeared distant from the first, consistent with spatially and temporally distinct second MTOC activation. Thus, NBs regulate MT nucleation and not just MT retention (Rusan, 2007).
To examine how the new nucleation center forms, centrosomes were imaged using a PCM protein, GFP-Cnn. NBs contain a single detectable centrosome during interphase. When NBs reenter mitosis, a second Cnn-positive centrosome appears distant from the first, mimicking activation of the second MTOC. To verify that these occur simultaneously, NBs expressing mCherry-Tubulin (chTub) and GFP-Cnn were imaged. This revealed perfect temporal and spatial correlation between the appearance of the second centrosome and activation of the second MTOC. Physical separation of two centrosomes/MTOCs was never seen. This is the first example of asynchronous and physically distant centrosome maturation, suggesting that NBs use a novel centrosome cycle (Rusan, 2007).
Higher temporal/spatial resolution imaging revealed that two GFP-Cnn spots separate during mitotic exit. One GFP-Cnn spot persists as the NB interphase centrosome, forming the dominant MTOC, whereas the other spot disappears. The persistent Cnn spot (centrosome) remains relatively stationary in interphase, consistent with the hypothesis that coarse spindle alignment begins in interphase by anchoring the dominant centrosome (Rusan, 2007).
Centrosome fate was examined in the two daughters (new NB and GMC). They differ dramatically in PCM retention, in contrast to mammalian cells, where both daughters' centrosomes retain PCM. The GMC centrosome sheds all PCM. The new NB centrosome (that becomes the dominant MTOC) retains PCM throughout interphase and further accumulates PCM during the next mitosis. The complete shedding of PCM in GMCs appears to be the normal behavior of interphase centrosomes in most fly cells, whereas in syncytial early embryos, both daughters retain PCM foci through the cell cycle. In contrast to both cell types, the NB daughters exhibit differential PCM retention (Rusan, 2007).
These data suggest that NBs have a novel centrosome cycle in which the second centrosome matures distant from the dominant centrosome/MTOC. One hypothesis to explain this would be the distal positioning of a differentially regulated centriole that is blocked from recruiting PCM in interphase and thus cannot form an MTOC until 'activated' during mitosis. If this centriole is always inherited by the GMC, it might also explain complete PCM loss as GMCs exit mitosis (Rusan, 2007).
Centrioles were examined live to test this hypothesis, using the centriole marker GFP-PACT and Histone-GFP Mother/daughter centrioles disengaged in late telophase, as in mammalian cells and fly embryos. Thus, two NB centrioles are present throughout interphase despite the presence of only one MTOC (Rusan, 2007).
The two centrioles then exhibit different behaviors. One remains fairly stationary, whereas the second moves to roughly the other side of the nucleus. Disengagement perfectly correlates with separation of Cnn spots, suggesting that the stationary centriole retains PCM to form the dominant MTOC and the mobile centriole completely sheds PCM. To test this, NBs were imaged expressing chTub and GFP-PACT. The stationary centriole retained MTs, whereas the mobile centriole did not. Upon reentering mitosis, the mobile centriole regained nucleation activity, forming the second MTOC (Rusan, 2007).
This suggests that full separation of the MTOCs that organize the spindle is biphasic. It begins in interphase, when one centriole retains PCM, remains stationary, and forms the dominant MTOC, whereas the second centriole sheds PCM and becomes mobile. Movement of the second centriole away from the dominant MTOC in interphase accounts for ~70% of the separation needed to form a spindle. Mechanisms of transporting the mobile centriole remain to be identified, but it is nonrandom, as in 26/30 NBs, the second MTOC emerged more than 90° from the dominant MTOC. After the second MTOC is activated, the two separate the last 30%, most likely via MT sliding forces. This might explain defects in lissencephaly1 mutants, where MTOCs are only separated by 124° at NEB. Perhaps interphase centriole movement is normal, but MT-based MTOC separation is defective (Rusan, 2007).
These data suggest that NBs differentially regulate the activity of their two centrioles within the same cytoplasm. Interestingly, a similar observation was made in clam eggs, which have three centrosomes just after fertilization. The sperm centrosome is functionally inactivated, whereas female centrosomes organize the meiotic spindle (Rusan, 2007).
Next NB centrosome regulation was examined. In preprophase, one centriole (marked by anti-DPLP) formed the dominant MTOC, whereas the second centriole had no associated MTs and was randomly positioned, confirming the live-cell data. Thus whether γtub is recruited asymmetrically was examined. Fixed preprophase NBs had two centrioles; only that opposite the GMCs accumulated γtub. Further, both γtub and Cnn are absent from the NB centriole nearest the GMCs in interphase/preprophase (Rusan, 2007).
Polo kinase promotes centrosome maturation by promoting γtub recruitment during mitotic entry. Differences in Polo localization/activity might underlie differences in timing of NB centrosome maturation. NBs expressing Polo-GFP and the centriole marker mCherry-DSAS-6 were examined. Only the centriole pair that forms the dominant centrosome was Polo-GFP positive during preprophase. Polo-GFP accumulated on the mobile centriole pair as the NB entered mitosis, increased on both centriole pairs through prophase, and moved on to kinetochores. When Polo-GFP was examined in cells exiting mitosis, it was retained at low levels on the dominant centrosome. In the future, it will be interesting to examine the localization of Aurora A, another centrosome regulator (Rusan, 2007).
Unlike the distal appendages of mammalian mother centrioles, fly mother and daughter centrioles have been thought to have no known ultrastructural or molecular differences. These data suggest that differences exist. It is unlikely that this differential regulation is a result of location, as both centrioles are initially adjacent after disengagement. The differences may be due to centriole age or procentriole maturation state (Rusan, 2007).
The NB spindle is largely aligned by NEB. Based on the current data, the hypothesis was tested that the dominant centrosome helps define one spindle pole before prophase. The angle between the dominant centrosome/MTOC axis and the anaphase axis was calculated, using the nuclear centroid as a fixed reference.
This revealed two phases in defining the future spindle axis. Through prophase onset, the dominant centrosome remains fairly stationary roughly opposite the GMCs, agreeing with fixed images, whereas the second centriole moves to a distal position (to within 46 ± 33° of the anaphase axis). This is consistent with the hypothesis. The dominant centrosome may be immobilized by aster–cortex interactions or by absence of an active displacement mechanism. In the second phase, alignment is refined in prophase and prometaphase (the angle between the NB centrosome and anaphase axes decreases from 31 ± 29° to 15 ± 12° (Rusan, 2007).
To further test whether anchoring the dominant centrosome helps roughly align the spindle, asl mutant NBs were imaged live. They lack functional centrosomes and astral MTs. Mutant NBs lack a dominant interphase centrosome, allowing assessment of its role in spindle orientation and asymmetric cell division. Live imaging revealed robust chromatin-mediated MT nucleation and spindle assembly producing fairly normal spindles. Spindle poles emerge from a disorganized MT array near the chromosomes that focuses as the spindle lengthened. Spindles do not rotate during formation, always forming along the initial pole separation axis, but do rotate during metaphase, suggesting that rotation can occur without astral MTs or that asl mutants have a reduced astral array sufficient for rotation. Surprisingly, consecutive divisions in asl mutants usually produce adjacent or near-adjacent daughters, as in wild type. In a few cases, however, spindles form parallel to the GMC cap and, presumably, the polarity axis (2/13; ~15%); these NBs divide symmetrically. This suggests that the second phase of spindle alignment can occur without a dominant centrosome and can rescue misalignment, as long as it is not too extreme, but occasional atypical symmetric divisions occur. This results in defective brain anatomy, with ectopic paired, smaller NBs, presumably progeny of symmetric divisions (Rusan, 2007).
These data reveal two new aspects of asymmetric division in this stem cell model. First, cells can adjust the canonical centrosome cycle to allow novel cell behaviors, as was observed during clam meiosis. Central brain NBs also alter this cycle: rather than both centrosomes maturing in synchrony and proximity, the two centriole pairs are differentially regulated, maturing asynchronously and distant from one another. One retains MT nucleating activity throughout the cell cycle, forming the dominant MTOC during interphase, whereas the second is initially inactive, only forming a functional centrosome and nucleating MTs at mitotic entry. One speculative possibility is that these are mother and daughter centrioles and that one is preferentially retained in the stem cell, a hypothesis that will now be tested. It is also of interest to ask whether other stem cells use this mechanism (Rusan, 2007).
Second, the data suggest that this novel centrosome cycle helps ensure high-fidelity spindle positioning and thus asymmetric division. A model is proposed in which NB mitotic spindles are aligned in two phases to ensure that GMC daughters are born next to the previous GMC. Rough alignment is achieved by confining the dominant MTOC to a relatively fixed position from the previous division and moving the second centriole to the other side of the cell. As spindles form, a second process refines this initial alignment. In asl mutants, without centrosomes, the first mechanism is inactive, but the second mechanism can align the spindle unless initial alignment is wildly off axis. In mud mutants, centriole separation must occur normally, as prophase MTOCs are nearly fully separated, but alignment of spindle poles to cortical polarity cues is defective. The normal two-step process is a robust mechanism ensuring successful asymmetric divisions and reproducible brain anatomy (Rusan, 2007).
The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. This study has tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. This work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin, an
eight-subunit complex that increases the number of spindle MTs, apparently by binding to preexisting MTs and recruiting γ-TuRC, in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules. Therefore clear evidence has been provided that all three pathways to spindle formation (centrosomal, chromatin, and acentrosomal MT organizing center driven are dependent on a fourth: Augmin. Together with recent work demonstrating that new MTs can be produced in an Augmin-dependent manner using preexisting ones generated in vitro in Xenopus egg extracts, this evidence suggests that this conserved protein complex, once active, works on all existing mitotic MTs (Hayward, 2014).
During mitosis, microtubules (MTs), dynamic polymers of α and β
tubulin, are nucleated in sufficient number so that they form a
bipolar spindle apparatus, generating the force required for
accurate alignment and segregation of duplicated chromosomes.
Work in different model organisms has shown that the
route to spindle formation can vary; for example, the MTs that
constitute spindles in Xenopus egg extracts are initially nucleated
by condensed chromatin, the assembly
of the Drosophila early embryonic spindle is regarded as centrosome
directed, while mammalian
oocytes generate MTs in the cytoplasm that gradually coalesce
to bipolarity In addition, MT-dependent MT generation, catalyzed by the
Augmin complex, provides an additional pathway that contributes
to overall spindle MT density (Goshima, 2007; Goshima, 2008;
Uehara, 2009; Wainman, 2009). Given that most
animal mitotic cells possess centrosomes and chromatin within
a substantial cytoplasm and that Augmin is functionally conserved,
one important question is whether all individual MT-generating
pathways coexist in a single cell. If they do, and if their
functions are integrated, it could explain why mature spindles are
so robust when challenged with physical, genetic, and chemical
perturbations (Hayward, 2014).
Although previous research has addressed the relationship
between centrosomal and chromatin-generated MTs these studies were undertaken prior to discovery
of Augmin (Goshima, 2007), and in tissue culture
cells. The relationship between all the major defined MT generating
pathways and their significance within a developmental
context therefore remains unclear. This study used the Drosophila
syncytial blastoderm embryo in order to comprehensively
address how a mitotic spindle forms. This tissue, in which
many hundreds of mitotic spindles form simultaneously in a
common cytoplasm, allows manipulation of MT-generating
pathways not only through genetics but also through immediate
inactivation of proteins facilitated by interfering antibody injections.
These advantages were combined with a live cold-treatment assay that allows mature embryonic mitotic spindles to be deconstructed and rebuilt and with the development
and implementation of image analysis software that
allows quantitative data to be extracted simultaneously from
multiple spindles (Hayward, 2014).
The results demonstrate that MTs can be
generated in this system by mitotic chromatin, in addition to
centrosomes, using a molecular pathway dependent on the
Drosophila homolog of the spindle assembly factor, HURP (FlyBase name: Mars).
By disrupting the accumulation of two pericentriolar material
(PCM) proteins, Spindle defective 2 (DSpd-2) and Centrosomin (Cnn), to the centrosome,
it was also found that Drosophila embryos can form bipolar
spindles from multiple cytosolic acentrosomal MT organizing
centers (aMTOCs). This study shows that all these routes to spindle
formation are supplemented by Augmin-generated MTs; inactivation
of Augmin abrogates chromatin-generated and
aMTOC-dependent MTs and substantially delays and reduces
astral MT input. It was also demonstrated that integration does,
indeed, exist between pathways. A reduction in centrosome-generated
MTs leads to an increased rate of MT nucleation
around chromatin, while a loss of chromatin- or Augmin-dependent
MT nucleation increases the growth rate of remaining
astral MTs. It was also shown that this effect is synergistic. Thus,
mitotic MT generation in a cell within a developing organism
comprises coordinated inputs from multiple MT-nucleating
pathways, providing inherent robustness and flexibility to the
mature mitotic spindle (Hayward, 2014).
These experiments demonstrate that the mature mitotic spindle in
Drosophila embryos, far from being formed via a single MT-generating
pathway dependent on centrosomally derived MTs,
is composed of MTs whose origins are, or can be, diverse. By
disrupting the molecular basis of the individual pathways, this study has assessed their relative contributions to spindle formation.
In doing so, the underlying flexibility inherent
within the system, in which removal of one MT-generating
pathway causes the cell to respond by increasing its use of
another, is demonstrated (Hayward, 2014).
The evidence supports a model in which normal, cycling
embryonic centrosomes are preprimed with PCM and γ-Tubulin
and exposed to α/β Tubulin dimer prior to the onset of mitosis
so that they, together with amplification via Augmin, nucleate
enough astral MTs to capture kinetochores quickly and efficiently
within 30 s of NEB. In contrast, the chromatin, which
is not exposed to α/β Tubulin dimer or Augmin until after NEB,
cannot participate in MT generation to any significant extent.
However, by reversing MT nucleation after mature spindle formation
through cold treatment, 'rebooting' the system in
midmitosis when both centrosomes and mitotic chromatin are
equally exposed to Tubulin and Augmin, and quantitatively
analyzing MT regrowth, it was shown that a chromatin-dependent
pathway exists and, indeed, dominates over the centrosomes
that are still present. This is not due to redistribution of
γ-Tubulin from centrosomes, as γ-Tubulin-GFP intensity is not
reduced at the centrosome in cold-treated embryos. Instead, it
appears to be a consequence of sequestering and activating
the SAF, D-HURP, around mitotic chromatin. Interestingly, in
contrast to some other biological systems, including Drosophila
S2 cells, the predominant site of new MT growth following cold
treatment is not restricted to kinetochores but occurs
throughout the region of the mitotic chromatin. Human HURP
generates and stabilizes MTs in a Ran-dependent manner. In the Drosophila embryonic
scenario, it is envisaged that cold treatment of mitotic embryos
after NEB leads to cell cycle arrest in which mitotic kinases
and the Ran gradient are fully active, allowing the association
of D-HURP with condensed chromatin where it nucleates short
MT seeds. Subsequent removal of the temperature restriction
will provide the necessary conditions for MT growth. That the
chromatin-dependent pathway is also part of the normal complement
of spindle-forming pathways in cycling embryos, but
that its input is limited until later in mitosis, is supported by the
observations that removal of D-HURP in cycling embryos results
in shorter mature spindles that have a higher likelihood of failing
in chromosome segregation (Hayward, 2014).
In addition to astral and chromatin-dependent MT generation,
this study has revealed an alternative pathway to spindle formation. A
failure to stably incorporate either DSpd-2 or Cnn to the centrosome
results in cytosolic MT asters that coalesce into mature
bipolar spindles. These aMTOCs are quite distinct from chromatin-
dependent MTs, appearing within 10 s following NEB in
regions of the cytoplasm devoid of chromosomes, and are qualitatively
similar to those reported for acentriolar Drosophila cell
lines (Moutinho-Pereira, 2009) and mouse oocytes. They may, therefore,
reflect a general mechanism of animal cell spindle formation in
the absence of functioning centrosomes, where the nucleation
and organization of MTs are achieved through concentration of
nucleating activity at multiple cytosolic sites and bipolarity follows
through their interaction and self-organization (Hayward, 2014).
This study has also provided clear evidence that all three pathways
to spindle formation (centrosomal, chromatin, and aMTOC-driven)
are dependent on a fourth: Augmin. Together with recent
work demonstrating that new MTs can be produced in an
Augmin-dependent manner using preexisting ones generated
in vitro in Xenopus egg extracts (Petry, 2013), the current evidence
suggests that this conserved protein complex, once active,
works on all existing mitotic MTs. However, whereas in Xenopus
extracts, TPX2 is required for Augmin-generated MTs (Petry, 2013),
the current in vivo analysis of spindle formation in the
absence of either D-TPX2 (using mei-38 null mutants) or
D-HURP supports a model in which D-HURP is the dominant
chromatin-directed MT nucleator in Drosophila embryos, generating
MT seeds that can then be amplified by Augmin. This likely
reflects either a difference in function between the Drosophila
and Xenopus proteins-for example, D-TPX2 shares homology
with TPX2 only in its C-terminal domain and does not possess
elements such as Aurora A targeting (Goshima, 2011), or a difference
in the usage of TPX2- and HURP-dependent pathways
by different biological systems (Hayward, 2014).
The current work also demonstrates that astral MT nucleation is
dramatically reduced in cycling embryos lacking Augmin, in a
D-TPX2- and D-HURP-independent manner. Under these conditions,
the remaining astral MTs are eventually able to search and
capture kinetochores, producing kinetochore-kMT interactions
that allow Rod poleward streaming. However, the spindle
assembly checkpoint remains unsatisfied, and Rough deal Rod-GFP movement
is perturbed, suggesting that some aspect of the interaction
is incorrect. One possibility is that Augmin binds to and
amplifies the initial kMTs, resulting in stable kMT bundles that
can stream Rod poleward. Alternatively, the effect on the checkpoint
may reflect the requirement of Augmin for generating many
short non-kMT spindle MTs. It has recently been demonstrated
that the viscoelastic properties of the Xenopus spindle, and its
ability to transmit force as a unit, can be altered by reducing
the density of such short, non-kMTs (Hayward, 2014).
It is therefore possible that Augmin-dependent non-kMTs transmit
force exerted on individual kinetochores by kMTs throughout
the spindle as part of a spindle-scale sensing mechanism, intrinsically
linked to the checkpoint. Whatever the molecular mechanism
at work, this study supports a model in which Augmin binds
indiscriminately to preexisting MTs to generate the bulk of the
embryonic mitotic spindle, placing Augmin at the heart of MT
generation during spindle formation. Interestingly, this scenario
was predicted by mathematical models of Drosophila embryonic
spindle organization, generated approximately 10 years ago. In order for their model to recapitulate
the dynamics of the anaphase spindle, the authors required
the presence of multiple short MTs with origins that were distinct
from centrosomes. Augmin fulfills such a role and, as such,
incorporating its precise mode of action into future models of
Drosophila spindle dynamics may well reveal additional features
of spindle formation (Hayward, 2014).
Although clearly essential for robust spindle formation in
mitotic systems, Augmin and, indeed, γ-Tubulin have been
shown to be dispensable for the bulk of MTs that form the initial
Drosophila female meiosis I spindle (Colombie, 2013;
Hughes, 2011). Instead, the Drosophila oocyte appears
to rely on the Chromosomal Passenger Complex (CPC), the
MT-stabilizing protein Minispindles, and the crosslinking motor
Subito to organize stable cytoplasmic MTs, generated prior to
meiosis onset, into an initial spindle structure. This likely
reflects the peculiarity of the pathways regulating formation of
the meiotic spindle in this system. Nonetheless, it does suggest
yet additional mechanisms by which a bipolar spindle can form,
further highlighting the robustness of this structure (Hayward, 2014).
Finally, importantly, this study has shown that a reduction of astral
input to spindle formation leads to an increase in chromatin-dependent
MT generation, while removal of chromatin- or
Augmin-dependent MT generation results in an increased accumulation
of EB1-GFP at MT plus ends and an increase in
the growth rate of the remaining astral MTs. The effect on
EB1-GFP accumulation is accentuated if the number of remaining
astral MTs is further reduced. These results suggest a coordinated
and synergistic cellular response to perturbing mitotic
MT-generating pathways. It is not known whether the increase
in astral MT dynamics is a passive response to availability of resources,
such as Tubulin.GTP dimer, or an active self-regulation,
driven by monitoring of MT generation by the cell. In the simplest
(passive) scenario, removing the MTs generated by one pathway
could result in an increase in the available local concentration of
Tubulin.GTP, shifting the dynamic equilibrium of remaining MTs
further toward growth. Although in vitro studies have shown that
increasing the concentration of Tubulin in solution increases MT
growth rates and that this correlates with
increased accumulation of EB1 at the growing tips,
the high diffusion rate of Tubulin-GFP in the early
embryo essentially rules out local depletion of resources close
to individual MT tips as a source of variability.
Therefore, if resource depletion is responsible for limiting
MT growth, it must be a global (spindle-scale) depletion. However,
the increase in EB1 comet length and MT dynamics that
occurs upon the loss of MT-generating pathways was measured
in the early stages of spindle regrowth. At similar time points in
embryos possessing all MT-generating pathways, the EB1 fluorescence
(i.e., MT growth) in the region of the chromatin continues
to increase dramatically over the following 2 min. Therefore, at these early time points,
Tubulin.GTP, EB1, or any other cytoplasmic molecule that stimulates
MT growth, cannot be depleted and therefore cannot be
limiting growth. This leaves open the intriguing possibility that
the cell somehow actively monitors the overall level of MT generation
during spindle formation and alters flow through available
pathways accordingly. Given this possibility, an important future
goal will be to identify MAPs whose association with MTs
changes upon inhibition of particular MT-generating pathways (Hayward, 2014).
In summary, by revealing the presence of all the major mitotic
MT-generating pathways described in animal cells within a
single system, the Drosophila syncytial embryo, and by demonstrating
a coordinated regulation between them, this work
highlights the remarkable flexibility inherent in mitotic spindle
formation. It implies that the key to building a successful spindle
lies in activating a set of MT generators that together provide
sufficient MTs to allow crosslinking and movement in relation
to one another, regardless of how and where the MTs were
initially generated. By subsequently limiting the nucleation and
growth of these MTs to balance depolymerization, a steady-state
spindle of defined length and physical properties is
ultimately formed. Understanding the way in which a cell determines
such a 'Goldilocks zone' of MT generation will undoubtedly
help lead to an understanding of the overall self-regulation of this
fundamental cellular structure (Hayward, 2014).
Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4; see Drosophila SAK) counterpart in other organisms. Although Plk4's centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. This study shows that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent (Dzhindzhev, 2014).
Asymmetric division of adult stem cells generates one self-renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. This study shows that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells (GSCs) are oriented within their niche and this ensures asymmetric division. GSCs containing misoriented centrosomes accumulate with age, and these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re-enter the cell cycle on correction of centrosome orientation. On the basis of these findings, it is proposed that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. It was also shown that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia (Cheng, 2008).
GSCs with misoriented centrosomes accumulate as flies age. Since such misoriented GSCs divide less frequently as compared to oriented GSCs, accumulation of misoriented GSCs contributes to the decline in spermatogenesis that occurs with age. Although misoriented GSCs rarely divide, they are not permanently arrested (or senescent) and are correctly oriented when they divide. Whether correction of GSC orientation is an active process that is part of the acquisition of stem cell identity remains to be determined. The low cell cycle activity of misoriented GSCs may also suggest that mechanisms are in place to detect misorientation and induce cell cycle arrest in response to this change, although the underlying mechanisms remain to be identified (Cheng, 2008).
It was also demonstrated that misoriented GSCs originate, at least in part, from dedifferentiation of spermatogonia. Although dedifferentiated GSCs have high frequency (>40%) of centrosome misorientation, they can function as stem cells by resuming the cell cycle, with correctly oriented mitotic spindles just like as constitutive GSCs. GSC numbers do not decrease as quickly as expected from the calculated GSC half-life, suggesting that a mechanism to compensate for the loss of GSCs exists. Since misoriented spindles, or symmetric stem cell division, was rarely observed, it is speculated that dedifferentiation is the major mechanism to replace stem cells over time in the Drosophila male germ line (Cheng, 2008).
A decline in GSC number in older males (day 50) was reported recently (Boyle, 2007) This decrease in stem cell number is likely due to failure of the niche function (via decreased signal from the niche as well as decreased E-cadherin-based attachment between the niche and GSCs. However, the decrease in the production of spermatogonia and testis involution precede the loss of GSCs such that decreasing GSC numbers cannot explain the testis involution that is observed at younger ages (Cheng, 2008).
The present results provide a novel mechanistic link between the control of stem cell polarity and the age-related decline in tissue regenerative capacity. Mechanisms responsible for monitoring stem cell orientation with respect to the niche not only prevent overproliferation of stem cells by ensuring the asymmetric outcome of the stem cell division, but they contribute to the decline in tissue regenerative capacity during aging. Many of the misoriented GSCs originate from the dedifferentiation of spermatogonia, a mechanism thought to be responsible for maintaining the stem cell population over extended periods of time. Therefore, although GSCs produce less progeny over time, the system appears to maximize the number of progeny produced throughout life, while maintaining asymmetric stem cell division (Cheng, 2008).
In summary, it is proposed that the GSCs with misoriented centrosome divide less frequently and that a combination of such a decreased stem cell division and a higher frequency of the GSC misorientation in aged testes leads to a decline in spermatogenesis with age (Cheng, 2008).
Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. This study shows that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex (Wang, 2009).
Radial glia cells constitute a major population of neural progenitor cells that occupy the proliferative VZ in the developing mammalian neocortex. In addition to their well-characterized function as a scaffold in supporting neuronal migration, radial glia cells display interkinetic nuclear oscillation and proliferate extensively at the luminal surface of the VZ (that is, the VZ surface). During the peak phase of neurogenesis [around embryonic day 13-18 (E13-E18) in mice] they predominantly undergo asymmetric division to self-renew while simultaneously giving rise either directly to a neuron, or to an intermediate progenitor cell which subsequently divides symmetrically to produce neurons. Whereas differentiating progeny progressively migrate away from the VZ to form the cortical plate (CP) (the future neocortex) renewing radial glia progenitors remain in the VZ for subsequent divisions. The distinct migratory behaviour of radial glia progenitors and their differentiating progeny is fundamental to the proper development of the mammalian neocortex; however, little is known about the basis of these behavioural differences (Wang, 2009).
Centrosomes, the main microtubule-organizing centres in animal cells, have an important role in many cell processes, particularly during cell division. All normal animal cells initially inherit one centrosome, consisting of a pair of centrioles surrounded by an amorphous pericentriolar material. The two centrioles differ in their structure and function. The older 'mother' centriole, which is formed at least one-and-a-half generations earlier, possesses appendages/satellites that bear specific proteins, such as cenexin (also known as Odf2), and anchor microtubules and support ciliogenesis. In contrast, the younger 'daughter' centriole, which is formed during the preceding S phase, lacks these structures. Full acquisition of appendages/satellites by the daughter centriole is not achieved until at least one-and-a-half cell cycles later. During each cell cycle, the centrosome replicates once in a semi-conservative manner, resulting in the formation of two centrosomes: one of which retains the original old mother centriole (that is, the mother centrosome) while the other receives the new mother centriole (that is, the daughter centrosome). This intrinsic asymmetry in the centrosome has recently been demonstrated to be important for proper spindle orientation during the division of male germline stem cells and neuroblasts in Drosophila, although female germline stem cells appear to divide normally in the absence of centrioles/centrosomes. These studies indicate a critical role for the differential behaviour of centrosomes with differently aged mother centrioles in asymmetric division of the progenitor/stem cells, although it remains unclear whether proper behaviour and development of the progenitor/stem cells and their differentiating daughter cells depend on centrosome asymmetry. Asymmetric division of radial glia progenitors accounts for nearly all neurogenesis in the developing mammalian neocortex. Three out of four autosomal recessive primary microcephaly (MCPH) genes identified so far encode centrosomal components, suggesting that proper neocortical neurogenesis and development entail a tight regulation of the centrosome, which is so far poorly understood. To address these issues, centrosome regulation during the peak phase of mammalian neocortical neurogenesis was investigated (Wang, 2009).
To examine centrosome behaviour, a plasmid encoding centrin 1, a central component of the centriole, fused with enhanced green fluorescent protein (EGFP-CETN1) was introduced into the developing neocortex of E13.5 mouse embryos by in utero electroporation. As expected, EGFP-CETN1 formed pairs of dots that co-localized with γ-tubulin, a centrosomal marker, suggesting that transient expression of EGFP-CETN1 reliably labels the two centrioles of individual centrosomes in the developing neocortex in vivo. Moreover, it was observed that at the onset of peak neurogenesis (E13-E14), EGFP-CETN1-labelled centrosomes were predominantly located at the VZ surface with a small subset located in the subventricular zone (SVZ) and the intermediate zone (IZ) (Wang, 2009).
To identify the cell types harbouring EGFP-CETN1-labelled centrosomes, a plasmid encoding DsRedexpress (DsRedex), a red fluorescent protein that diffuses throughout cells and thereby reveals their morphology, was co-electroporated . In bipolar radial glia progenitors in the VZ the centrosome was located in their ventricular endfeet at the VZ surface, whereas in multipolar cells in the IZ and the SVZ the centrosome was harboured in their cell bodies. Moreover, dividing radial glia progenitors were observed that possess a pair of centrosomes together with condensed chromosomes at the VZ surface. Consistent with this differential centrosome localization between radial glia progenitors and their differentiating daughter cells, a progressive increase was observed in the appearance of EGFP-CETN1-labelled centrosomes in the IZ and the CP as development proceeded, in addition to some that remained at the VZ surface. This gradual increase in centrosome localization in the IZ and the CP coincided with the production and migration of differentiating cells such as neurons to these regions during this period (Wang, 2009).
The distinct positioning of the centrosome in radial glia progenitors versus their differentiating progeny prompted asking whether the centrosomes in these two cell populations/types are different. To explore this, a plasmid encoding ninein, a mature centriole-specific protein that localizes to appendages/satellites, fused with EGFP (EGFP-Nin) together with a plasmid encoding CETN1 fused to DsRedex (DsRedex-CETN1) were electroporated into the developing mouse neocortex at E13.5. As expected, both EGFP-Nin and DsRedex-CETN1 formed dot-like structures and co-localized to the centrosomes, especially those at the VZ surface, as identified by an antibody to the integral centrosomal protein pericentrin 1. Notably, EGFP-Nin was preferentially concentrated at one of the two centrioles marked by DsRedex-CETN1 in individual centrosomes, suggesting that the two centrioles in interphase radial glia progenitors are not identical. Given that Nin specifically associates with mature centrioles, these results indicate that the centriole with abundant EGFP-Nin is the more mature mother centriole, whereas the one with little EGFP-Nin is the less mature daughter centriole. A similar inequity in the recruitment of EGFP-Nin by the duplicated centrosomes was also observed in dividing radial glia progenitors at the VZ surface, indicating that the duplicated centrosomes are not identical during late mitosis. The centrosome with abundant EGFP-Nin is probably the centrosome that retains the mature old mother centriole and the centrosome with little EGFP-Nin is probably the centrosome that bears the relatively immature new mother centriole (Wang, 2009).
Having found that the centrosomes in dividing radial glia progenitors exhibit asymmetry in their maturity, it was next asked whether this centrosome asymmetry is related to the distinct behaviour of radial glia progenitors and their differentiating progeny in the developing neocortex during neurogenesis. To address this, the relative distribution of centrosomes labelled by DsRedex-CETN1 versus those labelled by EGFP-Nin in the developing neocortex were compared as development proceeded. Interestingly, whereas DsRedex-CETN1-labelled centrosomes progressively occupied the IZ and the CP, where differentiating cells are situated, EGFP-Nin-labelled centrosomes were mostly found in the VZ, where radial glia progenitors are located. Given that DsRedex-CETN1 labels all centrosomes whereas EGFP-Nin selectively labels mature centrosomes, these results point to an intriguing possibility that the duplicated centrosomes in dividing radial glia cells are differentially inherited depending on their age and maturity during neocortical neurogenesis. It is known that during each cell division one centrosome retains the old mature mother centriole and the other bears the new less mature mother centriole. Thus, these results suggest that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors (Wang, 2009).
To test this, an assay was developed to distinguish explicitly between the centrosome containing the old mother centriole and the centrosome containing the new mother centriole in the developing neocortex in vivo. The assay takes advantage of the photoconvertible fluorescent protein Kaede, which changes from green to red fluorescence on exposure to violet light. Centrioles in the developing neocortex were labelled by transient expression of CETN1 fused with Kaede (Kaede-CETN1). Photoconversion was then performed to switch labelled centrioles from green to red fluorescence. This green-to-red fluorescence conversion of Kaede proteins is irreversible and the red protein is very stable, thus allowing long-term tracking of the existing photoconverted proteins and the structures with which they are associated. Moreover, all newly synthesized Kaede proteins are green fluorescent. It is known that centriole duplication requires new protein synthesis of centrin. As a result, newly duplicated centrioles that contain newly synthesized Kaede-CETN1 are green fluorescent, whereas previously existing centrioles are red fluorescent. Hence, in the first cell cycle after photoconversion, both centrosomes contain a red fluorescent mother centriole and a green fluorescent daughter centriole. However, in the second and subsequent cell cycles, centrosomes with the new mother centriole contain only green fluorescent centrioles, whereas centrosomes retaining the original old mother centriole harbour both red and green fluorescent centrioles, thus distinguishing between centrosomes with differently aged mother centrioles (Wang, 2009).
To carry out this assay in the developing neocortex in vivo, an in utero photoconversion procedure was developed and and combined with in utero electroporation. Kaede-CETN1, which localized specifically to the centrosomes, was introduced into the developing mouse neocortex at E13.5. One day later, that is, E14.5, the forebrain of electroporated embryos was treated with a short exposure of violet light while still in the uterus, which effectively converted nearly all Kaede-CETN1 proteins and their labelled centrosomes from green to red fluorescence [E13.5-E14.5(PC)]. The uterus was replaced and the embryos continued to develop in vivo. The localization and inheritance of centrosomes were analysed at different developmental stages thereafter (Wang, 2009).
It was found that one day after photoconversion [E13.5-E14.5(PC)-E15.5], around 95% of centrosomes contained both red and green fluorescent centrioles (indicated by yellow colour in the merged image), consistent with the notion that the labelled cells have undergone one round of division and have duplicated their centrioles during the 24-h period after photoconversion. This was shown directly by imaging centrosomes at high magnification, revealing that each centriole was mostly only red or green fluorescent. This also demonstrates that there is little diffusion of centrin proteins between duplicated centrioles, or between the centrioles and a cytoplasmic pool which was confirmed by fluorescence recovery after photobleaching (FRAP) experiments. Moreover, it was found that more than 30% of centrosomes possessed only green fluorescent centrioles 2 days after photoconversion [E13.5-E14.5(PC)-E16.5]. The appearance of the solely green fluorescent centrosomes 48 h after photoconversion indicates that the initially labelled radial glia progenitors have undergone two rounds of division during this period; this is consistent with the previous observation that the duration of the neocortical progenitor cell cycle is about 12 to 20 h around this developmental stage. The ongoing division of labelled radial glia cells at a normal rate suggests that expression of Kaede-CETN1 and the photoconversion procedure had no effect on their cell cycle. In addition, no obvious DNA damage or cell death was induced by the photoconversion treatment. Besides the green and yellow fluorescent centrosomes, about 4% of solely red fluorescent centrosomes were observed, indicating that a few labelled cells do not undergo cell division during this period (Wang, 2009).
Having successfully distinguished the centrosomes with differently aged mother centrioles in the developing neocortex in vivo, their distribution was examined to determine whether they are asymmetrically segregated. Remarkably, it was found that more than 76% of centrosomes with the new mother centriole (that is, only green fluorescent) were located in the IZ and the CP, whereas around 78% of centrosomes with the old mother centriole (that is, both green and red fluorescent) were located in the VZ in addition to the SVZ. These results demonstrate that the centrosomes with differently aged mother centrioles are asymmetrically segregated in the developing neocortex during the peak phase of neurogenesis. It is worth noting that a small fraction of both green and red (that is, yellow) fluorescent centrosomes was found in the IZ and the CP and that these centrosomes probably originated from the first cell cycle after photoconversion (Wang, 2009).
The asymmetric segregation of centrosomes suggests differential regulation of the duplicated centrosomes in dividing radial glia progenitors. To gather further evidence for this, time-lapse imaging experiments were carried out to monitor the behaviour of centrosomes with differently aged mother centrioles in dividing radial glia progenitors at the VZ surface in situ. Kaede-CETN1 was introduced into radial glia cells together with mPlum, a far-red fluorescent protein, to label cell morphology. Around 24 h later, cortical slices were prepared. Photoconversion of the existing Kaede-CETN1 proteins was then performed in individual slices, which were then cultured for another 24 h before being subjected to time-lapse imaging. Labelled dividing radial glia cells with enlarged and rounded cell bodies possessing a pair of centrosomes at the VZ surface were monitored at 10-min intervals over a period of 5 to 8 h. In six out of seven dividing radial glia cells that proceeded through mitosis at the VZ surface and reached the two-cell stage, the centrosome retaining the old mother centriole in both red and green fluorescence stayed at the VZ surface, whereas the centrosome containing the new mother centriole in solely green fluorescence migrated away from the VZ surface. These results demonstrate that the centrosomes with differently aged mother centrioles in dividing radial glia progenitors exhibit distinct behaviour during the peak phase of neurogenesis (Wang, 2009).
The distinct behaviour of the centrosomes suggests that they are differentially inherited by the two daughter cells embarking on different routes of fate specification and development. On the basis of their behaviour, it is postulated that the centrosome with the new mother centriole is largely inherited by differentiating cells, such as neurons, whereas the centrosome with the old mother centriole that remains located at the VZ is mostly inherited by radial glia progenitors. Indeed, it was found that 2 days after photoconversion [E13.5-E14.5(PC)-E16.5] the centrosomes with the new mother centriole, marked by green fluorescence alone, were mostly associated with cells expressing TUJ1, a differentiating neuronal marker, in the CP and the IZ. In contrast, the centrosomes that retained the old mother centriole in yellow (that is, both green and red) fluorescence at the VZ were largely associated with cells expressing Pax6, a radial glia progenitor marker. These results show that the centrosomes with differently aged mother centrioles in dividing radial glia cells are asymmetrically inherited by the two daughter cells: whereas the renewing radial glia progenitor inherits the centrosome with the old mother centriole, the differentiating daughter cell inherits the centrosome with the new mother centriole (Wang, 2009).
These data thus far show that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors in the developing neocortex. Next, whether the selective inheritance of the centrosome with the old mature mother centriole by radial glia progenitors is necessary for their maintenance at the VZ was tested. Should this be the case, given that Nin is an essential component of the appendage/satellite structures specific to the mature centriole, it was predicted that removal of Nin, which prevents centriole maturation, would disrupt asymmetric segregation of centrosomes with differently aged mother centrioles and impair the maintenance of radial glia progenitors in the developing neocortex (Wang, 2009).
To test this, short hairpin RNA (shRNA) sequences were developed against Nin, that effectively suppressed its expression (Nin shRNAs). Consistent with the prediction, expression of Nin shRNA, but not control shRNA, disrupted asymmetric segregation of centrosomes with differently aged mother centrioles labelled with Kaede-CETN1 in the developing neocortex, suggesting that Nin is necessary for centriole maturation, thereby generating asymmetry between duplicated centrosomes. The presence of solely green fluorescent centrosomes in Nin shRNA-expressing cortices indicates that centrosome duplication and segregation and cell division are not severely affected by removal of Nin. More importantly, it was found that removal of Nin caused a premature depletion of cells from the VZ, where radial glia progenitors reside. This effect of Nin shRNAs correlated with their efficacy in suppressing Nin protein expression and was rescued by a shRNA-insensitive Nin plasmid, suggesting that the effect of the Nin shRNA is due to a specific depletion of the endogenous Nin protein. A similar reduction in cells in the VZ was observed when Nin expression was suppressed using small interfering RNA (siRNA) (Wang, 2009).
To characterize further the extent to which removal of Nin leads to a depletion of radial glia progenitors, the fate specification was examined of cells expressing either control or Nin shRNA. When compared with the control, expression of Nin shRNA led to a marked reduction in the percentage of cells positive for Pax6 and glutamate transporter (GLAST), two radial glia progenitor markers, and a significant increase in the percentage of cells positive for TUJ1, a differentiating neuronal marker. These results suggest that removal of Nin leads to a depletion of radial glia progenitors and a concomitant increase in differentiating neurons. Consistent with this, a significant reduction was observed in phospho-histone 3 (P-H3)-labelled mitotic cells at the VZ surface and a marked increase in cell cycle exit. No obvious change in the cleavage plane orientation of late stage mitotic cells at the VZ surface was observed (Wang, 2009).
Previous studies showed that the carboxy-terminus of Nin is responsible for its localization to the centriole and expression of this region displaces endogenous protein at the centriole. Interestingly, it was found that, similar to removal of Nin, expression of the carboxy-terminus of Nin (Nin-Cter) led to a premature depletion of radial glia progenitor cells from the VZ, suggesting that centriolar Nin is critical for maintaining radial glia progenitor cells in the VZ. Taken together, these results indicate that preferential inheritance of a centrosome containing the mature mother centriole is required for the maintenance of radial glia progenitors in the proliferative VZ of the developing neocortex (Wang, 2009).
The results presented in this study suggest that the centrosomes with differently aged centrioles in asymmetrically dividing radial glia progenitors exhibit different behaviour and are differentially inherited by the two daughter cells during the peak phase of mammalian neocortical neurogenesis. Whereas the centrosome with the less mature new mother centriole migrates away from the VZ surface and is largely inherited by differentiating cells, the centrosome with the more mature old mother centriole stays at the VZ surface and is predominantly inherited by renewing radial glia progenitors. Recently, asymmetric behaviour of centrosomes has been observed during asymmetric division of Drosophila male germline stem cells and neuroblasts. The findings of this study suggest that this type of asymmetric centrosome regulation may be a general feature of asymmetric cell division across species. Furthermore, the findings provide new insight into centrosome regulation in the developing mammalian neocortex, which has been linked to the pathogenesis of human microcephaly (Wang, 2009).
Centrosomes with differently aged mother centrioles differ in their protein composition and thereby in their biophysical properties, such as microtubule anchorage activity. In this study, it was found that Nin, an appendage/satellite-specific protein required for centriole maturation, localized differently to the duplicated centrosomes in radial glia progenitors in late mitosis. Notably, another appendage/satellite-specific protein cenexin was recently found to be asymmetrically localized to centrosomes in sister cells after mitosis; moreover, the cell receiving the more mature old mother centriole usually grew a primary cilium first (Anderson, 2009). The asymmetric inheritance of centrosomes with distinct biophysical properties may thereby differentially regulate the behaviour and development of the daughter cells that receive them. For example, given that primary cilia have essential roles in a number of signal transduction pathways, including Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signalling, the asynchrony in cilium formation could differentially influence the ability of the two daughter cells to respond to environmental signals and thereby their behaviour and fate specification. Furthermore, the strong microtubule anchorage activity associated with the centrosome retaining the older mother centriole would facilitate its anchorage to a specific site (for example, the VZ surface), thereby tethering the cell that inherits it. Indeed, it was found that disruption of centriole maturation by removing Nin not only impairs asymmetric segregation of centrosomes, but also depletes radial glia progenitors from the VZ, a proliferative niche in the developing mammalian neocortex. Aside from their participation in microtubule organization and ciliogenesis, centrosomes associate with messenger RNAs (mRNAs) and membrane-bound organelles such as the Golgi and recycling endosomes and regulate protein degradation, thereby raising the possibility that asymmetric centrosome inheritance might contribute to proper segregation of cell fate determinants to the two daughter cells of asymmetrically dividing progenitor/stem cells (Wang, 2009).
Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. This has been addressed by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. In Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, this study showed that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species (Smyth, 2015).
Cells face a significant challenge each time they divide, because not only must they faithfully replicate and partition their genomic DNA, they must also expand and partition their cytoplasmic contents and organelles as well. It is particularly important that cells inherit sufficient quantities of functionally competent organelles with each division as cells cannot generate many of their organelles de novo. Despite this, mechanisms that ensure proper partitioning of organelles during cell division, particularly membrane-bound organelles like mitochondria and the endoplasmic reticulum (ER), are poorly understood. Delineating these mechanisms is key to understanding how organelle-specific functions regulate proper development, tissue homeostasis and injury repair.
The ER is the largest membrane-bound organelle in the cell, and its functions include folding and trafficking of secretory proteins, lipid synthesis and transport, and regulation of cytoplasmic Ca2+. During interphase, the ER is continuous with the nuclear envelope (NE) and is distributed throughout the cytoplasm as a network of broad sheets, or cisternae, and thin tubules. This interphase ER distribution depends in large part on numerous associations with the microtubule (MT) cytoskeleton, which involve MT motor-dependent transport, connections with growing MT tips and stable attachments along MT filaments. Importantly, the roles of particular ER morphologies and MT associations in specific ER and cellular functions are poorly understood. It is also unclear whether specific regulation of ER morphogenesis or distribution is required during cell division for the proper execution of mitosis or to ensure functional ER partitioning to progeny cells (Smyth, 2015).
Two hypotheses have been proposed to explain ER partitioning and inheritance during cell division. The first proposes that the ER is actively segregated during division, probably through interactions with cytoskeletal elements. This would provide a mechanism for specific regulation of ER partitioning to progeny cells. In support of this, and consistent with the association of the ER with MTs during interphase, the ER localizes to the MT-based mitotic spindle in a variety of cell types from different species including sea urchin and Drosophila embryos and mammalian tissue culture cells (Smyth, 2015).
Thus, it is expected that disruption of ER-spindle interactions would disrupt ER functions in progeny cells. However, the specific factors that physically link the ER with spindle MTs have not been identified in any animal cell type, and this has precluded a direct test of whether the ER-spindle association is required for functional ER partitioning. Further, several recent studies showing that the ER remains mostly peripheral to the mitotic spindle with no obvious MT contacts, particularly in cultured human cells, have challenged the idea that spindle association is a universal requirement for ER partitioning. These findings support the second hypothesis, which proposes that stochastic distribution of the ER throughout a dividing cell is sufficient to ensure adequate partitioning to progeny cells. Thus, although the ER is associated with MTs in some dividing cells, this active segregation may not be strictly required as long as each progeny cell acquires enough organelle material. However, it is notable that dissociation of the ER from spindle MTs is most readily apparent in cultured cells such as HeLa and Cos-7, and these cells may not have strict requirements for precise ER inheritance. By contrast, when cells divide in the context of a developing organism in which spatial and temporal coordination of cellular events is crucial, small alterations to ER partitioning may have far-reaching effects. This illustrates the critical importance of studying mitotic ER partitioning in cells dividing within intact, developing tissues, in order to understand how the partitioning mechanisms function within physiological cellular processes (Smyth, 2015).
A striking example of how active segregation of cellular components during cell division can have significant consequences for progeny cells within a developing or functional tissue is asymmetric stem cell division. During asymmetric stem cell division, differential partitioning of specific factors results in two progeny cells with different identities or fates, most commonly with one cell programmed to remain a stem cell and the second cell becoming a tissue-specific effector. The establishment of asymmetry in these dividing cells raises an important question that has never been addressed: is the ER asymmetrically partitioned during asymmetric stem cell division? If so, then this would strongly support the hypothesis that highly regulated, active segregation of the ER is required during in vivo cell division. Further, by integrating ER dynamics with known mechanisms that establish asymmetry in these cells, it may be possible to glean novel insights into ER partitioning mechanisms. This approach was taken in the current study by analysing ER partitioning in asymmetrically dividing Drosophila neural stem cells known as neuroblasts (NBs). Asymmetric NB divisions produce a large cell that retains NB identity, and a much smaller ganglion mother cell (GMC) that differentiates to form a functional neuron or glial cell. The analyses define an asymmetric segregation of the ER to the mitotic spindle poles that results in a larger proportion of the organelle being partitioned to the future stem cell. It was also shown that active, MT-dependent spindle pole segregation is required in vivo for proper ER partitioning in both asymmetrically and symmetrically dividing cells, as well as in human culture cells. Thus, active spindle pole segregation may be a highly conserved mechanism of ER partitioning that can be subject to precise regulation during specific developmental processes, such as asymmetric stem cell division (Smyth, 2015).
The mechanisms that regulate ER partitioning in dividing animal cells are far from clear, and the fundamental issue of whether the organelle is actively partitioned or stochastically distributed remains controversial. These controversies may be due to several factors in previous studies that have addressed the issue of ER partitioning: first, several recent studies have relied on cultured, transformed cell lines such as HeLa cells that may not recapitulate physiologically relevant processes [9,16,28]; second, potential active partitioning mechanisms, such as spindle MT interactions, have not been directly tested; and third, most analyses have focused on ER interactions with the inner spindle MTs while largely neglecting astral MTs. The current study addresses these issues by combining genetic and pharmacological manipulations with analysis of ER partitioning in vivo in intact Drosophila tissues, as well as in cultured cells. The results show that in the Drosophila cell types examined, the ER is recruited to centrosomes early in cell division, probably through interactions with centrosomal MTs. This recruitment in prophase is concomitant with centrosome maturation, the process whereby more pericentriolar material is recruited to the centrosome to afford greater MT nucleation and anchorage in preparation for spindle formation (Smyth, 2015).
Several models could explain this increase in recruitment of ER to mature centrosomes and astral MTs. One model is that cells simply use the same mechanism of linking the ER and MTs in both interphase and mitosis. Therefore, by increasing MT density at the centrosome in mitosis, more ER is recruited and concentrated at the developing poles. An alternative model is that a new ER-MT linking mechanism is engaged, or activated specifically in mitosis, potentially through regulation by mitotic cyclin/cdks. There is precedence for controlling the ER-MT linkage in a cell-cycle-specific manner-STIM1 phosphorylation in mitosis disengages the ER from spindle MTs. Although this is a form of negative regulation, it is not unreasonable to hypothesize the presence of a parallel positive regulatory mechanism. In either model, it is still puzzling as to why the ER is not recruited to all MTs-why is it specific to astral and peripheral interpolar MTs? One hypothesis is that the MT density, or the viscosity of the spindle, forms a physical barrier that prevents ER entry to the interpolar region. This hypothesis is not favored because it is known that artificially linking the ER to MTs using a non-phosphorylatable STIM1 construct can force the ER deep into the spindle region. An alternative hypothesis is favored, whereby specific subpopulations of MTs convey ER-binding capability, while others do not. How might this occur? The complexity of MT modifications, which can have dramatic effects on MT behaviour and function, is beginning to be appreciated. A very relevant finding is the presence of detyrosinated tubulin exclusively within the spindle and not in astral MTs. One plausible hypothesis is that an unknown ER-MT linker protein cannot bind detyrosinated MTs, which would result in ER exclusion from the spindle region. This would be an exciting future direction because of its implication in asymmetric stem cell divisions-one might envision that unique MT modifications could exist on the apical versus basal spindle poles. In addition, MT motor-dependent ER sliding events preferentially occur on acetylated MTs, further supporting a role for MT modifications in dynamic ER regulation (Smyth, 2015).
As the cell proceeds through subsequent stages of mitosis, the ER remains associated with astral MTs, resulting in active partitioning of the organelle to the two progeny cells. This study further showed that disruption of centrosomes and astral MTs leads to ER partitioning defects, confirming the obligate role of these cytoskeletal structures. These mechanisms are not limited to Drosophila cells, as it is shown that mitotic ER positioning also depends on astral MTs in human cells. The important and novel finding is also presented that the ER is asymmetrically partitioned in asymmetrically dividing neural stem cells. This ER asymmetry is probably dependent on centrosomal and MT asymmetry, and results in a higher concentration of ER in the regenerating stem cell compared with the differentiating GMC. Thus, the data suggest that association of the ER with astral MTs may be a universal mechanism of ER partitioning that can be adapted within specific cellular or developmental contexts such as during asymmetric stem cell division. Further, the NB results present the provocative possibility that ER partitioning may have a specific role in the mechanism of asymmetric cell division or tissue development (Smyth, 2015).
Identification of the molecular factors that link the ER with spindle MTs is the key to further understanding the role of ER partitioning in organismal physiology and development. Importantly, it is likely that the molecular features of ER partitioning, including the specific factor that links the ER to astral MTs, are highly conserved as similarities are seen in systems as disparate as mitotic HeLa and meiotic Drosophila spermatocytes. It is believed that Drosophila, with its powerful genetic tools and in vivo analyses, is an ideal system in which to further investigate these mechanisms. A distinct possibility is that MT motors are involved, and systematic analysis of all known Drosophila MT motors is a clear and tenable approach. Other proteins such as spastin, Climp-63 and REEPs have also been shown to associate the ER with MTs in various cell types, and possible roles of these proteins in spindle MT attachment should be studied further. Most significant among these, it was recently shown that REEP3 and REEP4 are required for spindle pole focusing in HeLa cells, suggesting a potential role for these proteins in mitotic ER partitioning (Smyth, 2015).
In conclusion, the results demonstrate that interaction of the ER with spindle MTs is a conserved mechanism that controls the distribution of the organelle during animal cell division. This facilitates equal partitioning of the organelle during symmetric cell divisions, but also allows for specific regulation of ER distribution during specialized processes such as asymmetric cell division. Drosophila will be a powerful system moving forward to identify the specific molecular mechanisms involved and the functional roles of ER partitioning in organismal physiology (Smyth, 2015).
In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP-orthologues of ZYG-1, SAS-6, and SAS-4, respectively-are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. This study finds that of eight candidate duplication factors identified in cultured fly cells, only two, anastral spindle 2 (Ana2) and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. This study shows that Ana2 is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. It is proposed that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery (Stevens, 2010).
The centriole is composed of a radial array of nine microtubule (MT) triplets, doublets, or singlets depending on species and cell type. Centrioles are required to make two important cellular structures: centrosomes and cilia. The centrosome consists of a pair of centrioles surrounded by pericentriolar material (PCM) and is the major MT organizing center in many animal cells. Cilia are formed when the centriole pair migrates to the cell cortex, and the older, mother, centriole forms a basal body that nucleates the ciliary axoneme. Many different cell types possess cilia, and they have multiple roles in development (Stevens, 2010).
To ensure their inheritance by each daughter cell, centrioles duplicate precisely once per cell cycle. This process must be tightly regulated. Failure in centriole duplication leads to catastrophic errors during embryogenesis in both worms and flies, and an increasing number of human diseases have been linked to defects in centrosome and/or cilia function. Centriole overduplication can be equally damaging, as excess centrioles are frequently observed in human tumors, and there appears to be a direct causative relationship between centriole overduplication and tumorigenesis in flies (Stevens, 2010).
In canonical centriole duplication, a new daughter centriole grows at a right angle to the mother centriole. A series of genome-wide RNAi and genetic screens in worms have found just five proteins essential for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. SPD-2 is required to recruit the kinase ZYG-1 to the centriole, and both proteins then recruit a complex of SAS-5 and SAS-6. SAS-5 and SAS-6 are mutually dependent for their centriolar localization and are in turn needed to recruit SAS-4 (Stevens, 2010 and references therein).
Although DSpd-2 is not essential for centriole duplication in flies, and no SAS-5 homologues have been identified outside worms, proteins related to ZYG-1, SAS-6, and SAS-4 have a conserved role in centriole duplication in other systems. In Drosophila, for example, the kinase Sak, which is related to ZYG-1, and the homologues of SAS-6 (DSas-6) and SAS-4 (DSas-4) are required for centriole duplication. Recently, however, several additional proteins have been identified in cultured fly cells that are potentially involved in centriole duplication. This study set out to identify which of these potential duplication factors are likely to function as upstream regulators of centriole formation (Stevens, 2010).
Genome-wide RNAi screens in cultured fly cells identified just 18 proteins that, when depleted, gave a reduced number of centrioles. This list includes Sak, DSas-6, and DSas-4, as well as eight other proteins that specifically localize to centrosomes (Ana1, Ana2, Ana3, Asl, DCP110, DCep135/Bld10, DCep97, and Rcd4): these eight are therefore good candidates to play a direct role in centriole duplication (Stevens, 2010).
GFP-Sak, GFP-DSas-6, and DSas-4-GFP share the unique ability to drive de novo formation of centriole-like structures in unfertilized eggs when highly overexpressed from the upstream activation sequence (UAS) promoter. UAS-GFP-Sak and UAS-GFP-DSas-6 induce these structures in ~95% of unfertilized eggs, whereas UAS-DSas-4-GFP does so in ~60% of unfertilized eggs. It was asked if this assay could be used to identify other components likely to function upstream in the centriole duplication pathway. Transgenic lines were generated carrying GFP fusions to all eight potential duplication factors under the control of the UAS promoter, which allowed overexpression in unfertilized eggs. Strikingly, only Ana2 (in 97% of eggs) and Asl (in 33% of eggs) were able to drive de novo formation of centriole-like structures (Stevens, 2010).
Asl has recently been shown to be essential for centriole duplication in flies, whereas, of the six proteins unable to induce de novo centriole formation, two, DCep135/Bld10 and Ana3, are now known not to be essential for centriole duplication in flies. These findings indicate that the overexpression assay can identify those proteins likely to be most intimately involved in centriole duplication. Since Asl has already been shown to be required for centriole duplication, focus was placed on investigating the function of Ana2 (Stevens, 2010).
Ana2 can drive de novo formation of centriole-like structures as efficiently as DSas-6 and Sak. It was important to verify, however, that Ana2 also has a role in canonical centriole duplication. Overexpressing GFP-Sak or GFP-DSas-6 from the ubiquitin (Ubq) promoter induces centriole overduplication in brains and embryos, respectively. Surprisingly, however, overexpression of Sak, DSas-6, or DSas-4 cannot drive centriole overduplication in primary spermatocytes, which suggests that another duplication protein is limiting. To test if Ana2 might be this limiting factor, Ubq-GFP-Ana2 transgenic lines were generated. Strikingly, it was found that in spermatocytes expressing Ubq-GFP-Ana2, in addition to the normal centriole pairs (doublets), centriole triplets, quadruplets, and even quintets were observed. The extra centrioles in these clusters appeared to be fully functional; they separated from one another by the end of meiosis I (as centriole doublets normally do), and the extra centrioles inherited by secondary spermatocytes recruited PCM and nucleated MT asters, and so formed multipolar spindles during meiosis II (Stevens, 2010).
It was important to compare the localization of Ana2 with that of the other Drosophila centriole duplication factors. DSas-4-GFP, GFP-DSas-6, and GFP-Sak are all enriched at the proximal and distal ends of the large spermatocyte centrioles. It was found that, likewise, Ana2-GFP localized preferentially to the proximal and distal centriole tips. Strikingly, however, Ana2-GFP (and GFP-Ana2) also exhibited a unique asymmetric distribution, consistently localizing preferentially along one centriole barrel (Stevens, 2010).
In primary spermatocytes, it is possible to distinguish mother and daughter centrioles, as the daughter can often be observed associating end-on with the side of the mother. In 25 centriole pairs where mother and daughter centrioles could unambiguously distinguished, Ana2-GFP was always enriched on the daughter. Mother and daughter centrioles can show important differences in their behavior in vertebrate cells and during asymmetric stem cell divisions in Drosophila. Although mother and daughter centrioles are morphologically and molecularly distinguishable in vertebrates, this is not the case in Drosophila. Ana2-GFP is the first fly protein shown to localize asymmetrically to mother and daughter centrioles in this manner (Stevens, 2010).
Interestingly, as spermatocytes progressed through meiosis I, this centriolar asymmetry became less pronounced, and this appeared to reflect the selective loss of GFP-Ana2 from the daughter centriole, bringing its levels down to that of the mother. Since overexpression of Ana2 can lead to centriole overduplication, Ana2 levels presumably must normally be tightly regulated to prevent the formation of extra centrioles (Stevens, 2010).
After exit from meiosis II, each spermatid inherits a single centriole, which acts as a basal body to nucleate the flagellar axoneme. Structural components of the centriole, like Ana3 and Drosophila pericentrin-like protein (D-PLP), continue to localize along the basal body. In contrast, Ana2, like the conserved duplication proteins, was undetectable along the basal body. Ana2 did, however, colocalize with GFP-DSas-6 at the proximal centriole-like structure, a small nodule adjacent to the basal body that has been proposed to be an early intermediate in centriole formation (Stevens, 2010).
Intriguingly, Drosophila homologues have been identified for all the C. elegans centriole duplication factors except SAS-5, which has no clear homologues outside worms. Ana2 and SAS-5 are similar in size and have a single central coiled-coil domain, leading to a suggeston that Ana2 could be the Drosophila equivalent of SAS-5. As SAS-5 interacts with SAS-6 in worms, genetic interaction between Ana2 and DSas-6 was tested in flies (Stevens, 2010).
A small percentage of eggs laid by mothers carrying two copies of a Ubq-GFP-DSas-6 transgene assemble centriole-like structures. To see if this effect could be enhanced, flies were generated carrying one copy of Ubq-GFP-DSas-6 and one copy of Ubq-Ana2-GFP, neither of which alone (as a single copy) induces the assembly of centriole-like structures. Strikingly, almost all the unfertilized eggs laid by these females contained hundreds of large structures that stained for centriole markers, recruited PCM, and nucleated asters. Importantly this interaction was specific to Ana2 and DSas-6. In eggs from mothers carrying one copy of either Ubq-Ana2-GFP or Ubq-GFP-DSas-6 together with one copy of either Ubq-GFP-Sak, Ubq-Asl-GFP, or Ubq-DSas-4-GFP, at most a very small number of asters were observed in very few eggs (Stevens, 2010).
Interestingly, the centriole-like structures produced by overexpressing UASp-GFP-DSas-6 differ significantly from those resulting from the overexpression of GFP-Sak, DSas-4-GFP, Asl-GFP, or Ana2-GFP in that they are much larger and often appear ring-shaped, and that only one structure is contained within each aster. The structures in the eggs from females expressing both Ubq-GFP-DSas-6 and Ubq-Ana2-GFP were similar to this DSas-6 type. This suggests that Ana2-GFP acts to promote the assembly of GFP-DSas-6 into these structures (Stevens, 2010).
Having shown that Ana2 functionally interacts with DSas-6, physical interaction was sought. Using a yeast two-hybrid (Y2H) assay, it was found that Ana2 and DSas-6 interact and that the N-terminal region of DSas-6 and the C-terminal region of Ana2 are necessary and sufficient for this interaction. Moreover, like SAS-5, Ana2 also interacts with itself. Attempts to test whether Ana2 and DSas-6 associate in vivo were hindered by their low abundance. However, it was found that DSas-6 antibodies coimmunoprecipitated Ana2-GFP from S2 cells overexpressing Ana2-GFP. Collectively, the evidence of a specific functional and physical interaction between Ana2 and DSas-6 indicates that Ana2 likely represents the Drosophila functional orthologue of SAS-5 (Stevens, 2010).
Having shown that Ana2 is the likely SAS-5 functional orthologue in Drosophila, Ana2/SAS-5 orthologues were sought in other species. Using an iterative BLAST search, significant homology was found between Ana2 and the STIL or SIL protein family. Moreover, the reciprocal iterative BLAST search starting with zebrafish STIL identified Ana2 as the most similar Drosophila protein. Although vertebrate STIL family members are larger than Ana2 or SAS-5, all of these proteins share a short, central, coiled-coil domain. In addition, a particularly conserved region of ~90 aa toward the C terminus of Ana2 and STIL was identified, that was called the STil/ANa2 (STAN) motif. The STAN motif of Ana2 is 31% identical (48% similar) to that of zebrafish STIL. A divergent STAN motif can be detected in SAS-5, which is 12% identical (26% similar) to that of zebrafish STIL. Importantly, the STAN motif is within the regions of SAS-5 and Ana2 that interact with SAS-6 and DSas-6, respectively (Stevens, 2010).
Data from studies of STIL in mice, zebrafish, and humans are consistent with a function in centriole duplication, although this was not appreciated at the time of these studies. First, mitotic spindles often lack centrosomes in stil mutant zebrafish. Second, STIL mutant mice show defects characteristic of aberrant cilia function, such as randomized left-right asymmetry and neural tube abnormalities. Most importantly, it has recently been shown that mutations in human STIL cause primary microcephaly (MCPH), a congenital disorder characterized by reduced brain size. Mutations in four other genes, MCPH1, CDK5RAP2, ASPM, and CPAP/CENPJ, are known to cause MCPH, and all are centrosomal proteins, strongly suggesting that STIL is required for efficient centrosome function in humans (Stevens, 2010).
This study has show that of eight centrosomal proteins identified as potential duplication factors in Drosophila tissue culture cells, only two, Asl and Ana2, appear to be able to induce de novo formation of centriole-like structures in unfertilized eggs. Asl has recently been shown to be essential for centriole duplication, and this study provides evidence that Ana2 is also a key centriole duplication factor. Thus, Ana2 and Asl join Sak, DSas-6, and DSas-4 to make up a module of just five proteins known to drive centriole duplication in flies (Stevens, 2010).
These data strongly suggest that the Ana2/STIL family of centrosomal proteins are the long-sought functional orthologues of SAS-5. Thus, four of these five components (Sak/ZYG-1, DSas-6/SAS-6, Ana2/SAS-5, and DSas-4/SAS-4) are functionally conserved between flies and worms. Moreover, three of these proteins are required for centriole duplication in humans, whereas the fourth, SAS-5/Ana2/STIL, also appears likely to be required for this process in vertebrates (Stevens, 2010).
Both flies and worms have an additional protein (SPD-2 in worms, Asl in flies) that appears to be essential for centriole duplication. Intriguingly, both SPD-2 are not only required for centriole duplication, but also for PCM recruitment. There is evidence that the PCM promotes centriole duplication, so SPD-2 and Asl could play a more indirect role in centriole duplication via their ability to recruit PCM. Alternatively, both proteins may act directly in centriole duplication, with the function of SPD-2 in worms perhaps being performed by Asl in flies (Stevens, 2010).
In summary, this study shows that Ana2 acts as a centriole duplication factor in Drosophila and is likely to have a conserved role in other species. Overall, centriole duplication appears to be a highly conserved process, at the heart of which is a small number of key proteins. The challenge will now be to tease apart how these components cooperate to build a centriole of the right size, in the right place, and at the right time (Stevens, 2010).
Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila Anastral spindle-2 (Ana2) is a conserved centriole duplication factor. While recent work demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. This study examined the Ana2-LC8 interaction and mapped two LC8-binding sites within Ana2's central region, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 mμM) while site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 mμM). Both LC8-binding sites flank a predicted ~34-residue alpha-helix. Two independent atomic structures are presented of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form beta-strands that extend a central composite LC8 beta-sandwich. LC8 recognizes the signature TQT motif in Ana2's first LC8 binding site, forming extensive van der Waals contacts and hydrogen bonding with the peptide, while the Ana2 site 2 TQC motif forms a uniquely extended beta-strand, not observed in other dynein light chain-target complexes. Size-exclusion chromatography coupled with multi-angle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization likely enhances Ana2's avidity for centriole binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis (Slevin, 2014).
Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules. In interphase, it promotes microtubule catastrophe; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux. This study shows that Klp10A also acts as a microtubule depolymerase on centriolar microtubules to regulate centriole length. Thus, in both cultured cell lines and the testes, absence of Klp10A leads to longer centrioles that show incomplete 9-fold symmetry at their ends. These structures and associated pericentriolar material undergo fragmentation. It was also show that in contrast to mammalian cells where depletion of CP110 leads to centriole elongation, in Drosophila cells it results in centriole length diminution that is overcome by codepletion of Klp10A to give longer centrioles than usual. How loss of centriole capping by CP110 might have different consequences for centriole length in mammalian and insect cells is discussed, and also these findings are related to the functional interactions between mammalian CP110 and another kinesin-13, Kif24, that in mammalian cells regulates cilium formation (Delgehyr, 2012).
To study Klp10A's roles at the spindle poles, flies with reduced Klp10A expression were examined that showed male sterility resulting from a P element insertion. Meiotic cells in Klp10A testes had supernumerary asters and abnormal, frequently multipolar meiotic spindles. Consistently, 79% of elongating spermatid bundles contained fewer than the normal 64 nuclei, and flagella were immotile and shorter than in control flies. Electron microscopy revealed that elongating spermatids had missing or incomplete axonemes. Moreover, Klp10A adults were uncoordinated and needed increased time to recover from mechanical shock, a hallmark of centriole defects (Delgehyr, 2012).
These findings led to an examination of centrioles in 16-cell cysts of primary spermatocytes in late G2 or in meiosis. Most such cells (>70%) were found to have more than two centrosomes together with additional rod- and dot-like structures that stained with anti-Dplp (Drosophila pericentrin-like protein). Moreover, whereas wild-type spermatocytes had only a few centrioles (2.2%) with no detectable Sas6 (spindle assembly abnormal protein 6) at their distal part, Klp10A spermatocytes showed an increase of such centrioles, suggesting they were incomplete or fragmented. Spd2 (spindle defective 2), which is closely associated with spermatocyte centrioles otherwise lacking pericentriolar material (PCM), also associated with centriolar fragments in Klp10A mutants, often fraying out from their ends. By the end of G2, Klp10A mutant centrioles displayed discontinuities in the outer microtubules of both basal body-like structures and the short primary cilia that they nucleate. Finally, Klp10A was present along the length of wild-type centrioles, being more concentrated near both ends, but was barely detectable in mutant centrioles (Delgehyr, 2012).
To understand how defective centrioles and centrosomes might arise in Klp10A mutant testes, earlier mitotic stages of spermatogenesis were examined. These showed significantly more cells with either more than (>20% of the cells) or fewer than two Dplp (centriole marker) bodies. Not only the number but also the size of Dplp bodies was compromised: whereas in wild-type spermatogonia, Dplp bodies were of uniform size, Klp10A mutants showed a significant increase in both shorter and longer Dplp bodies. Similar findings were obtained using anti-Spd2 labeling. Examination of spermatogonia by electron microscopy confirmed that Klp10A centrioles were both longer and shorter than wild-type; longer centrioles were generally not uniformly elongated, and shorter ones often appeared tenuously attached to them. This excessive elongation did not, however, seem to prevent formation of procentrioles, indicating that centriole duplication could occur. To better correlate the immunofluorescence observations with electron microscopy, structured illumination microscopy was carried out on anti-Spd2-stained spermatogonial cells. This revealed both shorter and longer centrioles 'frayed' at their ends in Klp10A spermatogonia. Apparent segments of centriolar walls were found both associated with the frayed ends or free in the cytoplasm, suggesting that the elongated structures could become fragmented (Delgehyr, 2012).
To test Klp10A's role in somatic centriole biogenesis, RNA interference (RNAi) was used to efficiently deplete the protein from cultured Dmel cells. Previous studies of the functional requirements for Klp10A for spindle microtubules have not examined centrosome behavior per se. After Klp10A RNAi, cells either lacking or having numerous Dplp bodies were detected. The increase in cells without Dplp bodies was however quite modest, around twice the control. Moreover, the proportion of such cells did not increase following several rounds of Klp10A depletion. This was a quite different outcome from that following depletion of Plk4, a core component of the centrosome duplication pathway, where virtually 100% of cells lose their centrosomes. Accordingly, it was found that centrosomes could reform after Plk4 RNAi treatment in either the presence or absence of Klp10A. Under these conditions, depletion of Sas6, which is known to be essential for centriole duplication, would block de novo centriole formation. Together, these results indicate that the centriole duplication pathway is independent of Klp10A function (Delgehyr, 2012).
It was noticed that Klp10A-depleted cells contained both weakly and brightly fluorescing Dplp bodies. Because it was difficult to measure the length of these bodies (centrioles are less than 0.2 μm in these cells), their fluorescence intensity was measured. Dplp is found at both centrioles and PCM, giving a combined indication of PCM size and centriole length. It was found that Klp10A RNAi resulted in a doubling of both very bright and very weak dots. By contrast, cells depleted for the microtubule depolymerases Klp59C or Klp59D (kinesin-13 family) or Klp67A (kinesin-8) did not show an increase in brightly fluorescent Dplp bodies, suggesting that changes in centrosome size are not a general consequence of cytoplasmic microtubule depolymerization (Delgehyr, 2012).
To understand how these different centrosomal bodies might be generated, video microscopy was carried out to follow centrosomes in cells constitutively expressing GFP-Spd2 from a weak promoter. Because centrosome number is notoriously variable in Dmel cells, cells with two GFP-Spd2 punctae were examined at interphase. In control cells, the two punctae became brighter on mitotic entry as centrosomes recruited PCM and separated to form the spindle poles. Centriole disengagement occurred in telophase, and the daughters had two centrosomal punctae. In Klp10A-depleted cells, the two centrosomes coalesced upon mitotic entry with the apparent collapse of the spindle and bipolarity was recovered; the coalesced centrosomes split into several punctae at one pole, whereas there were none at the other. Centrosomes then dispersed into numerous scattered dots. Thus, in the absence of Klp10A, fragmentation in M phase appears to account for the weak Dplp bodies (Delgehyr, 2012).
It is considered that the weak Dplp bodies could arise by PCM dispersion or centriole fragmentation per se. The former possibility was addressed by staining to reveal Spd2, which makes a greater contribution to PCM, in addition to Dplp. Klp10A depletion led to an increase in small punctae positive for Spd2 but not Dplp, consistent with some PCM fragmentation. In the absence of good antibodies to label centrioles, the possibility of centriole fragmentation was addressed by applying structured illumination immunofluorescence microscopy and electron microscopy to examine centrioles in control and Klp10A-depleted cells. Structured illumination microscopy resolved the punctae staining in control cells into circular structures of ∼0.4 μm outer diameter that, when rotated through 90°, revealed the rod-like structures of centrioles. In Klp10A-depleted cells, these rods could be the same length or longer than in control cells. What appeared to be segments of the centriolar wall was also observed, suggesting that centriole fragmentation could also contribute to the weak Dplp bodies seen by conventional immunofluorescence (Delgehyr, 2012).
Electron microscopy revealed an increase in centrioles longer than 0.18 μm from 15.4% in control cells to 72.1% in Klp10A-depleted cells and a less pronounced increase in centrioles shorter than 0.15 μm (from 12.8% to 17.6%). Moreover, 10.5% of centrioles in Klp10A-depleted cells had an incomplete complement of microtubules. It is possible that the small centrioles represent intermediates in centriole duplication. If so, the relatively small increase in their number suggests that centriole duplication cannot account for the almost doubling of weak Dplp bodies after Klp10A RNAi. Together, these results therefore suggest that Klp10A depletion leads to centriole elongation and fragmentation associated with dispersion of the PCM (Delgehyr, 2012).
Centrosome separation during mitosis is mainly asymmetric in Klp10A-depleted cells: one cell inherits both centrosomes, and the other inherits none. Because the latter cell might be expected to engage in de novo centriole formation, it was wondered whether formation of longer centrioles following Klp10A depletion could be a consequence of this. Therefore centriole reformation was examined after inducing their loss by extensive Plk4 depletion, and it was found that such centrioles were no longer than in control cells. This accords with the normal morphology of centrioles formed de novo in unfertilized eggs overexpressing Plk4. Thus, the long fragmented centrioles seen after Klp10A depletion are unlikely to be a consequence of de novo centriole formation (Delgehyr, 2012).
It was previously found that injection of anti-polyglutamylated tubulin antibody into HeLa cells in G2 resulted in centriole loss and scattering of PCM in a dynein-dependent manner. This has been widely taken to indicate that polyglutamylation might stabilize centrosomes to forces exerted upon them in mitosis. Unlike in mammalian cells, tubulin is not polyglutamylated in most Drosophila tissues, and accordingly this modification could not be detected in Drosophila centrosome preparations or on centrioles of primary spermatocytes or Dmel cells. It was therefore wondered whether centrosomes might scatter following Klp10A downregulation in a dynein-dependent process. To test this, it was decided to partially release tension on centrosomes by depleting dynein (Dhc64, dynein heavy chain 64) in the presence or absence of Klp10A. Because dynein depletion leads to inequitable heritance of centrosomes, their detachment from spindle poles, and cell death, around 50% of the protein was depleted. This did not lead to defects in centrosome number or staining intensity. When Klp10A was extensively depleted and dynein was partially depleted, numbers of weak Dplp bodies decreased at the expense of an increase in bright Dplp bodies as compared to cells depleted for Klp10A alone. Thus, partial depletion of dynein apparently rescues centrosome fragmentation resulting from Klp10A depletion. Electron microscopy revealed that codepletion of Klp10A and dynein resulted in long centrioles, some exceeding the size observed after Klp10A depletion alone. Although the possibility cannot be completely excluded that dynein depletion directly affects the centriole per se, it is more likely that dynein depeletion results in reduced forces upon the centriole, thus protecting the long centrioles of Klp10A-depleted cells from fragmentation (Delgehyr, 2012).
To determine whether Klp10A's microtubule-depolymerizing properties are needed for its centrosomal functions, mutations were generated in two conserved class-specific motifs of its motor domain shown to be essential for microtubule depolymerization in other organisms. These mutations changed residues KVD (aa 317–319) or KEC (aa 546–548) of Klp10A each to three alanines. In accord with the known properties of Klp10A, it was found that high levels of wild-type protein depolymerized interphase microtubules, whereas cells expressing similar levels of the mutant forms had interphase microtubules of normal length. Thus, both mutations abolish Klp10A's microtubule depolymerization activity. Lines expressing either wild-type or mutant forms of GFP-tagged Klp10A were also generated from the endogenous Klp10A promoter that had the 5′ but not the 3′ untranslated region (UTR) of Klp10A. Both wild-type and mutant forms were expressed at levels comparable to the endogenous protein and were associated with microtubules and centrosomes or spindle poles in interphase and mitosis. Expression of the tagged wild-type protein at these low levels did not result in depolymerization of cytoplasmic microtubules. Then endogenous Klp10A was depleted using RNAi directed at the 3′ UTR and the effects upon centrosomes were assessed. This led to an increase in cells either without centrosomes or with both weak and bright Dplp bodies in RNAi-treated cells expressing GFP (control) or GFP-tagged mutant forms of Klp10A, but not in cells expressing GFP-tagged wild-type protein. Thus, generation of both larger centrosomes and centrosome fragments requires loss of the microtubule-depolymerizing activity of Klp10A (Delgehyr, 2012).
Because human CP110 or Centrobin (required for CP110's centriolar localization) results in centriole elongation, it was intriguing to find Drosophila CP110 enriched at the distal ends of the centrioles of primary spermatocytes in a region similar to Klp10A. Moreover, CP110 colocalized with Klp10A in interphase Dmel cells, and other evidence suggested that CP110 and Klp10A can physically interact. It was found that several rounds of CP110 depletion led to an increase in Dmel cells lacking centrosomes, CP110 depletion affects centrosome biogenesis. However, centrosomes could reform after their elimination by Plk4 RNAi treatment, even in the absence of CP110, although this was prevented by further depletion of Sas6, required for bona fide centriolar duplicatio. Thus, there is no absolute requirement for CP110 for centriole duplication. Interestingly, however, there was an increase in weakly fluorescing Dplp bodies in CP110-depleted cells, and electron microscopy revealed that 50% of centrioles were shorter than 0.15 μm, compared to 12.8% in control cells, with most being about 0.11 μm long. This is unlikely to represent an increase in procentrioles, because total centriole number decreased. Centrioles of about 0.11 μm are similar in length to the unit cartwheel made mainly of three or four tiers in Chlamydomonas reinhardtii and may represent the smallest stable centriole structures. Thus, it is concluded that, in contrast to depletion of CP110 in mammalian cells , depletion of CP110 in Drosophila leads to centriole shortening and destabilization (Delgehyr, 2012).
It was then asked whether centriole shortening following CP110 depletion might be rescued by sequentially codepleting Klp10A. It was found that this resulted in the reappearance of long centrioles: 62.5% of the centrioles exceeded 0.18 μm, compared to 15.4% in control cells. When Klp10A was depleted first, followed by codepletion with CP110, the reappearance of longer centrioles was observed. Thus, in the absence of Klp10A, centrioles increase in length regardless of whether CP110 is present or absent. The destabilization of Dplp bodies after CP110 depletion appeared to be enhanced by overexpression of GFP-Klp10A that led to an increased proportion of cells lacking centrosomes after 3 days of CP110 RNAi. Together, these results suggest that CP110 might provide a barrier to prevent Klp10A-mediated depolymerization of centriolar microtubules, and indeed, it forms a plug-like structure at the distal part of the centriole. However, CP110 has no effect on microtubule elongation in the absence of Klp10A. Thus, Klp10A can restrict centriole length regardless of whether or not CP110 is present, and so their physical interaction is not required for Klp10A's recruitment and/or microtubule-depolymerizing activity. Indeed Klp10A may be recruited directly on the centrioles, because it is known to have affinity for the microtubule lattice in vitro (Delgehyr, 2012).
Klp10A is the first kinesin-13 demonstrated to regulate centriole length. Other family members have, however, been shown to regulate the length of flagella and more recently formation of cilia. In contrast to Klp10A, however, the mammalian kinesin-13 Kif24 is further limited to acting from the mother centriole on microtubules of cilia. Thus, Kif24 depletion leads to aberrant cilia formation in cycling cells but does not promote growth of centrioles in nonciliated cells. Although Drosophila Klp10A and mammalian Kif24 act differently, both are able to interact with CP110. However, the interaction of CP110 and Kif24 in mammalian cells affects only the process of cilium formation. These functional differences between kinesin-13:CP110 complexes could reflect a regulative adaptation by mammals associated with evolution of the ability to generate primary cilia in many cell types. Thus, whereas in Drosophila, where few cells are ciliated, the capping function of CP110 might help to fix the length of the centriole by blocking Klp10A-mediated microtubule depolymerization, in mammals it has the additional function of blocking cilium formation until the correct phase of the cell cycle. Whether in mammals another kinesin-13 might play the role of Klp10A in regulating centriole length remains an open question (Delgehyr, 2012).
Stem cells have remarkable self-renewal ability and differentiation potency, which are critical for tissue repair and tissue homeostasis. Recently it has been found, in many systems (e.g. gut, neurons, and hematopoietic stem cells), that the self-renewal and differentiation balance is maintained when the stem cells divide asymmetrically. Drosophila male germline stem cells (GSCs), one of the best characterized model systems with well-defined stem cell niches, were reported to divide asymmetrically, where centrosome plays an important role. Utilizing time-lapse live cell imaging, customized tracking, and image processing programs, this study found that most acentrosomal GSCs have the spectrosomes reposition from the basal end (wild type) to the apical end close to hub-GSC interface (acentrosomal GSCs). In addition, these apically positioned spectrosomes were mostly stationary while the basally positioned spectrosomes were mobile. For acentrosomal GSCs, their mitotic spindles were still highly oriented and divided asymmetrically with longer mitosis duration, resulting in asymmetric divisions. Moreover, when the spectrosome was knocked out, the centrosomes velocity decreased and centrosomes located closer to hub-GSC interface. It is proposed that in male GSCs, the spectrosome recruited to the apical end plays a complimentary role in ensuring proper spindle orientation when centrosome function is compromised (Bang, 2015).
During interphase in Drosophila neuroblasts, the Centrobin (Cnb)-positive daughter centriole retains pericentriolar material (PCM) and organizes an aster that is a key determinant of the orientation of cell division. This study shows that daughter centrioles depleted of CNB cannot fulfill this function whereas mother centrioles that carry ectopic CNB can. CNB co-precipitates with a set of centrosomal proteins that include gamma-Tub, Ana2, Cnn, Sas-4, Asl, DGRIP71, Polo and Sas-6. Following chemical inhibition of Polo or removal of three Polo phosphorylation sites present in Cnb, the interphase microtubule aster is lost. These results demonstrate that centriolar Cnb localization is both necessary and sufficient to enable centrioles to retain PCM and organize the interphase aster in Drosophila neuroblasts. They also reveal an interphase function for Polo in this process that seems to have co-opted part of the protein network involved in mitotic centrosome maturation (Januschke, 2013).
Since first described in germline stem cells, unequal mother-daughter centriole behaviour has become a major issue in stem cell self-renewing asymmetric division that has now been reported to occur in mice and in other cell types in Drosophila melanogaster including neuroblasts. Drosophila neuroblasts are neural precursors that generate the flys central nervous system through repeated rounds of self-renewing asymmetric divisions. Neuroblast asymmetric division is driven by the polarized localization of protein complexes at the apical and basal cortex and by the orientation of the mitotic spindle along the apico-basal axis. As a result, the apical and basal sides of the cortex are cleaved apart into the renewed neuroblast and the smaller differentiating ganglion mother cell (GMC), respectively. Dysfunction of several of the molecules involved in these processes results in tumour growth (Januschke, 2013).
Neuroblast cortical polarity and mitotic spindle alignment are tightly linked to the centrosome cycle of these cells in which centriole splitting is a very early interphase event and mother and daughter centrosomes exhibit significant differences in structure, function and fate. In Drosophila neuroblasts, the mother centriole, which has no significant microtubule organizing activity and migrates extensively during interphase, is inherited by the GMC after mitosis. In contrast, the daughter centriole, which retains high microtubule organizing activity and remains rather stable near the apical cell cortex throughout interphase, is retained by the neuroblast at mitosis. Thus, unlike most Drosophila cells where centrosomes are rather feeble microtubule organizing centres (MTOCs) during interphase, Drosophila neuroblasts posses a prominent interphase microtubule aster that is organized by the cortex-bound daughter centriole. Drosophila centrioles do not seem to have age-dependent ultrastructural attributes such as the satellites and appendages observed in vertebrates. However, Drosophila CNB, similarly to its homologues in mouse and human cells, binds only to the daughter centriole, revealing a molecular dimorphism that could contribute to daughter-centriole-specific behaviour in Drosophila larval neuroblasts (Januschke, 2013).
This study shows that in Drosophila neuroblasts, daughter centrioles without CNB behave like mother centrioles, and mother centrioles with ectopically localized CNB behave like daughter centrioles. Moreover, by co-immunoprecipitation and tandem mass-spectroscopy (MS/MS) this study shows that CNB co-precipitates with γ-Tub, Ana2, Cnn, Sas-4, Asl, Asl, DGRIP71, Polo and Sas-6. These centriolar and PCM proteins are part of a well-characterized network that drives PCM accumulation during mitotic centrosome maturation (see Gopalakrishnan, 2011). This study also shows that CNB phosphorylation by Polo is essential to maintain the interphase aster, hence revealing a requirement for Polo function during interphase (Januschke, 2013).
In neuroblasts expressing YFP-CNB-PACT (The PACT domain is a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin), both mother and daughter centrioles carry CNB and behave as daughter centrioles do in wild-type neuroblasts (see Centrobin function and graphical summary). Interestingly, ectopic activation of the interphase PCM retention program on both centrioles forces a canonical centrosome cycle in these cells whereby one centrosome-aster complex splits in two that segregate away from each other. In these cells, spindle assembly frequently occurs at an angle with respect to the apico-basal axis, but eventually rotates to align with it as in delaminating neuroblasts in the Drosophila embryo. In neuroblasts mutant for Cnb or expressing YPF-CNBT4A T9A S82A, the two centrioles behave as mother centrioles do in wild-type neuroblasts; they retain little or no PCM during interphase, but centrosomes mature at mitosis entry. In all three cases the spindle aligns with the apico-basal axis. CNB depletion, similarly to CNB ectopic localization on the two centrioles, randomizes age-dependent centriole fate. Apico-basal orientation can change from one cycle to the next in CNB-mutant cells, but not in neuroblasts that express CNB-PACT, strongly suggesting that the orientation of cortical polarity is critically dependent on daughter centriole behaviour and that whether this is provided by one or two centrioles seems to be irrelevant as long as a cortex-bound microtubule network is maintained during interphase (Januschke, 2013).
The results show that interphase PCM retention in neuroblasts requires Polo to act on CNB-decorated centrioles to mediate Cnn and PCM recruitment. Which centriole and where exactly on the centriole CNB is positioned do not seem to be essential because ectopic localization of CNB by the PACT domain works just as well. Indeed, the interaction of CNB with proteins such as Ana2 and Sas6 that are essential to build the centriolar cartwheel, and with Cnn and Polo that are key regulators of PCM assembly, strongly suggests that CNB might serve its function by providing a critical bridge between centrioles and PCM. It also suggests that the neuroblast and daughter-centriole-specific process of PCM retention by the CNB-containing centriole has co-opted at least some of the proteins that drive PCM enrichment on centrosomes during mitosis in many cell types. Nevertheless, the fact that mitotic centrosome maturation does not require CNB and is hardly affected by 20 nM of the Polo kinase inhibitor BI2536 strongly suggests that important differences apply between interphase PCM retention in Drosophila neuroblasts and the ubiquitous process of mitotic centrosome maturation (Januschke, 2013).
Interphase PCM retention by CNB-containing centrioles also requires Pins, but seems to require neither Mud, nor the Khc-73/Dlg pathway. Centriole migration to the cortex shortly after mitosis in neuroblasts mutant for pins suggests that Pins function is required at a later stage. Such a function is unlikely to require cortical Pins because CNB-decorated centrioles detached from the cortex by microtubule poisons efficiently retain PCM and, indeed, no evidence of Pins cortical localization during interphase has been reported in Drosophila larval neuroblasts. The seemingly normal mitotic centrosome maturation in >pins-mutant neuroblasts further substantiates that despite shared molecular factors, important differences apply between interphase PCM retention in Drosophila neuroblasts and mitotic centrosome maturation (Januschke, 2013).
Neither cell size asymmetry nor Mira cortical polarization seem perturbed by CNB depletion or by CNB ectopic localization in both centrioles. However, given the extraordinary complexity of cell fate determination, which is only partially understood, these observations cannot substantiate any solid conclusion on the effect that CNB dysfunction might have on cell fate determination and brain development. CNB is a ubiquitous protein that is one of the two centrioles in different cell types in Drosophila. This study also found that some CNB allelic combinations are lethal, which strongly suggests that CNB might have functions in other tissues. In vertebrates, Centrobin has been reported to bind to the tumour suppressor BRCA2, and some common genetic variants of human Centrobin have been associated with breast cancer susceptibility (Januschke, 2013 and references therein).
A recent study has revealed that in human neuroblastoma cell lines, the sister cell that retains the NuMA crescent, which is thought to have more self-renewal potential, frequently inherits the daughter centrosome. These results suggest the tantalizing possibility that the age-dependent centrosome segregation demonstrated in Drosophila neuroblasts might also occur in mammals and may have relevance in human disease (Januschke, 2013).
Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila) inhibits basal body function. These data open the question of CNB's role in spermatocytes, where daughter and mother centrioles become basal bodies. This study reports that in these cells, CNB localizes equally to mother and daughter centrioles and is essential for C-tubules to attain the right position and remain attached to B-tubules as well as for centrioles to grow in length. CNB appears to be dispensable for meiosis, but flagellum development is severely compromised in Cnb mutant males. Remarkably, three N-terminal POLO phosphorylation sites that are critical for CNB function in neuroblasts are dispensable for spermatogenesis. These results underpin the multifunctional nature of CNB that plays different roles in different cell types in Drosophila, and they identify CNB as an essential component for C-tubule assembly and flagellum development in Drosophila spermatogenesis (Reina, 2018).
Centrobin was initially identified in humans through a yeast two-hybrid screen for proteins that interact with the tumor suppressor BRCA2. Like human Centrobin (referred to as CNTROB in humans), its homologues in other species are components of daughter centrioles in different cell types. In mammals, CNTROB has been reported to be required for centriole duplication and elongation as well as for microtubule nucleation and stability through its binding to tubulin and its effect in stabilizing centrosomal P4.1-associated protein (CPAP) (Reina, 2018 and references therein).
Drosophila melanogaster Centrobin is a key determinant of mother/daughter centriole functional asymmetry in larval neuroblasts and type I sensory neurons. In neuroblasts, CNB functions as a positive regulator of microtubule organizing center (MTOC) activity during interphase: its depletion impedes daughter centrioles to assemble an MTOC, whereas mother centrioles carrying ectopic CNB become active MTOCs. This activity is dependent on CNB phosphorylation by PLK1/POLO on three conserved sites and involves the function of pericentrin-like protein (PLP) and BLD10/CEP135. In Drosophila type I sensory neurons, CNB functions as a negative regulator of ciliogenesis: CNB depletion enables daughter centrioles as functional basal bodies that template ectopic cilia, and mother centrioles modified to carry CNB cannot function as basal bodies (Reina, 2018).
The function of a daughter centriole protein like CNB as a negative regulator of ciliogenesis opens the question of CNB's role in Drosophila primary spermatocytes where after centrosome duplication all four centrioles, mothers and daughters alike become basal bodies that assemble axoneme-based cilium-like structures, which are the precursors of sperm flagella. To investigate this issue, this study examined CNB localization and function in spermatogenesis (Reina, 2018).
As in other cell types in Drosophila and other species, CNB localization in spermatogonial cells was asymmetric, restricted to only one of the two centrioles that were labeled with anti-SAS4 antibodies. However, after the last (fourth) round of spermatogonial mitosis, the resulting early primary spermatocytes presented CNB equally distributed on the two centrioles of each pair (Reina, 2018).
After centrosome duplication, all four centrioles of each spermatocyte grow significantly in length, migrate to the cell surface, and become basal bodies that template a short axoneme that forms a protrusion covered by the cell membrane. Recent studies refer to this protrusion as the cilium-like region (CLR) or the ciliary cap (Reina, 2018).
To determine CNB localization in the basal body-CLR complex, CNB was immunostained together with PLP, ANA1, and UNC. PLP signal was strongest on the proximal end of the basal body. ANA1 labelled the entire length of the basal body. UNC overlapped with ANA1 except on the PLP-positive proximal end, was strongest at the transition zone, and extended further distally into the axoneme. CNB signal was equal in all basal bodies; most concentrated on the proximal end of the ANA1 domain, largely overlapping with PLP. Like PLP, a very weak CNB signal could also be detected all along the basal body. These observations reveal some remarkable differences regarding CNB localization, which in turn suggest important functional differences as far as ciliogenesis is concerned between early spermatocytes and sensory neurons in Drosophila (Reina, 2018).
To determine whether CNB has a function in the basal body-CLR complex, trans-heterozygous PBac(RB)Cnb[e00267]/Df(3L)ED4284 (henceforth referred to as Cnb mutant) spermatocytes were examined. The length of YFP-Asterless (ASL) signal (a proxy for centriole length) was significantly shorter in Cnb mutant than in WT males. Longitudinal EM sections from centriole pairs in which both mother and daughter were cut along their entire length confirmed this observation and revealed that daughter basal bodies were shorter than their mothers in Cnb mutant spermatocytes. This was not the case in WT spermatocytes, where it was found that mother and daughter basal bodies were of equal length. EM sections also revealed that 10 out of 16 mutant axonemes sectioned longitudinally were irregularly shaped and had abnormal axonemes with doublets that were aberrantly interrupted halfway through the CLR (Reina, 2018).
Ultrastructural details were revealed by transversal EM sections. Unlike basal bodies in sensory neurons that are made of doublets, basal bodies in WT spermatocytes are made of triplets. At the transition zone, the outermost tubule (C-tubule) progressively lost protofilaments, hence becoming an open longitudinal sheet that extended beyond the transition zone along the axoneme. At the point where C-tubule remnants and B-tubules intersect, short radial projections could be observed (Reina, 2018).
In addition to this basic layout of triplets, doublets, and open sheaths, about half of basal body-CLR complexes in Drosophila WT spermatocytes present a luminal singlet microtubule independently of the Z position of the cross-section. The presence of this luminal singlet is as erratic in Cnb mutant as it is in WT males and does not represent a Cnb mutant phenotype (Reina, 2018).
Eleven basal body-CLR complexes were serially sectioned from two Cnb mutant males. In all 11 samples, C-tubules were either misplaced outwards, away from the nearly straight line defined by the A/B doublet, or lost, leaving only duplets behind. These abnormalities extended distally along the axoneme, where C-tubule-derived longitudinal sheets were often lacking in Cnb mutant spermatocytes. The short radial projections observed in WT axonemes also appeared to be lacking in Cnb mutant CLRs, but this observation must be interpreted with caution because of the technical difficulties of detecting such small structures that are located in regions of electron-dense material (Reina, 2018).
These observations reveal that CNB is required in Drosophila spermatocytes for centrioles to properly position and stabilize C-tubules, grow in length up to full-sized basal bodies, and template normal axonemes. The results identify CNB as one of the very few proteins known so far to be required for C-tubule assembly, including Δ-tubulin in Paramecium tetraurelia and Chlamydomonas reinhardtii along with UNC and SAS4 in Drosophila. Notably, as in Cnb mutant spermatocytes, basal bodies are also shorter than normal in Sas4F112A and Unc mutant spermatocytes, hence suggesting that short centrioles and lacking or mispositioned C-tubules may be causally linked (Reina, 2018).
Drosophila SAS4 and its human homologue CPAP are closely functionally related to the corresponding Centrobin homologues CNB/CNTRB. Mutants in the N-terminal helical motif of the PN2-3 domain of CPAP and SAS4 cause overelongation of newly formed centriolar/ciliary microtubules, and so does CNTRB overexpression by stabilizing and driving the centriolar localization of CPAP. Moreover, the mutants in PN2-3 domain of CPAP referred to above result in biciliated cells, and so does Cnb loss of function in Drosophila sensory neurons. In Drosophila, SAS4 and CNB coimmunoprecipitate from embryo extracts. These data strongly suggest that the role of CNB on basal body length may be SAS4 dependent (Reina, 2018).
The ultrastructural phenotypes that were observed in basal body-CLR complexes in Cnb mutant spermatocytes persist through meiosis and are present in early postmeiotic cells. However, no observable defects were observable in meiosis. Cnb mutant males assembled fairly normal meiotic spindles and generated normal cysts of postmeiotic onion-stage spermatids that presented a uniform nuclear size, which is a very sensitive readout of faithful meiotic chromosome segregation. These results strongly suggest that shortened centrioles and lacking C-tubules have no major observable consequences on meiosis progression (Reina, 2018).
However, soon after meiosis, when the basal body attaches to the nucleus and the axoneme starts to grow, pushing the old CLR caudally, Cnb mutant spermatids presented CLRs that were much longer than those from WT cells. In WT cells at this stage, CNB signal was strongest on the basal body proximal to UNC and was also detectable over the perinuclear plasm, a cap over the nuclear hemisphere where the basal body is engaged (Reina, 2018).
Cnb mutant phenotypes were also conspicuous at later stages of elongation. Transversal sections through the tails of WT elongating spermatids showed two mitochondrial derivatives flanking the axoneme, which was almost completely surrounded by a double membrane. Among the sample of 32 Cnb mutant elongating spermatids that were analyzed in detail, a wide range was found of phenotypic variability. Five presented a fairly WT 9 + 2 configuration, whereas the other 27 included cells that had no axoneme or axonemes with missing or highly disarrayed duplets and in which the surrounding membrane was widely open. Among the latter, six had some duplets that carried C remnants, and 16 did not. Interestingly, not all duplet-bound open sheaths observed in Cnb mutant cells were C remnants. Four axonemes were found that presented one to three triplets with an attached open sheath. No such structures have been reported in WT cells. The presence of a fraction of Cnb mutant spermatids with axonemes that presented C remnants suggests that a certain fraction of Cnb mutant primary spermatocytes retain C-tubules (Reina, 2018).
In addition to lacking and disarranged axonemes, Cnb mutant elongating spermatids also presented ectopic nuclei at sections where only tails were present in WT cysts. Scattered nuclei have also been reported in other Drosophila mutants like Unc and Sas4. Head-to-tail attachment defects have been documented in the sperm of a rat CNTROB mutant strain (hd rats) that was male sterile (Reina, 2018).
The results reveal that CNB depletion brings about a spectrum of mutant phenotypes at different stages of spermatogenesis that includes shorter centrioles, mispositioned or absent C-tubules, lack of C-tubule derivatives, abnormal or absent axonemes, and scattered nuclei along elongating spermatid bundles. It cannot be discarded that CNB may have independent functions accounting for each of these phenotypes. However, the apparent pleiotropic effect of CNB loss may simply reflect the downstream consequences of centrioles that are shorter than normal and present C-tubule defects (Reina, 2018).
The different, and in some aspects opposite, roles of CNB in basal body structure and ciliogenesis in type I sensory neurons and spermiogenesis are remarkable. In neurons, CNB inhibits basal body function on the centriole to which it binds that in WT cells is only the daughter centriole. In primary spermatocytes, however, CNB localizes to both mother and daughter centrioles, which in these cells are equally able basal bodies, and acts as a positive regulator of ciliogenesis. One conspicuous difference between these two cell types is that basal bodies in spermatocytes contain C-tubules, whose assembly, positioning, and stability requires CNB. In vertebrates, where centrioles also have C-tubules, a recent study has found that in addition to its primary localization to daughter centrioles, CNTROB associates with mother centrioles at the onset of ciliogenesis, and CNTROB loss causes defective axonemal extension. These results suggest that the different effect that CNB depletion has on ciliogenesis in Drosophila neurons and spermatocytes may reflect the doublet versus triplet structure of the corresponding basal bodies (Reina, 2018).
Many stem cells, including Drosophila germline stem cells (GSCs), divide asymmetrically, producing one stem cell and one differentiating daughter. Cytokinesis is often asymmetric, in that only one daughter cell inherits the midbody ring (MR) upon completion of abscission even in apparently symmetrically dividing cells. However, whether the asymmetry in cytokinesis correlates with cell fate or has functional relevance has been poorly explored. This study shows that the MR is asymmetrically segregated during GSC divisions in a centrosome age-dependent manner: male GSCs, which inherit the mother centrosome, exclude the MR, whereas female GSCs, which is shown in this study to inherit the daughter centrosome, inherit the MR. It is further shown that stem cell identity correlates with the mode of MR inheritance. Together these data suggest that the MR does not inherently dictate stem cell identity, although its stereotypical inheritance is under the control of stemness and potentially provides a platform for asymmetric segregation of certain factors (Salzmann, 2014).
This the MR is inherited asymmetrically during GSC divisions
in the Drosophila germline and that this correlates with centrosome age and
depends on a functional centrosome. Interestingly, inheritance of the MR by the cell
containing the daughter centrosome is opposite to a recent observation in
mammalian cells (Kuo, 2011). Further studies are required to determine
whether the asymmetrically inherited MR, or factors associated with it, regulates
stem cell behavior, and whether this regulation occurs in a species- or cell type-dependent
manner. Importantly, mutations that randomize MR inheritance (cnn and
dsas-4) do not drastically modulate stem cell identity, and cnn and
dsas-4 mutants show apparently normal progression of differentiation regarding the cell fate. Furthermore, the MR is inherited by the differentiating daughter in the male germline, whereas it is inherited by the stem cell in the female germline. Therefore, it is unlikely that the MR harbors an
inherent fate determinant. However, it is tempting to speculate that certain fate
determinants 'hitchhike' the MR in certain cell types, taking advantage of its
stereotypical inheritance. Additionally, it is possible that the MR regulates an aspect
of stem cell behavior rather than identity per se; for example, the MR could regulate
the rate of stem cell division. The fact that multiple MRs are never seen in a single cell
(GSC or CySC) may indicate that removal of the MR is a prerequisite of cell cycle
progression into the next cell cycle. Moreover, the MR that is transferred from the
GB to the CySC/CC might function as a messenger to coordinate the division
frequency between GSCs and CySCs (Salzmann, 2014).
The reports by Kuo and Ettinger are seemingly contradictory in
that Kuo reported that stem cells are characterized by the accumulation of
MRs, whereas Ettinger reported that they are characterized by the high
capacity for MR release into the extracellular space (Ettinger, 2011; Kuo,
2011). This study using male and female GSCs demonstrates that MR fates are highly
stereotypical yet strikingly distinct depending on the cell type. This finding indicates
that each cell type handles MRs with its own elaborate cellular program. The reason
why MR must be handled in such an elaborate manner awaits future investigation.
Nonetheless, this study reveals that a basic cellular asymmetry such as MR
inheritance correlates with asymmetry during stem cell division (Salzmann, 2014).
Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. This study investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. It was further shown that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, it was shown that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells (Moutinho-Pereira, 2013).
This study has identified eight genes involved in spindle assembly
in S2 cells. Mitotic spindle organization in the
presence/absence of centrosomes is driven by a common set of
genes. However, a specific cohort of genes was identified that
differentially affect the formation of a bipolar spindle depending
upon whether the centrosomes are present or not. In particular,
knockdown of γ-TuRC, 26S proteasome, and the chaperone complex
t-complex polypeptide-1 (TCP-1) subunits (involved in folding various proteins, including actin and tubulin) all produced a much more obvious pole-focusing
defect specifically when centrosomes are absent. Finally, it was also
found that spindle assembly in S2 cells is not affected by >95%
depletion of the RanGTP effector RCC1 (Moutinho-Pereira, 2013).
These results suggest that either Drosophila S2 cell spindles are
very robust to a decrease in RanGTP or that, alternatively, Ran-independent
pathways compensate for the loss of RanGTP. The
recently discovered Aurora B phosphorylation gradient along the
spindle (Tan, 2011) may provide the necessary spatiotemporal cues for
centrosome-independent MT stabilization and bipolar spindle
formation. Indeed, previous findings have implicated the human
CPC in spindle assembly and in SAC response. The
present data link both processes and support that the CPC [see Incenp, an essential subunit of the CPC (the Aurora B complex)] has an
impact on mitotic spindle assembly/maintenance, in part, by
regulating the duration of mitosis, regardless of the presence/
absence of functional centrosomes (Moutinho-Pereira, 2013).
Collectively, this study's RNAi screening results in Drosophila S2 cells
suggest that a centrosome-independent spindle pathway operates
constitutively during spindle assembly and does not require a distinct
backup genetic mechanism. These data are fully consistent with recent
transcriptome profiling studies of acentrosomal cells in Drosophila
brains and wing discs. However, certain cell systems
demonstrate a greater dependence on centrosomes for spindle
assembly and furrow positioning during early embryonic divisions
or centrosomes might enhance cell division fidelity in
mammalian somatic cells. Nevertheless, the conclusions support
the view that centrosomes are not main drivers of spindle assembly
during mitosis (Moutinho-Pereira, 2013).
Novak, Z. A., Conduit, P. T., Wainman, A. and Raff, J. W. (2014). Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos. Curr Biol 24(11): 1276-82. PubMed ID: 24835456
Centrioles form centrosomes and cilia, and defects in any of these three organelles are associated with human disease. Centrioles duplicate once per cell cycle, when a mother centriole assembles an adjacent daughter during S phase. Daughter centrioles cannot support the assembly of another daughter until they mature into mothers during the next cell cycle. The molecular nature of this daughter-to-mother transition remains mysterious. Pioneering studies in C. elegans identified a set of core proteins essential for centriole duplication, and a similar set have now been identified in other species. The protein kinase ZYG-1/Sak/Plk4 recruits the inner centriole cartwheel components SAS-6 and SAS-5/Ana2/STIL, which then recruit SAS-4/CPAP, which in turn helps assemble the outer centriole microtubules. In flies and humans, the Asterless/Cep152 protein interacts with Sak/Plk4 and Sas-4/CPAP and is required for centriole duplication, although its precise role in the assembly pathway is unclear. This study shows that Asl is not incorporated into daughter centrioles as they assemble during S phase but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, requires DSas-4, and, crucially, is essential for daughter centrioles to mature into mothers that can support centriole duplication. Therefore a 'dual-licensing' model of centriole duplication is proposed, in which Asl incorporation provides a permanent primary license to allow new centrioles to duplicate for the first time, while centriole disengagement provides a reduplication license to allow mother centrioles to duplicate again (Novak, 2014).
This study demonstrates that Asl recruitment to disengaged new centrioles has a critical role in allowing these centrioles to mature into mothers that can duplicate for the
first time. During all subsequent duplication cycles, however,
mother centrioles already contain a pool of immobile Asl,
and this appears to be sufficient to allow subsequent rounds
of duplication, because anti-Asl antibodies block the recruitment of the mobile fraction of Asl to mother centrioles but do not block their duplication. For an
old centriole to duplicate again, therefore, disengagement of
the daughter centriole appears to be the crucial licensing event
that allows reduplication, because immobile Asl
incorporation has already occurred. Taken together, these
findings suggest a dual-licensing model in which the recruitment of the immobile fraction of Asl by DSas-4 provides an irreversible primary license to allow newly formed centrioles
to duplicate for the first time, while centriole disengagement
provides a reduplication license to allow older centrioles
to duplicate again (Novak, 2014).
How might Asl perform this primary licensing function? In
flies, Asl localizes Sak to centrioles, probably explaining
why Asl incorporation is a crucial step in converting a
disengaged daughter centriole into a mother centriole that
can duplicate. Cep152 (human Asl) is also required for the
efficient loading of Plk4 (human Sak) onto centrioles in verte-
brate cells, although it appears to share this function
with Cep192 (human SPD-2). This model is consistent
with superresolution microscopy studies on fixed cells, which
show that Asl/Cep152 is associated with the mother centriole
in an engaged centriole pair, suggesting that a
similar model may operate in vertebrates. Although the
primary and reduplication licensing steps are mechanistically
different, it is suspected that they share a common purpose: to
provide an Asl platform that is competent to recruit Sak to
initiate daughter centriole assembly (Novak, 2014).
The model can explain why only mother centrioles can
support certain types of experimentally induced centriole
reduplication, including that induced by Sak overexpression or by ablation of one of the engaged centrioles during an arrested S phase. It can also explain why daughter
centrioles appear to have to be 'modified' before they can
support any duplication; the results strongly suggest that
this modification, at least in flies, is Asl incorporation.
How is Asl recruited to centrioles? It is speculated that
DSas-4 initially recruits the immobile fraction of Asl, which
then recruits the mobile fraction. This would explain the 50:50
ratio of immobile to mobile Asl. The finding that anti-Asl antibodies strongly block the
recruitment of the mobile fraction of Asl to mother centrosomes also supports this possibility. It is tempting to speculate that the mobile fraction of Asl may be important
for the previously described role of Asl in mitotic PCM recruitment. It is also interesting to note that only very low levels of Asl seem to be required at new mother centrioles to
allow duplication (Novak, 2014).
It remains to be determined what regulates the interaction
between DSas-4 and Asl such that Asl is only recruited to
daughter centrioles at about the time they separate from their
mothers. It is speculated that the phosphorylation state of either
or both proteins could be altered at the end of mitosis, perhaps
increasing the affinity of their interaction. Polo/Plk1 seems to
play a crucial part in resetting the reduplication license at old
centrioles through the regulation of centriole disengagement; perhaps it also has an important role in the primary licensing of new centrioles by regulating the interaction
between DSas-4 and Asl (Novak, 2014).
Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates and Drosophila neuroblasts, the neural precursors of the fly's central nervous system, contain molecularly and physically asymmetric centrosomes, established through differences in pericentriolar matrix (PCM) retention. For instance, the daughter centriole maintains PCM and thus microtubule-organizing center (MTOC) activity through Polo-mediated phosphorylation of Centrobin (Cnb). The mother centriole, however, quickly downregulates PCM and moves away from the apical cortex, randomly migrating through the cytoplasm until maturation sets in at prophase. How PCM downregulation is molecularly controlled is currently unknown, but it involves Pericentrin (PCNT)-like protein (plp) to prevent premature Polo localization and thus MTOC activity. This study reports that the centriolar protein bld10, the fly ortholog of Cep135, is required to establish centrosome asymmetry in Drosophila neuroblasts through shedding of Polo from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM, generating two active, improperly positioned MTOCs. Failure to shed Polo and PCM causes spindle alignment and centrosome segregation defects, resulting in neuroblasts incorrectly retaining the older mother centrosome. Since Cep135 is implicated in primary microcephaly, it is speculated that perturbed centrosome asymmetry could contribute to this rare neurodevelopmental disease (Singh, 2014).
In a gene candidate approach to identify molecules required for centrosome asymmetry in Drosophila neuroblasts, this study identified bld10/Cep135 as a potential centrosome dematuration regulator. bld10 is a ubiquitous centriolar protein, localizing to centrioles in Drosophila larval neuroblasts and other cell types.
To investigate centrosome asymmetry, live imaging experiments were performed in intact third-instar larval brains, labeling centrosomes with the centriolar markers DSas6::GFP or DSas4::GFP and mCherry::Jupiter. jupiter encodes for a microtubule binding protein, sharing properties with several structural microtubule-associated proteins (MAPs), and is ideally suited to visualize microtubule dynamics and microtubule-organizing center (MTOC) activity. In agreement with previous findings, it was found that wild-type (WT) interphase neuroblasts contained one apical MTOC only. The second MTOC appeared during prophase in close proximity to the basal cortex. By prometaphase, both MTOCs reached maximal activity and intensity. However, in bld10 mutant interphase neuroblasts (bld10c04199/Df(3L)Brd15, two centrosomes of similar size and MTOC activity were observed close together on the apical cortex. The two centrosomes progressively separated from each other until they reached their respective positions on the apical and basal cortex by prometaphase. Thus, in contrast to the wild-type, bld10 mutant neuroblasts show symmetric centrosome behavior. bld10's centrosome asymmetry defect could be rescued with bld10::GFP, and immunohistochemistry experiments confirmed the live imaging results (Singh, 2014).
The bld10c04199 allele is predicted to produce a truncated protein, retaining bld10's N terminus. A new N-terminal deletion allele (bld10ΔN was generated that showed the same centrosome asymmetry phenotype. In addition, neuroblasts were found containing monopolar and multipolar spindles, which are not observed with the bld10c04199 allele. This suggests that bld10c04199 is a separation-of-function allele, specifically disrupting centrosome asymmetry. Unless otherwise noted, all of the experiments described in the following sections were performed with the bld10c04199 allele (Singh, 2014).
The lack of centrosome asymmetry in bld10 mutant neuroblasts could be due to aberrant centriole migration. For example, the mother centriole could either fail to migrate through the cytoplasm or migrates back to the apical cortex to mature. This hypothesis was tested, measuring centriole migration as a function of time, and it was observed that centriolar migration in wild-type and bld10 mutant neuroblasts occur in two distinct phases: (1) centrioles steadily separated from each other, followed by (2) a sudden increase in intercentriolar distance, which peaked when centrioles reached a separation distance of ~4-6 μm in the wild-type and bld10 mutants. Centrioles in bld10 mutants did not require more time to reach this threshold distance and did not return to the apical cortex to mature. It is concluded that bld10's centrosome asymmetry defect is not due to aberrant centriole migration (Singh, 2014).
To get mechanistic insight into the bld10 phenotype, live imaging was used to measure the dynamic localization of three GFP-tagged pericentriolar matrix (PCM) markers: γ-tubulin (γ-Tub), Mini spindles (Msps; CKAP5 in vertebrates) and centrosomin (Cnn; CDK5Rap2 in vertebrates). Wild-type neuroblasts showed robust localization of γ-Tub, Msps, and Cnn to the apical centrosome during interphase. After centrosome splitting, all three PCM markers were downregulated from the basal centrosome (shedding phase) but reaccumulated during prophase (maturation phase. bld10 mutant neuroblasts also correctly localized γ-Tub, Msps, and Cnn to the apical interphase centrosome. However, similar to the MTOC marker Jupiter, γ-Tub, Msps, and Cnn were not downregulated from the separating centriole. Centrosome size was measured and a centrosome asymmetry index was plotted, starting at centrosome splitting until metaphase. Wild-type centrosomes developed a clear size asymmetry during the shedding phase and reduced it during the maturation phase. bld10 mutant centrosomes stayed similar in size, manifested in an asymmetry index below 1.5. Centrosome size and intensity measurements also revealed that in most wild-type neuroblasts, γ-Tub, Msps, and Cnn were removed from the basal centrosome ~15 min after centrosome splitting. Basal wild-type centrosomes were essentially devoid of γ-Tub after that time, whereas bld10 mutants contained equal amounts of this PCM marker. Changes in centrosome size were further compared, and it was found that wild-type apical centrosomes predominantly grew, whereas basal centrosomes increased (maturation phase) and decreased (shedding phase) their size to almost the same extent. bld10 mutant centrosomes were able to enlarge but showed very little size reduction, comparable to apical wild-type centrosomes. It is concluded that bld10 mutant centrosomes are able to mature but fail to downregulate the PCM markers γ-Tub, Msps, and Cnn (Singh, 2014).
The results suggest two possible mechanisms for centrosome asymmetry: (1) bld10 could prevent premature mother centrosome maturation by blocking the precocious accumulation of PCM proteins. (2) Alternatively, bld10 could promote PCM shedding right after centrosomes separate, thereby preventing the basal mother centrosome to prematurely become an MTOC. An in vivo pulse-chase labeling experiment was devised to distinguish between these two possibilities. To this end, Cnn at its endogenous locus was tagged with the photoconvertable fluorescent protein mDendra2. If bld10 blocks premature PCM accumulation, mother centrioles should quickly shed photoconverted Cnn and prematurely reaccumulate unconverted Cnn in bld10 mutants. Vice versa, if PCM shedding is compromised, it should be possible to follow the photoconverted centrosomes from the moment they separate until telophase. It was found that apical wild-type daughter centrioles retained the majority of photoconverted Cnn::mDendra2 from early interphase until prophase (possibly longer), indicating that very little Cnn protein gets exchanged. The basal mother centriole, however, lost photoconverted Cnn::mDendra2 within approximately 10-15 min after centriole separation, confirming that Cnn is shed quickly. Interestingly, bld10 centrioles retained photoconverted Cnn::mDendra2 for at least 45 min after separation. In some cases, one of the centrioles decorated with photoconverted Cnn::mDendra2 was even inherited by the newly formed GMC. It is conclude that (1) on the apical centrosome, Cnn protein turnover is absent or significantly reduced during interphase, that (2) on the basal centrosome, Cnn is shed quickly and replaced with new Cnn when maturation sets in, and that (3) bld10's centrosome asymmetry defect is not due to premature centrosome maturation. Instead, separating basal centrioles fail to shed Cnn in particular and possibly PCM proteins in general (Singh, 2014).
To elucidate the molecular mechanism underlying PCM shedding, the relationship was analyzed between bld10, Centrobin (Cnb), and Pericentrin (PCNT)-like protein (plp). Recently, it was shown that Cnb is necessary and sufficient for PCM retention on the apical daughter neuroblast centrosome. However, gain- and loss-of-function experiments with Cnb did not perturb bld10's localization. Similarly, as in the wild-type, Cnb was localized asymmetrically in bld10 mutants. plp mutants fail to downregulate γ-Tub on the mother centrosome. In plp mutant neuroblasts, the basal centrosome retained Cnn and MTOC activity during interphase. Interestingly, photoconversion experiments showed that similar to bld10, Cnn shedding from the basal centrosome was compromised in plp mutants. However, plp localization is not perturbed in bld10 mutant neuroblasts, and bld10 was normally localized in plp mutants. Knockdown of plp in bld10 mutants did not enhance bld10 PCM shedding phenotype, but due to the occurrence of additional phenotypes (fragmented or multiple centrosomes), the shedding phenotype could also be partially masked. In sum, it is concluded that bld10 is regulating centrosome asymmetry independently of Cnb and that plp is also required to shed Cnn (Singh, 2014).
Since the mitotic kinase Polo has been implicated in PCM retention during interphase, Polo localization dynamics were analyzed in wild-type and bld10 mutant neuroblasts. Recently, it was reported that Polo localizes to the apical centrosome during interphase and is only detectable at the basal centrosome during prophase, when maturation sets in. A Polo::GFP protein trap line was used and it was confirmed that Polo is stably localized to the apical interphase centrosome. Surprisingly, weak Polo was also found on the separating mother centrosome. Subsequently, Polo disappeared from the basal mother centriole within 10 min, comparable to Cnn, γ-Tub, and Msps shedding times. With a genomic Polo::GFP transgene, showing lower fluorescence intensity, Polo was found to be localized on both centrosomes in bld10 mutants. These data suggest that in wild-type neuroblasts, Polo is not just recruited onto the basal mother centrosome by prophase as previously reported, but is also subject to shedding during interphase. Polo is required for PCM retention since in bld10 mutant neuroblasts treated with the Polo inhibitor BI2536, both centrosomes lose MTOC activity. Thus, it is concluded that shedding of Polo is a requirement for the subsequent shedding of Cnn, γ-Tub, and Msps, enabling basal mother centrosome dematuration and the establishment of centrosome asymmetry (Singh, 2014).
Finally, the consequences were analyzed of disrupted centrosome asymmetry. The daughter centriole was labeled with Cnb::YFP ] and centrosome segregation was assayed. It was confirmed that wild-type neuroblasts faithfully retain the Cnb* daughter centriole, whereas the Cnb- centrosome segregates into the GMC. bld10 mutants showed correct asymmetric Cnb localization, but ~45% of bld10 mutant neuroblasts wrongly retained the mother centriole and segregated the daughter centriole into the GMC. Cnb+ centrosomes are usually bigger in wild-type and bld10 mutant neuroblasts, but centrosome segregation is independent of MTOC activity and size since bld10 mutant neuroblasts often retained the smaller centrosome. It is concluded that centrosome asymmetry is required for faithful centrosome segregation (Singh, 2014).
Since bld10 mutant neuroblasts have mispositioned MTOCs in relation to the apical-basal division axis, spindle orientation was analyzed. Immunohistochemistry experiments showed that bld10 mutant neuroblast spindles deviate from the regular orientation range, with isolated cases of extreme misalignment. Metaphase spindles, aligned orthogonally to the apical-basal polarity axis, can induce symmetric neuroblast divisions, resulting in an increase of the neural stem cell pool. However, neuroblast numbers were unchanged in bld10 mutants compared to control brains, and symmetric neuroblast divisions were not found with live imaging. Instead, time-lapse experiments showed that bld10 mutant centrosomes prematurely formed misaligned bipolar spindles. Spindle rotation during metaphase corrected this misalignment. Apical-basal polarity is a prerequisite for correct spindle orientation, but apical and basal polarity markers localized normally in bld10 mutants. Similarly, the spindle orientation regulators, Partner of Inscuteable (Pins; LGN/AGS3 in vertebrates) and the NuMA ortholog Mud, were also correctly localized. It is concluded that controlled PCM shedding and maturation is required for correct centrosome positioning but backup mechanisms exist, correcting for misaligned metaphase spindles (Singh, 2014).
Many cell types, including stem and progenitor cells, contain asymmetric centrosomes and segregate them nonrandomly, suggesting a connection between centrosome asymmetry and cell fate. How centrosome asymmetry is regulated is currently not understood, but centrosome dematuration is a critical step in establishing centrosome asymmetry. It was found that the centriolar protein bld10/Cep135, known as a centriole duplication and elongation factor, is required to establish centrosome asymmetry. On the basis of these data, it is proposed that plp and bld10 induce Polo's removal from the mother centriole, triggering the shedding of PCM proteins such as Cnn, γ-Tub, and Msps. Polo has been reported to be closely associated with centrioles, ideally positioned to phosphorylate both centriolar and PCM proteins. Thus, it is proposed that Polo-mediated phosphorylation of PCM proteins maintains a stable interaction between the centriole and surrounding PCM (Singh, 2014).
How Polo localization is regulated is currently not known, and no direct molecular interaction was detected between bld10 and Polo. Although no centriolar markers were found to be mislocalized inbld10 mutants (at the resolution level of confocal microscopy), it is possible that structural centriole defects, as detected in bld10 mutant spermatocytes and wing disc cells, could affect PCM turnover rates. However, since bld10 is not asymmetrically localized, it is difficult to conceive how such defects specifically compromise the behavior of the mother but not the daughter centrosome (Singh, 2014).
Although perturbed centrosome asymmetry does not seem to undermine neuroblast polarity, the cell cycle, or physical and molecular asymmetric cell division, the possibility cannot be excluded that centrosome asymmetry could have long-term consequences currently beyond the ability to detect. Interestingly, defects in centrosome maturation or mutations in Cep135 can cause neurodevelopmental disorders such as primary microcephaly. It will be interesting to address the question whether lack of Cep135 is causing microcephaly due to compromised centrosome asymmetry and dematuration (Singh, 2014).
Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. This study shows that the centriole protein Asterless (Asl) initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn mitotic PCM assembly is diminished; in the absence of both proteins it appears to be abolished. DSpd-2 helps incorporate Cnn into the PCM and Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies (Conduit, 2014).
Several hundred proteins are recruited to the PCM that expands around the centrioles during centrosome maturation in mitosis, but how so many proteins are organised into a functional mitotic centrosome has remained mysterious. Remarkably, this study shows that the assembly of the mitotic PCM in flies appears to depend on just two proteins, Cnn and DSpd-2. Both proteins appear to form scaffolds that initially assemble around the mother centriole and then spread outward, forming a dynamic platform upon which most, if not all, other PCM proteins ultimately assemble. DSpd-2 and Cnn partially depend on each other for their centrosomal localisation, and both proteins are required to ensure robust centrosome maturation. In the absence of one of these proteins, reduced levels of the other protein still localise around the 2 centrioles and can support the partial assembly of the mitotic PCM. In the absence of both proteins mitotic PCM assembly appears to be abolished (Conduit, 2014).
How are DSpd-2 and Cnn recruited to mother centrioles? The results strongly suggest that in fly embryos Asl initially helps recruit DSpd-2 to centrioles and DSpd-2 then helps to recruit Cnn. Cnn does not appear to be required to
recruit either Asl or DSpd-2 to centrosomes, but it is required to properly maintain DSpd-2 within the PCM. It is speculated that this interaction between DSpd-2 and Cnn creates a positive feedback loop that drives the dramatic expansion of the PCM scaffold around mother centrioles during mitosis. Although direct interactions between Asl and DSpd-2 and between DSpd-2 and Cnn have been identified by Y2H, and the endogenous proteins can all co-immunoprecipitate with one another in fly embryo extracts, it is stressed that it is uncertain that these interactions are direct in vivo. The requirement for Asl to initiate the mitotic recruitment of DSpd-2 and Cnn probably explains why these proteins are specifically recruited to mother centrioles. It has been recently shown that although Asl is essential for centriole duplication, it is not incorporated into daughter centrioles until they have passed through mitosis and matured into new mother centrioles, and Asl/Cep152 proteins mainly localise to mother centrioles in several species. The PCM appears to be preferentially associated with mother centrioles in many systems. The current findings provide a potential explanation for why this is so, and raise the intriguing possibility that all the mitotic PCM 0 may be organised exclusively by mother centrioles. Although DSpd-2 seems to be the major recruiter of centrosomal Cnn in embryos, there must be an alternative recruiter, as the centrosomal localisation of Cnn is not abolished in the absence of DSpd-2. Asl is an attractive candidate as anti-Asl antibodies perturb Cnn recruitment to centrioles (although this could be an 7 indirect consequence of their effect on DSpd-2 recruitment), and Asl and Cnn interact in Y2H analysis. Moreover, human Cep152/Asl has a role in the centrosomal recruitment of human Cdk5Rap2/Cnn (Conduit, 2014).
Interestingly, in flies this alternative pathway appears to be stronger in larval brain cells than in eggs/embryos: in the absence of DSpd-2, Cnn levels are reduced by only ~35% in brains but by ~80% in eggs. Thus, the detail of the mitotic PCM assembly pathway may vary between different cell types even in the same species. The data suggest that after DSpd-2 and Cnn have been recruited to centrioles they rapidly assemble into scaffolds that then move slowly away from the centrioles. For Cnn, there is strong data indicating that scaffold assembly is regulated by phosphorylation. Cnn contains a phospho-regulated multimerization (PReM) domain that is phosphorylated by Polo/Plk1 in vitro and at centrosomes during mitosis in vivo. Mimicking phosphorylation allows the PReM domain to multimerise in vitro and Cnn to spontaneously assemble into cytosolic scaffolds in vivo that can organise MTs. Conversely, ablating phosphorylation does not interfere with Cnn recruitment to centrioles, but inhibits Cnn scaffold assembly 06. It is speculated that, like Cnn, DSpd-2 can assemble into a scaffold and that this assembly is regulated in vivo so that it only occurs around mother centrioles. It remains unclear, however, whether DSpd-2 itself can form a scaffold, or whether it requires other proteins to do so (Conduit, 2014).
It is striking that both DSpd-2 and Cnn exhibit an unusual dynamic behaviour at centrosomes. Both proteins incorporate into the PCM from the inside out, and are in constant flux, as the molecules that move slowly outward away from the centrioles are replaced by newly incorporated molecules close to the centriole surface. This inside out assembly is likely to have important consequences, as it means that events close to the centriole surface, rather than at the periphery of the PCM, can ultimately regulate mitotic PCM assembly. This may be particularly important in cells where centrioles organise centrosomes of different sizes, as is the case in certain asymmetrically dividing stem/progenitor cells. Fly neural stem cells, fo example, use centrosome size asymmetry to ensure robust asymmetric division, and there is strong evidence that new and old mother centrioles differentially regulate the rate of Cnn incorporation in these cells. Moreover, mutations in human Cdk5Rap2/Cnn have been implicated in microcephaly, a pathology linked to a failure in neural progenitor cell proliferation, although the precise reason for this is unclear. Although DSpd-2 and Cnn have a major role in centrosome maturation, it is stressed that other PCM components are likely to make important contributions (Conduit, 2014).
Pericentrin, for example, has been implicated in PCM recruitment in several systems, and the fly homologue, D-plp, forms ordered fibrils in cultured S2 cells that extend away from the centriole wall and support PCM assembly in interphas. These centriolar fibrils, however, cannot explain how centrioles organise such a vastly expanded PCM matrix during mitosis, and D-plp appears to have an important, but more minor, role in mitotic PCM 1 assembly in vivo (Martinez-Campos, 2004). Nevertheless, proteins like 2 D-plp will certainly help recruit other PCM proteins and help form structural links within the PCM, thus strengthening the mitotic PCM matrix. The 4 important distinction is that, in flies at least, proteins like D-plp are recruited into the PCM by an underlying PCM scaffold, whereas DSpd-2 and Cnn appear to form this scaffold. Homologues of Asl, DSpd-2 and Cnn have been implicated in PCM assembly in many species suggesting that the mechanism of mitotic PCM recruitment identified in this study may have been conserved in evolution. To date, no PCM component has yet been shown to assemble from the inside out and to flux away from the centrioles in any other system. Nevertheless, although the precise molecular details will likely vary from cell type to cell type and from species to species, it is suspected that this unusual dynamic behaviour of an underlying mitotic PCM scaffold will prove to be a general feature of mitotic centrosome assembly in many systems (Conduit, 2014).
This study reports that two distinct centriole lineages exist in Drosophila: somatic centrioles usually composed of microtubule doublets and germ line centrioles characterized by triplets. Remarkably, the transition from doublets to triplets in the testis occurs within the stem cell niche with the formation of the C-tubule. It was demonstrated that the old mother centriole that stays in the apical cytoplasm of the male germline stem cells (GSCs) is invariably composed by triplets, whereas its daughter is always built by mixed doublets and triplets. This difference represents the first documentation of a structural asymmetry between mother and daughter centrioles in Drosophila GSCs and may reflect a correlation between the architecture of parent centrioles and their ability to recruit centrosomal proteins. It was also found that the old mother centriole is linked to the cell membrane by distinct projections that may play an important role in keeping its apical position during centrosome separation (Gottardo, 2015).
Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues within any given tissue, their size is essentially constant. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel, how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates. This study examine Cep97 function in Drosophila melanogaster. Cep97 is shown to be essential for formation of full-length centrioles in multiple tissues of the fly. The microtubule deacetylase Sirt2 was identified as a Cep97 interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells, and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila, Cep97 appears to be only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, these results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis (Dobbelaere, 2020).
The results indicate that Cep97 forms part of a capping structure that is recruited to fully elongated, mature centrioles. Removal of Cep97 need not occur to allow initiation of the ciliary axoneme. Rather, Cep97 is required for proper basal body function in ciliogenesis. Consistent with its late loading onto centrioles, loss of Cep97 does not affect procentriole assembly up to and including formation of the CPAP/Sas4-containing centriole wall. However, failure to assemble the Cep97 cap structure leaves centriolar microtubules exposed, which can result in abnormal extension or shrinkage, depending on cytoplasmic context. Instead of counteracting the activity of centriole elongation factors, such as CPAP, CEP120, or SPICE1, Cep97 therefore acts to limit microtubule dynamics once the proper length has been reached (Dobbelaere, 2020).
This work provides novel mechanistic insight into how this might occur. A hallmark feature of centrioles is their remarkably constant size and stability, with no detectable turnover of tubulin subunits after their incorporation, enabling individual centrioles to be traced through many cell divisions and indeed the lifetime of an animal. The extensive post-translational modification of centriolar microtubules, including by acetylation, is likely key for this stability, as memorably demonstrated by the dissolution of centrioles after microinjection of antibodies against poly-glutamylated tubulin. This study identified the microtubule deacetylase Sirt2 as a Cep97 interactor. It was further shown that loss of Cep97 results in loss of centriolar microtubule acetylation. Remarkably, perturbation of components of the microtubule acetylation machinery (Sirt2, Hdac6, and Atat1) largely phenocopies loss of Cep97. All three components have been localized to stable microtubules, including those at centrioles. This localization could be recapitulated by overexpression of GFP transgenes in Drosophila S2 cells. The simplest explanation then is that Cep97 regulates this machinery at centrioles to stabilize centriolar microtubules and confer on them their remarkable lack of dynamics. Consistent with this, bimolecular fluorescence complementation analysis shows an interaction between Cep97 and Sirt2 at centrioles. Finally, it bears remarking that the effects of Cep97 perturbation are much more severe than for CP110. Cep97 is also more widely conserved across eukaryotes than CP110, which is found exclusively in metazoans. Cep97 is therefore clearly more than just a CP110 recruitment factor. The work presented in this study should serve as a foundation for studies examining Cep97 function also in other experimental models (Dobbelaere, 2020).
To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy, but AJs are dynamic during epithelial development and remodeling, and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation. This study shows that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer, and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization. Par-1 was found to promote the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference (Jiang, 2015).
The identification of Par-1 as an inducer of Baz-centrosome co-recruitment is surprising given its well-established role in inhibiting Baz complex formation in Drosophila, C. elegans, and mammalian systems. It is proposed that Par-1 contributes to both global inhibition and local promotion of Baz complex assembly, providing a simple and potent Baz polarization mechanism (Jiang, 2015).
The Baz-centrosome positive feedback loop is evident from the specific accumulation of Baz next to cortical centrosomes, the MT requirement for Baz accumulation, the Baz requirement for centrosome recruitment, and the dynein role for drawing Baz and centrosomes together. Significantly, Par-1 is also necessary and sufficient for the loop and seems to have two direct roles. One is promotion of astral microtubules around the centrosome, an effect consistent with known effects of Par-1 on MT regulators, but requiring further elucidation in the Drosophila embryo. The other is the phosphorylation of Baz at Ser-151 and Ser-1085. These modifications have well-characterized inhibitory effects on Baz cortical association, but strikingly, they are also enriched where the Baz-centrosome positive feedback loop occurs and appear necessary for Baz entry into the loop. It is speculated that phospho-regulated Baz-14-3-3 protein interactions mediate further protein interactions, or induce conformational changes, important for Baz-MT association. Indeed, 14-3-3 proteins can bridge MT motors, a Par-3 conformational change induces direct MT binding, Par-3 directly binds a dynein subunit, and other links to MTs are known (Jiang, 2015).
Although the Par-1-Par-3-centrosome pathway can be a potent Baz polarization mechanism, it is normally attenuated within a homeostatic system. During early cellularization, Par-1 localizes over the entire PM and presumably phosphorylates Baz and MT regulators. In response, it is proposed that Baz is continually displaced and diffuses over the PM but is additionally primed for MT interactions. Simultaneously, the two centrosomes found atop each nucleus would provide the positional information for localizing Baz around the apical circumference through dynein-mediated MT associations. As Baz accumulates, it recruits aPKC to the apical domain, from where aPKC then displaces Par-1. Normally, this Baz-aPKC-Par-1 negative feedback loop seems to keep the Par-1-Baz-centrosome pathway in check. In the absence of aPKC, the Par-1- Baz-centrosome pathway continues unabated, leading to excessive Baz and centrosome polarization, loss of AJ isotropy, and later epithelial dissociation (Jiang, 2015).
Intriguingly, focused accumulations of Par-3 and AJs colocalize with cortical centrosomes during C. elegans intestinal development and during zebrafish collective cell migration. Moreover, Par-1 induces centrosomal MT interactions with AJs during human liver lumen formation in vitro and is needed for Baz-centrosome associations during the asymmetric division of Drosophila germline stem cells. Thus, induction of the Par- 1-Par-3-centrosome pathway, with regulated shifts to aPKC or Par-1 activities, may be generally relevant to developmental transitions of animal tissues (Jiang, 2015).
Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, this study shows in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, it is proposed that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation (Gallaud, 2020).
Centrosome asymmetry has previously been described to occur in asymmetrically dividing Drosophila neural stem cells (neuroblasts), manifested in biased interphase MTOC activity and asymmetric localization of the centrosomal proteins Cnb, Plp, and Polo and PCM proteins like Centrosomin. This study has shown that neuroblast centrosomes become intrinsically asymmetric by dynamically enriching centriolar proteins such as Cnb and Polo on the young daughter centriole during mitosis. This establishment of centriolar asymmetry is tightly linked to centriole-to-centrosome also called mitotic centriole conversion. In early prophase, Cnb and Polo colocalize on the existing mother centriole of the apical centrosome but from late prometaphase onward, Cnb is exclusively and Polo predominantly localized on the daughter centriole. Mechanistically, these dynamic localization changes could entail a direct or indirect translocation of Cnb and Polo from the mother to the daughter centriole. This model is partially supported for Cnb with FRAP data. Interestingly, Cnb behaves differently on the basal centrosome: the existing mother centriole does not contain any Cnb, appearing only on the forming daughter centriole in late prophase. This suggests a direct recruitment mechanism, which could also apply to the apical centrosome from anaphase onward. 3D-SIM, FRAP, and live-cell imaging data combined are most consistent with a model proposing that on the apical centrosome, a small pool of Cnb transfers from the mother to the daughter centriole during early mitosis. From anaphase onward, and from late prophase onward on the basal daughter centriole, Cnb levels increase through the recruitment of Cnb that was not previously associated with the mother centriole (Gallaud, 2020).
Cnb is phosphorylated by the mitotic kinase Polo and Polo-dependent phosphorylation of Cnb is necessary for its timely localization during mitosis. Interestingly, the data further suggest that Polo, which also becomes enriched on the daughter centriole during mitosis, is co-dependent with Cnb, while also requiring Wdr62. Polo's involvement in mitotic centriole conversion further suggests that the same molecular machinery cooperatively converts a maturing centriole into a centrosome for the next cell cycle while simultaneously providing it with its unique molecular identity (Gallaud, 2020).
The mechanisms generating 2 molecularly distinct centrioles during mitosis seem to directly influence the centrosome's MTOC activity in interphase; the 'Cnb+, high Polo' daughter centriole will retain MTOC activity during interphase whereas the 'Cnb-, low Polo' mother centriole, separates from its daughter in early interphase and becomes inactive. This model is in agreement with bld10 (Cep135) or plp mutants, which fail to down-regulate Polo from the mother centriole, resulting in the formation of 2 active interphase MTOCs. This is further supported by mislocalization data, showing that optogenetic manipulation of Polo and Cnb asymmetry specifically during mitosis impacts MTOC activity in the subsequent interphase. However, it cannot be excluded that MTOC asymmetry is also controlled independently of mitotic centrosome asymmetry establishment because optogenetic interphase manipulations of Polo and Cnb alone can also perturb biased MTOC activity (Gallaud, 2020).
Loss of Wdr62 or Cnb also affects asymmetric centriolar Polo localization. Yet, interphase centrosomes lose their activity in these mutants. wdr62 mutants and cnb RNAi neuroblasts both show low Polo levels in interphase. It is thus hypothesized that in addition to an asymmetric distribution, Polo levels must remain at a certain level to maintain interphase MTOC activity; high symmetric Polo results in 2 active interphase MTOCs, whereas low symmetric Polo results in the formation of 2 inactive centrosomes. Indeed, the optogenetic experiment triggered an increase in centriolar Polo levels upon blue-light induction, suggesting that both Polo levels and distribution influence MTOC activity. This hypothesis is strengthened by Cnn's capacity to oligomerize and form a scaffold, supporting PCM assembly upon phosphorylation by Polo. The more Polo is recruited, the more stable is the Cnn scaffold, supporting MTOC activity (Gallaud, 2020).
Centrobin is also enriched on the daughter centriole in mammals and preferentially becomes incorporated into the newly assembled daughter centriole in late G1 or early S phase. Centrobin remains localized at the daughter centrioles throughout the cell cycle. It is of interest to note that in mammalian cells, Centrobin becomes enriched on daughter centrioles during the G1-S transition and not during centriole-to-centrosome conversion. It is tempting to speculate that other kinases and mechanism regulate this translocation (Gallaud, 2020).
It was also observed that Cnb and Plp localize in a mutually exclusive manner, with Cnb localizing to the daughter centriole and Plp remaining on the mother centriole. In mammalian cells, similar mutual exclusion between the centriolar proteins Cep120 or Neurl4 and PCM proteins has been observed (Gallaud, 2020).
Taken together, the results reported in this study are consistent with a model, proposing that the establishment of 2 molecularly distinct centrioles is primed during mitosis and contributes to biased MTOC activity in the subsequent interphase. Wild-type neuroblasts unequally distribute a given pool of Cnb and Polo protein between the 2 centrioles so that the centriole inheriting high amounts of Cnb and Polo will retain MTOC activity. Furthermore, the dynamic localization of Polo and Cnb provides a molecular explanation for why the daughter-centriole-containing centrosome remains tethered to the apical neuroblast cortex and is being inherited by the self-renewed neuroblast. It remains to be tested why neuroblasts implemented such a robust machinery to asymmetrically segregate the daughter-containing centriole to the self-renewed neuroblast. More refined molecular and behavioral assays will be necessary to elucidate the developmental and postdevelopmental consequences of biased centrosome segregation. The tools and findings reported in this study will be instrumental in targeted perturbations of intrinsic centrosome asymmetry with spatiotemporal precision in defined neuroblast lineages (Gallaud, 2020).
Finally, the observations reported in this study further raise the tantalizing possibility that centriolar proteins also dynamically localize in other stem cells, potentially providing a mechanistic explanation for the differences in centriole inheritance across different stem cell systems (Gallaud, 2020).
Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. This study reports instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. It was shown that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals (Carvajal-Gonzalez, 2016).
Taken together with observations that Fz/PCP signalling regulates basal body and cilia positioning in vertebrates (Song, 2010; Gray; 2011; Borovina, 2010), the current data on centriole positioning as a Fz/PCP readout in non-ciliated Drosophila wing cells indicate that centriole/MTOC (MT organizing centre)/basal body positioning is an evolutionarily conserved downstream effect of Fz/PCP signalling. Its link with actin polymerization (hair formation in Drosophila wing cells) suggests that actin polymerization effectors also affect cilia positioning, possibly through docking of the basal bodies to the apical membranes. Inturned, Fuzzy and Rho GTPases regulate apical actin assembly necessary for the docking of basal bodies to the apical membrane (Park, 2006; Pan, 2007) and this apical actin membrane accumulation is lost in Dvl1-3-depleted cells (Carvajal-Gonzalez, 2016).
In left-right asymmetry establishment of the Drosophila hindgut, which is not a Fz/PCP-dependent process, asymmetric centriole positioning is observed. During this so-called planar cell shape chirality process, which affects gut-looping and thus embryonic left/right asymmetry, centriole positioning is however still dependent upon actin polymerization downstream of Rho GTPases (Rac and Rho), via MyoD and DE-cadherin control. As Rho GTPases (Rac, Cdc42 and Rho) are downstream effectors of Fz-Dsh/PCP complexes, and their mutants cause PCP-like phenotypes including multiple cellular hairs or loss of hairs in wing cells. It is thus tempting to infer that both processes, planar cell shape chirality and Fz/PCP, regulate centriole positioning through a common Rho GTPase-mediated actin polymerization pathway, initiated by an upstream cellular communication system, although this assumption will require experimental confirmation. In the mouse, Fz/PCP signalling regulates cilia movement/positioning in cochlear sensory cells via Rho GTPase-mediated processes, suggesting a similar mechanism in a representative mammalian PCP model system. In conclusion, the positioning of centrioles appears to be a key and an evolutionary conserved downstream readout of Fz/PCP signalling, ranging from flies to mammals in both ciliated and non-ciliated cells (Carvajal-Gonzalez, 2016).
Stem cell homeostasis requires nuclear lamina (NL) integrity. In Drosophila germ cells, compromised NL integrity activates the ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 2 (Chk2) checkpoint kinases, blocking germ cell differentiation and causing germline stem cell (GSC) loss. Checkpoint activation occurs upon loss of either the NL protein emerin or its partner barrier-to-autointegration factor, two proteins required for nuclear reassembly at the end of mitosis. This study examined how mitosis contributes to NL structural defects linked to checkpoint activation. These analyses led to the unexpected discovery that wild-type female GSCs utilize a non-canonical mode of mitosis, one that retains a permeable but intact nuclear envelope and NL. The interphase NL is remodeled during mitosis for insertion of centrosomes that nucleate the mitotic spindle within the confines of the nucleus. Depletion or loss of NL components causes mitotic defects, including compromised chromosome segregation associated with altered centrosome positioning and structure. Further, in emerin mutant GSCs, centrosomes remain embedded in the interphase NL. Notably, these embedded centrosomes carry large amounts of pericentriolar material and nucleate astral microtubules, revealing a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that defects in centrosome structure are upstream of checkpoint activation, suggesting that these centrosome defects might trigger checkpoint activation and GSC loss. Connections between NL proteins and centrosome function have implications for mechanisms associated with NL dysfunction in other stem cell populations, including NL-associated diseases, such as laminopathies (Duan, 2021).
Nuclear structure is shaped by proteins resident in the nuclear lamina (NL). This extensive network is composed of lamins and hundreds of lamin-associated proteins that line the inner nuclear membrane. The NL confers nuclear rigidity and contributes to chromatin organization important for regulation of transcription, replication, and DNA repair. Additionally, NL proteins transmit regulatory information between cellular compartments through connections that link the nucleoskeleton with the cytoskeleton. Nuclear structure correlates with cell-type-specific changes in NL composition, differences that impact genome organization and function during development (Duan, 2021).
The LAP2-emerin-MAN1 domain (LEM-D) protein family has a prominent role in the NL. These proteins share an ~40-amino-acid domain (LEM-D) that interacts with Barrier-to-autointegration factor (BAF) (sometimes referred to as BANF1), a conserved chromatin protein. In non-dividing cells, interactions between LEM-D proteins and BAF link the genome with the nuclear periphery. In dividing cells, these interactions control mitotic spindle assembly and positioning, as well as nuclear reassembly at the end of mitosis. These properties highlight mechanisms wherein the LEM-D and BAF partnership contributes to nuclear architecture (Duan, 2021).
Physiological aging and many diseases are associated with changes in nuclear structure. Indeed, misshapen and lobulated nuclei are common features of laminopathies, diseases that result from mutations in genes encoding NL proteins. Laminopathies affect some cell types more than others, with primary defects found in skeletal muscle, skin, fat, and bone. Age-associated worsening of laminopathic diseases has been linked to failures in stem cell maintenance suggesting that the NL plays an important role in balancing stem cell proliferation with differentiation. Although contributions of the NL to stem cell function are being investigated, mechanisms that preserve healthy stem cell populations and promote tissue homeostasis remain poorly understood (Duan, 2021).
Studies in Drosophila melanogaster have identified roles for LEM-D proteins and BAF in adult stem cell maintenance. Drosophila encodes three NL LEM-D proteins that bind BAF, including two emerin orthologs (emerin also known as otefin and emerin2 also known as Bocksbeutel) and MAN1. Notably, loss of emerin compromises homeostasis of adult stem cells in the female and male germlines, blocking germ cell differentiation and causing germline stem cell (GSC) loss. These defects are coupled with GSC-restricted deformation of the NL and accumulation of DNA damage, phenotypes that mirror those found in laminopathic cells. Strikingly, gametogenesis of Drosophila emerin mutant germ cells is rescued by mutation of two DNA damage response (DDR) kinases, the responder kinase ataxia telangiectasia and Rad3-related (ATR) or the transducer kinase checkpoint kinase 2 (Chk2). Although emerin mutant GSCs carry DNA damage, genetic and molecular analyses suggest that ATR and Chk2 activation occurs independently of canonical DNA damage triggers and is linked to the NL structural deformation. Germ-cell-specific BAF depletion also causes NL deformation and GSC loss that is partially rescued by chk2 mutation. These findings indicate that emerin and BAF contribute to shared NL functions needed for GSC maintenance (Duan, 2021).
This study extends an investigations of events associated with activation of the NL checkpoint. Prompted by shared requirements for emerin and BAF in nuclear reassembly, this study examined whether defects in mitosis were responsible for NL deformation and checkpoint activation. To this end, the structure of the NL was followed throughout female GSC (fGSC) mitosis. These analyses led to the unexpected discovery that wild-type fGSCs use a non-canonical mode of mitosis, wherein a permeable but intact nuclear envelope (NE) and remodeled NL remain throughout mitosis. This mode of mitosis imposes requirements for NL components, evidenced by observations that depletion or loss of NL components causes defects in centrosome positioning and spindle structure. In emerin mutant fGSCs, centrosomes remain embedded in the interphase NL and retain large amounts of pericentriolar material (PCM) that nucleates astral microtubules. These observations reveal a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that PCM retention in emerin mutant GSCs is upstream of Chk2 activation, indicating that the altered structure of the interphase centrosome is linked with NL checkpoint activation. Based on these data, it is proposed that other stem cells might employ distinct modes of mitosis that sensitize these cells to defects in the NL. These findings have implications for mechanisms associated with NL dysfunction in other systems, including laminopathies (Duan, 2021).
The NL has a central role in establishing structures important for the homeostasis of diverse stem cells. Indeed, survival of Drosophila GSCs depends upon the integrity of the NL, wherein NL deformation is linked to activation of the ATR and Chk2 kinases that leads to GSC loss. This studu investigated mechanisms leading to NL deformation in fGSCs, examining the role of mitosis in shaping the NL (Duan, 2021).
Two main modes of mitosis exist, open and closed. In open mitosis, the NE and NL break down, enabling spindle microtubules nucleated from cytoplasmic centrosomes to capture and segregate chromosomes. In closed mitosis, mitotic spindles are nucleated by spindle pole bodies (centrosome equivalents) that are embedded in a retained NE. It is generally assumed that metazoan cells use open mitosis, whereas fungi use closed mitosis. This assumption is linked with metazoan-limited expression of lamin and BAF, two proteins that are required for nuclear reassembly at the end of open mitosis. However, several exceptions to this rule exist, indicating that open and closed mitoses represent extremes of a continuum of mitotic strategies. A classic example of an exceptional mode of mitosis occurs in the Drosophila early embryo. In these early divisions, limited nuclear breakdown occurs, wherein local NE and NL breakdown occurs near centrosomes, with large portions of the NE and NL remaining until metaphase. This NL has a stabilizing function on spindle microtubules, as disruption of the lamin-B delays prometaphase spindle assembly. However, progression to anaphase requires NL and NE dispersal, as increased stabilization of the lamin-B prevents spindle elongation. These mitotic events indicate that the regulation of NE and NL remodeling optimizes progression through mitosis (Duan, 2021).
Mitotic divisions of Drosophila female germ cells also deviate from open mitosis. In contrast to somatic cells in the ovary that show universal NL dispersal, germ cells change the mode of mitosis depending on developmental stage. In larval PCGs, nuclear breakdown begins in prometaphase, forming large, broken patches of NL, whereas in adult fGSCs, the NL remains intact, even into anaphase. As a result, spindle microtubules form within the mitotic fGSC nucleus, emanating from centrosomes embedded in the NL. Notably, features of this non-canonical mitosis are shared with fungi. For example, in Aspergillus nidulans, the never-in-mitosis kinase partially disassembles NPCs, increasing permeability of an otherwise intact mitotic NE to allow mitotic regulators access to the prophase nucleoplasm. Similarly, it was found that NPCs in mitotic fGSCs are partially disassembled, allowing for exchange of nuclear and cytoplasmic components. However, other features differ between fGCS and fungal mitosis. For example, Saccharomyces cerevisiae carry a centriole-less spindle pole body that is embedded in the NE, which allows nucleation of spindle and astral microtubules within the nuclear compartment. In contrast, the centriole-containing centrosomes of fGCSs move into the NE and NL to promote spindle microtubule assembly within the nucleoplasm. fGCS centrosomes are inserted into a cup-like structure composed of lamin-B and emerin, suggesting that localized remodeling of the NE and NL occurs. Based on these comparisons with fungi, it is proposed that fGSCs employ an intermediate form of mitosis, one that is not completely closed because the NE becomes permeable or completely open because the NE and NL remain intact. The current data add additional evidence that metazoans do not solely employ an open mode of mitosis. Indeed, in Drosophila, modes of mitosis are cell type and developmental stage specific (Duan, 2021).
Genetic studies suggest that NL proteins are required for execution of fGSC mitosis. Loss of emerin or depletion of lamin-B alters the structure and positioning of mitotic spindles. Both NL mutants increase the frequency of lagging chromosomes in anaphase, suggesting that the quality of fGSC mitosis is compromised upon NL dysfunction. Defects in the mitotic spindle might result from disruption of mitotic spindle assembly or mitotic matrix formation, as mammalian lamin B is a structural component of the spindle matrix that promotes microtubule organization in mitosis. Alternatively, mitotic spindle defects might result from an altered distribution of nuclear pores, as centrosome separation in mitotic prophase is linked to nuclear pore distribution. Further studies are needed to address these possibilities (Duan, 2021).
The significance of the developmental switch between PGCs and fGSCs mitosis is unclear. Notably, this switch correlates with the transition from symmetric to asymmetric division, suggesting that retention of a mitotic NL might contribute to acquisition of distinct cell fates that occur within a single cell division. Indeed, fGSC homeostasis is linked to the asymmetric inheritance of two nuclear factors that regulate rRNA transcription and maturation, Wicked/U3 small nucleolar RNA (snoRNA)-associated protein 18 and UnderdevelopedTAF1 (TATA-Box Binding Protein Associated Factor 1). The data indicate that asymmetric trafficking of these proteins occurs within an intact mitotic NL, which might help establish the distinct distribution of these proteins and possibly others. For example, microtubules direct the asymmetric distribution of pSmad to one centrosome for its degradation in cultured human embryonic stem cells. Although niche signaling has a dominant role in fGSC maintenance, it is suggested that retention of a mitotic NL might amplify mechanisms used in asymmetric division (Duan, 2021).
Although emerin and lamin-B are required for fGSC mitosis, only loss of emerin leads to fGSC death. Indeed, oogenesis in nos > lam RNAi females occurs without evidence of Chk2 activation (Duan, 2021).
Although the low levels of lamin-B that remain in nos > lam RNAi fGSCs might be sufficient to guide mitosis, it is also possible that emerin and lamin-B make distinct contributions. Observations that the structure of the interphase centrosome differs in the two mutant backgrounds provide support for the latter possibility. In emerin mutant fGSCs, centrosomes remain embedded in the NL, and these centrosomes retain increased amounts of PCM, defects not observed in nos>RNAi mutants. Embedded centrosomes might contribute to the extensive structural deformation of the NL found in these emerin mutant fGSCs, because the expanded PCM retains γ-tubulin, the major microtubule nucleating component of the PCM.82 As a result, emerin, but not nos > lam RNAi, mutants nucleate astral microtubules in interphase fGSCs. Epistasis studies demonstrate that both PCM expansion and microtubule nucleation remain in chk2, emerin double mutants, implying that these features are independent or upstream of checkpoint activation. It is predicted that differences in interphase centrosome structure are connected to NL checkpoint activation (Duan, 2021).
Mechanisms responsible for expanded PCM in emerin mutants are unknown. Centrosome maturation and disassembly involve regulated activities of kinases that promote PCM expansion and phosphatases that reverse phosphorylation of PCM proteins. Structural defects of the interphase emerin mutant centrosome might originate from incomplete or partial PCM disassembly or from premature recruitment of PCM. Effects on centrosome structure might be direct, as emerin is a component of mitotic centrosomes in Drosophila embryos. This association appears to be conserved, as human emerin is also found in mitotic centrosomes. Further, phosphorylation of Drosophila emerin by Aurora-A kinase is required for mitotic exit in SL2 cells, indicating that emerin has a regulatory function at the centrosome. Alternatively, effects of loss of emerin might be indirect, resulting from gene expression changes that alter levels of mitotic regulators. Regardless of mechanism, the current data suggest that emerin has a role in PCM regulation. Further investigations are needed to test contributions of emerin to the centrosome cycle in fGSC mitosis (Duan, 2021).
ATR and Chk2 kinases localize to mitotic centrosomes. Studies in human cells indicate that ATR associates with γ-tubulin and influences the kinetics of microtubule formation at centrosomes. Localization of ATR to the centrosome provides a link between mitosis and the DDR. However, DDR proteins localize to centrosomes even in the absence of DNA damage, raising the possibility that ATR is a general sensor of structure and function at centrosomes. Building from these observations, it is predicted that the structurally defective centrosome in emerin mutant fGSCs might be responsible for transmitting signals to ATR and Chk2 kinases, ultimately leading to fGSC loss (Duan, 2021).
Mutations in NL LEM-D proteins cause diseases linked to compromised stem cell homeostasis. This study links centrosome dysfunction with failures in stem cell homeostasis due to mutation of the Drosophila NL protein Emerin. These studies align with observations in human fibroblasts that emerin anchors interphase centrosomes to the nucleus through direct interactions with microtubules and that expression of mutant forms of emerin in HeLa cells causes aberrant nuclear shape and mislocalization of tubulin and centrosomes. Taken together, these observations reinforce connections between emerin and centrosomes. As mechanisms of stem cell homeostasis are shared between cell types and organisms, it is possible that other stem cell populations used non-canonical modes of mitosis to ensure robustness of the asymmetric division, which might sensitize division of these cells to defects in the NL composition (Duan, 2021).
The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. This study investigated Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. Sequences within the coding region and 3'UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy,an N-terminal fragment of Bsg25D was shown to bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. It is concluded that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila (Kowanda, 2016).
Accurate mitotic spindle assembly is critical for mitotic fidelity and
organismal development. Multiple processes coordinate spindle assembly and
chromosome segregation. Two key components are centrosomes and the spindle assembly checkpoint
(SAC), and mutations affecting either can cause human microcephaly. In
vivo studies in Drosophila melanogaster have found that loss of
either component alone is well tolerated in the developing brain, in
contrast to epithelial tissues of the imaginal discs. This study reveals
that one reason for that tolerance is the compensatory relationship
between centrosomes and the SAC. In the absence of both centrosomes and
the SAC, brain cells, including neural stem cells, experience massive
errors in mitosis, leading to increased cell death, which reduces the
neural progenitor pool and severely disrupts brain development. However,
data also demonstrate that neural cells are much more tolerant of
aneuploidy than epithelial cells. These data provide novel insights into
the mechanisms by which different tissues manage genome stability and
parallels with human microcephaly (Poulton, 2017). The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. This study analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, these results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication (Pavlova, 2019).
In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, it is hypothesized that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes revealed that dynein is required for ER concentration around centrosomes during late interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. In fact, there was a sudden onset of ER association with astral MTs in dynein RNAi cells, revealing activation of an M-phase specific mechanism of ER-MT association. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, these results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells (Karabasheva, 2019).
The endoplasmic reticulum (ER) cannot be formed by cells de novo and must be inherited during the process of cell division. While the essential roles of the ER in the biogenesis of proteins, lipids and steroid hormones, as well as calcium signaling, are well recognized, little is known about the molecular mechanisms that ensure proper partitioning of the ER to progeny cells. This knowledge is fundamental to understanding the role of the ER in cell division and stem cell biology, with important implications for proper development, tissue repair, and cancer. Specifically, recent findings indicate that asymmetric partitioning of the ER, and misfolded proteins that accumulate there, has a critical role in maintaining pluripotency in stem cells as they undergo rapid cycles of cell division. Related evidence that ER functions contribute to the proliferative capacity and drug resistance of cancer cells is under active investigation, with an objective of revealing novel therapeutic strategies for treating malignancies. The overall goal of the present study was to establish new understanding of the cellular mechanisms that guide the localization of the ER during cell division, bringing closer a full comprehension of the essential roles of the ER in normal physiology and disease (Karabasheva, 2019).
Recent data suggest that proper partitioning of the ER during cell division, or M-phase, depends on specific association of the organelle with astral microtubules (MTs) of the mitotic spindle in both symmetrically and asymmetrically dividing cells4. However, the specific factors that link the ER to astral MTs remain unknown. Identification of these factors is therefore an important next step in understanding mitotic ER partitioning. Most of the knowledge of ER-MT associations comes from non-dividing interphase cells, in which the ER is distributed throughout the cytoplasm with a distinct clustering or focus around centrosomes, the major MT organizing centers of cells. This distribution depends on MT motor-dependent movements of the ER toward both MT plus-ends and minus-ends, as well as stable attachments of the ER along MT filaments mediated by ER membrane proteins including REEPs, spastin, and CLIMP-6. Transport of the ER toward MT minus-ends is mediated by dynein motors, which are also responsible for focusing the ER around centrosomes where MT minus-ends are anchored. Conversely, MT plus-end transport is likely mediated by kinesins9, and also depends on association with growing MT tips mediated, at least in part, by ER membrane embedded STIM1 and STIM2 proteins10. Collectively these associations point to a carefully orchestrated interplay between MT plus-end and minus-end directed transport mechanisms that determine the cellular distribution of the ER (Karabasheva, 2019).
During the course of M-phase, there is a dramatic reorganization of the MT cytoskeleton, whereby the spindle apparatus forms with MT minus-ends anchored at the spindle poles by centrosomes. As this occurs, the majority of the ER becomes focused around the two centrosomes at the spindle poles and along astral MTs, and virtually none is found in the kinetochore region of the spindle where MT plus-ends reside. Thus, there appears to be a shift from the balanced MT plus- and minus-end directed ER distribution during interphase to predominantly minus-end directed localization around spindle poles during M-phase. Consistent with this conclusion, tracking of the ER with growing MT plus-ends is inhibited during cell division due to mitosis-specific phosphorylation of STIM1. This suppression of MT plus-end directed ER transport, and the totality of ER distribution toward MT minus-ends around spindle poles, suggests a predominant role for the MT-minus end motor dynein in M-phase specific ER distribution. Notably, dynein is highly localized to astral MTs and spindle poles in dividing cells and is required for the spindle pole localization of endosomes. However, despite this compelling case, a definitive role for dynein in M-phase ER distribution has never been directly established. Determining dynein's role in the dramatic redistribution of the ER to spindle poles in M-phase is therefore important to understanding the mechanisms that ensure proper ER partitioning to progeny cells (Karabasheva, 2019).
The primary goal of this study was to test the hypothesis that dynein is required for astral MT association and spindle pole focusing of the ER in dividing cells. This was accomplished by live timelapse imaging of Drosophila spermatocytes undergoing the first meiotic division of spermatogenesis in vivo. This experimental system is particularly well suited for this investigation, as Drosophila spermatocytes allow for the analysis of dividing cells in a physiologic environment and have contributed greatly to understanding of fundamental cell division mechanisms including spindle formation and cytokinesis. Moreover, Drosophila spermatocytes are large cells that exhibit clearly defined redistribution of the ER onto astral MTs early in meiosis. This study presents the surprising finding that although dynein is required for peri-centrosomal focusing of the ER during late interphase, it does not mediate the astral MT-dependent spindle pole focusing of the ER around centrosomes during M-phase. Surprisingly, the results further reveal that redistribution of the ER toward MT minus-ends in dividing cells is mediated by a mechanism of ER-MT association that is entirely specific to M-phase and does not operate during interphase. This report lays the groundwork for identification of this novel mechanism of ER-MT association that is essential for the process of ER inheritance (Karabasheva, 2019).
Proper distribution of the ER in dividing cells is critical, because it ensures that progeny cells receive necessary proportions of this essential organelle. Disruptions in this process may result in cells that are prone to accumulation of misfolded proteins and ER stress, as well as dysregulation of lipid homeostasis and calcium signaling, all of which are involved in a spectrum of diseases including cancer, neurodegeneration, and diabetes. However, the importance of mitotic ER distribution may extend beyond organelle inheritance. For example, it was recently demonstrated that the ER plays an essential role in restricting the distribution of damaged proteins in asymmetrically dividing stem cells. This mechanism, which depends on proper distribution of the ER around the mitotic spindle, may allow vital stem cells to protect themselves by asymmetrically shuttling damaged proteins to non-essential progeny cells. In addition, it has been suggested that the ER delivers highly localized calcium signals that are essential for proper function of the spindle apparatus. Thus, disruption of ER distribution around the mitotic spindle may impair the process of cell division itself with potentially devastating effects on development and tissue homeostasis. Conversely, therapeutic interventions that specifically target ER functions in cancer cells may prove to be effective treatments in neoplastic disease. Identification of the molecular mechanisms that regulate M-phase specific distribution of the ER is critical to understanding these multi-faceted roles for the organelle in dividing cells. The present investigation demonstrates the existence of a novel mechanism that mediates ER-MT association in a manner that is specific to M-phase and independent of dynein and other known MT motors (Karabasheva, 2019).
The first studies to examine ER distribution in dividing cells reported the organelle's distinct organization around the poles of the mitotic spindle, where MT minus-ends are clustered. Subsequent studies have confirmed this observation across multiple species, suggesting the existence of a unifying mechanism of ER partitioning that involves MT-dependent spindle pole association. Accordingly, involvement of a MT minus-end motor to move the ER toward spindle poles has been suggested in multiple studies, with dynein being the most likely candidate. Importantly, dynein associates with ER-rich microsomal fractions and is required for proper ER distribution in interphase cells and extracts, suggesting that the motor can associate with and transport ER membranes. Surprisingly however, the putative role for dynein in regulating ER distribution during cell division has not been directly tested. This question was addressed using meiotic Drosophila spermatocytes, which demonstrate a dramatic, near complete MT-dependent redistribution of the ER to spindle poles during cell division. The results clearly demonstrate a role for dynein in ER transport and localization in these cells, because ER distribution toward MT minus-ends and around centrosomes during late interphase was completely disrupted by dynein suppression. Surprisingly, however, dynein was dispensable for astral MT association and spindle pole focusing of the ER during meiosis. Importantly and consistent with previous reports, dynein suppression had dramatic effects on spindle formation and architecture in Drosophila spermatocytes including failure of centrosome migration to the nuclear envelope and complete dissociation of spindle poles from the focused kinetochore MT fibers. Nevertheless, despite these highly abnormal spindles, this study observed that early in meiosis the ER was still drawn onto the astral MTs and towards the centrosomes, despite the centrosomes' mislocalization at the cell cortex. Moreover, this redistribution of the ER occurred with the same kinetics and to the same extent as in control cells, suggesting that the mechanism of ER-astral MT association is completely independent of dynein. In this regard, data indicating that dynein association with membranous organelles is inhibited during M-phase of cell division suggests the existence of a dynamic mechanism that reciprocally regulates dynein association of the ER with MTs according to the stage of the cell cycle (Karabasheva, 2019).
An important outcome of dynein suppression in spermatocytes is that it allowed a clear separation of interphase versus M-phase mechanisms of ER association with centrosomal MTs, wherein the ER was completely excluded from centrosomal MTs during late interphase but suddenly moved along MTs toward the centrosomes early in M-phase. This reveals that an M-phase specific mechanism of ER-MT association that concentrates the ER around centrosomes is triggered at the onset of cell division, possibly due to specific cyclin/cyclin-dependent kinase activity. This conclusion is further supported by the observations that several of the mechanisms for ER-MT association that operate during interphase are in fact inhibited during M-phase, including those mediated by STIM1, CLIMP-63, and possibly dynein. Collectively, these findings suggest an enticing mechanism whereby most or all interphase mechanisms of ER-MT association are inhibited during M-phase, allowing an M-phase specific mechanism to predominate and ensure ER association with astral MTs and proper partitioning to daughter cells (Karabasheva, 2019).
The challenge moving forward is to identify the M-phase specific mechanism that associates the ER with astral MTs. Importantly, in dynein suppressed spermatocytes it was clear that the ER moved along astral MTs toward the minus-ends, suggesting that a MT minus-end motor distinct from dynein may be involved. However, the present findings indicate that neither ncd, the only other known bona fide MT minus-end motor in Drosophila, nor Klp61F, which generates pole-ward forces in the spindle, are involved in the unique M-phase distribution of ER with astral MTs. Thus, it is possible that an unidentified MT minus-end motor is required, or that a non-motor factor stably attaches ER membranes along MT fibers and tubulin flux then moves these membranes toward centrosomes. In this regard, it was recently demonstrated that human REEP3 and 4, members of the REEP1-4 family of ER membrane proteins, directly associate with MTs and play a role in spindle pole focusing of the ER37. However, whether REEP3/4 associate the ER specifically with astral MTs has yet to be determined. Drosophila have a single orthologue to human REEPs1-4, known as REEPA, and preliminary observations indicate that REEPA also is not required for astral MT association of the ER in meiotic Drosophila spermatocytes. It is also important to note that different mechanisms of ER-MT association may operate during cell division in different cell types. Thus, while the current results cannot rule out a role for dynein or other MT minus-end motors in ER partitioning in cells other than Drosophila spermatocytes, the findings do indicate that these factors are not universally required (Karabasheva, 2019).
In addition to identifying the molecular factors that link the ER to astral MTs during cell division, another important question is how does this association discriminate astral MTs from other MTs of the spindle apparatus? Certainly, suppression of interphase mechanisms plays a role, as expression of nonphosphorylatable STIM1, which remains associated with MTs during mitosis, results in mislocalization of the ER to kinetochore MTs. It is also possible that differences in tubulin post-translational modifications facilitate discrimination between different MT populations within the spindle, as demonstrated for kinesin-7 motors that carry chromosomes toward the spindle equator along detyrosinated MTs of the inner spindle (Karabasheva, 2019).
In conclusion, this study has demonstrated that an M-phase specific mechanism associates the ER with astral MTs and partitions the organelle to spindle poles in dividing cells. Surprisingly, and in contrast to many previous suggestions, this ER localization is not mediated by dynein or other known MT minus-end motors. Identification of the mechanisms involved is an important next step in better understanding the role of the ER in cell division, stem cell longevity and pluripotency, and tissue architecture. This knowledge will facilitate development of novel therapeutics for pathological processes underlying cancer and age-related tissue degeneration (Karabasheva, 2019).
Centrosomes are microtubule-organizing centers required for error-free mitosis and embryonic development. The microtubule-nucleating activity of centrosomes is conferred by the pericentriolar material (PCM), a composite of numerous proteins subject to cell cycle-dependent oscillations in levels and organization. In diverse cell types, mRNAs localize to centrosomes and may contribute to changes in PCM abundance. This study investigated the regulation of mRNA localization to centrosomes in the rapidly cycling Drosophila melanogaster embryo. RNA localization to centrosomes was found to be regulated during the cell cycle and developmentally. A novel role for the fragile-X mental retardation protein was identified in the posttranscriptional regulation of a model centrosomal mRNA, centrocortin (cen). Further, mistargeting cen mRNA is sufficient to alter cognate protein localization to centrosomes and impair spindle morphogenesis and genome stability (Ryder, 2020).
The centrosome is a multifunctional organelle that serves as the primary microtubule-organizing center of most animal cells and comprises a central pair of centrioles surrounded by a proteinaceous matrix of pericentriolar material (PCM). During mitosis, centrosomes help organize the bipolar mitotic spindle and function to ensure the fidelity of cell division. In interphase, centrosomes contribute to cell polarization, intracellular trafficking, and ciliogenesis (Ryder, 2020).
Cell cycle-dependent changes in PCM composition contribute to functional changes in centrosome activity. Upon mitotic entry, centrosomes undergo mitotic maturation, a process by which centrosomes augment their microtubule-nucleating capacity through the recruitment of additional PCM. This process is reversed upon mitotic exit by PCM shedding. These dynamic oscillations in PCM composition and organization are essential for centrosome function, and their deregulation is associated with developmental disorders, increased genomic instability, and cancer. Nonetheless, the regulation of PCM dynamics remains incompletely understood (Ryder, 2020).
Centrosomes are essential for early Drosophila embryogenesis, which proceeds through 14 rounds of rapid, synchronous, abridged nuclear cycles (NCs) consisting of S and M phases with no intervening gap phases before cellularization. From NC 10 to 14, the embryo develops as a syncytial blastoderm, wherein thousands of nuclei and their associated centrosome pairs divide just under the embryonic cortex. Nuclear migration and divisions are coordinated by the centrosomes, and mutations in centrosome-associated genes impair spindle morphogenesis, mitotic synchrony, genome stability, and embryonic viability. As in many organisms, the early development of the Drosophila embryo proceeds through a period of transcriptional quiescence and is supported by a maternal supply of mRNA and proteins. Thus, PCM dynamics apparent in early embryos rely on posttranscriptional mechanisms (Ryder, 2020).
More than a decade ago, a high-throughput screen for mRNAs with distinct subcellular locations in syncytial Drosophila embryos uncovered a subset of mRNAs localizing to spindle poles. Many of the centrosome-enriched transcripts identified in that screen encode known centrosome regulators, including cyclin B (cyc B) and pericentrin-like protein (plp). These findings raise the possibility that RNA localization, translational control, and other posttranscriptional regulatory mechanisms contribute to centrosome activity and/or function. Consistent with this idea, RNA is known to associate with centrosomes in diverse cell types, including early embryos (Drosophila, Xenopus, zebrafish, and mollusk), surf clams, and cultured mammalian cells. The functional consequences and the mechanisms that regulate centrosome-localized RNA remain little understood, however (Ryder, 2020).
This study reports that multiple RNAs dynamically localize to centrosomes in Drosophila early embryos. These RNAs localize in unique patterns, with some forming higher-order granules and others localizing to centrosomes as individual molecules. This study further demonstrates that some RNAs localize to centrosomal subdomains, e.g., centrosome flares, which extend from interphase centrosomes and define the PCM scaffold. This study has identified one centrosomal RNA, centrocortin (cen), which forms micrometer-scale granules that localize asymmetrically to centrosomes. This study further defines the mechanisms underlying cen mRNA granule formation and function. cen mRNA granules include Cen protein and the translational regulator fragile-X mental retardation protein (FMRP), the orthologue of the fragile X syndrome-related RNA-binding protein encoded by the Fmr1 gene. These data show that FMRP regulates both the localization and steady-state levels of cen RNA and protein. Moreover, this study found that reducing cen dosage is sufficient to ameliorate mitotic spindle defects associated with Fmr1 loss. Finally, mislocalization of cen mRNA was shown to prevent the localization of protein to distal centrosomes and is associated with disrupted embryonic nuclear divisions (Ryder, 2020).
This study systematically examined five transcripts shown to enrich near spindle poles to quantitatively define their common and unique localization patterns in Drosophila embryos. Subsets of mRNAs were identified showing centrosome enrichment in a cell cycle-regulated and developmentally regulated manner. These nonrandom variances in RNA distributions further imply biological relevance. Tests were performed to see if RNA localization contributes to normal centrosome functions through in-depth studies with a model transcript, cen mRNA. FMRP was identified as an RNA-binding protein required for regulation of cen RNA localization, organization, and translational control. Further, reducing cen dosage rescued Fmr1-dependent mitotic errors and embryonic lethality. The consequences of mistargeting cen mRNA were directly tested. Mislocalization of cen mRNA to the anterior abrogated the normal localization of Cen to more distal centrosomes and disrupted spindle organization. Anterior mitotic divisions were also severely disrupted due to the increased local concentration of cen mRNA, which also recruited FMRP. These studies suggest that a normalized local concentration of cen mRNA is essential for normal cell division and genome stability (Ryder, 2020).
FMRP is a multifunctional RNA-binding protein with roles in translational repression, activation, RNA localization, and RNA stability. In humans, mutations in the gene encoding FMRP, FMR1, are the leading cause of heritable intellectual disability and autism. Although high-throughput studies have identified putative RNA substrates, surprisingly few of these have been validated. The current studies demonstrate that cen mRNA is regulated by FMRP, either directly or indirectly, and that titrating cen dosage is sufficient to partially restore embryonic viability in Fmr1 mutants. Consistent with direct regulation of cen mRNA by FMRP, the cen coding sequence contains six putative binding motifs for FMRP, according to RBPmap, an RNA-binding motif predictor. Moreover, human orthologues of cen, CDR2 and CDR2L, were identified as direct FMRP targets (Ascano, 2012). Deregulation of CDR2 and CDR2L is associated with paraneoplastic cerebellar degeneration (Albert, 1998; Corradi, 1997). These studies suggest that Drosophila cen may serve as a valuable model to uncover mechanisms underlying FMRP-mediated regulation of CDR2 and CDR2L. Whether FMRP similarly regulates other centrosome-localized mRNAs is an interesting question for future study (Ryder, 2020).
The enhanced recruitment of cen mRNA to heterogeneously sized pericentrosomal granules, coupled with the increased production of Cen protein within Fmr1 mutants, led to a speculation that cen mRNA granules may be sites of local translation, as recently proposed (Bergalet, 2020). However, disruption of cen granule formation, as in cnnB4 mutants, does not impair total Cen protein levels. This finding raises the possibility that Cen may be translated at alternate sites or that maternal stores of Cen obscure changes resulting from cen mRNA granule loss. These models are not mutually exclusive, and cen mRNA may be translated at multiple locales. The data support a model in which centrosomes serve as platforms for translation control, which may be positive or negative depending on the specific transcript and/or cell cycle stage, consistent with the idea that cen mRNA granules are sites of Cen translational regulation (Ryder, 2020).
This study shows that cen mRNA preferentially localizes to interphase centrosomes; that the centrosome scaffold, Cnn, is required for cen mRNA granule formation and localization; and that FMRP functions as a negative regulator of cen mRNA, limiting cen mRNA stability and translation of Cen protein. It is speculated that FMRP represses Cen translation within cen mRNA granules, dampening the local Cen concentration. Consequently, cen mRNA enrichment at centrosomes is exaggerated in Fmr1 mutants. Other factors likely promote Cen translation. Translational repression or derepression may be coupled to cen mRNA granule centrosome proximity, which decreases as embryos enter mitosis. An imbalance of Cen levels at centrosomes, either too little (as in cen mutants) or too much (as in Fmr1 mutants or cen-bcd-3'UTR embryos), impairs centrosome function/spindle integrity and embryonic viability. As the cen 3'UTR recruits ik2 mRNA to centrosomes, the mitotic defects observed following cen perturbation may result from indirect effects via ik2 mRNA. Nonetheless, cen mRNA dosage must be properly regulated for mitotic fidelity (Ryder, 2020).
A common trend emerging from comparative analyses is the greater enrichment of mRNA at interphase versus metaphase centrosomes. One possible explanation is the differential size of interphase centrosomes, which are significantly larger in Drosophila embryos owing to the elaboration of extended centrosome flares, part of the architecture of the centrosome scaffold. This pattern contrasts with mammalian centrosomes, which are larger in mitosis. According to this size model, a larger centrosome might dock additional RNAs simply because of the increased volume it occupies in the cell. This model was discounted based on the finding that a highly expressed control transcript, gapdh, does not enrich at interphase centrosomes. This result also argues against the idea that centrosomes recruit RNA molecules spuriously. Relatively few RNAs localize to centrosomes. This study shows that the localization of centrosome-associated RNA is regulated in space and time (Ryder, 2020).
Why do RNAs localize to interphase centrosomes? Recent work in mammalian cells proposed that some lengthy transcripts may be cotranslationally transported to centrosomes. This model would account for contemporaneous recruitment and colocalization of centrosome mRNA and proteins and may be pertinent to cen mRNA localization. Of the RNAs overlapping with the centrosome surface, sov was unique in that it appeared to preferentially dock along centrosome flares, localizing to the outer PCM zone. However, thus stydt did not detect Sov protein at centrosomes. Instead, Sov resides in the nucleus during interphase and is undetectable after nuclear envelope breakdown. These findings suggest that Sov is rapidly translocated into the nucleus. Live imaging of RNA transport and nascent protein synthesis is required to rigorously test the dynamics of RNA localization and local translation (Ryder, 2020).
Another model that may account for enrichment of centrosome RNAs at interphase centrosomes is the possibility that RNA contributes to centrosome structure, perhaps by promoting phase transitions. A common principle of phase transitions is the association of intrinsically disordered proteins with specific RNA molecules to form non-membrane-bound organelles with unique biophysical properties. Might cen mRNA granules represent phase-separated domains? Congruous with phase separation, Cen protein contains multiple predicted intrinsically disordered domains. While the contribution of all centrosomal RNAs cannot be ruled out, the current studies do not suggest that cen mRNA contributes to centrosome structure. Mistargeting cen mRNA to the anterior cortex did not appear to disrupt the organization of distal centrosomes, for example (Ryder, 2020).
Critically, disrupting the PCM scaffold is sufficient to inhibit formation of the cen mRNA granule. Previous work has shown that the PCM scaffold becomes progressively more structured during the prolonged interphases of later NCs. Additionally, the mother centrosome organizes a larger PCM scaffold owing to inherently greater levels of Cnn and PLP (Conduit, 2010; Lerit, 2015). Collectively, these features may account for the asymmetric localization of cen mRNA to mother centrosomes in late-stage syncytial embryos. These data lead to the conclusion that the PCM scaffold organized by Cnn and PLP is upstream of the recruitment and organization of cen mRNA granules (Ryder, 2020).
Many types of RNP granules form within cells, including stress granules, germ granules, P-bodies, etc., which all have unique functions and modes of assembly. The spatial proximity of multiple RNA molecules may facilitate intermolecular RNA interactions subsequently recognized by RNA-binding proteins. The FMRP-containing cen mRNA granule represents one such RNP, and further understanding how it promotes mitotic integrity warrants further investigation. As the early Drosophila embryo is transcriptionally quiescent, posttranscriptional regulatory mechanisms, and especially translational control, are fundamentally important for proper centrosome regulation and function (Ryder, 2020).
Ploidy variations such as genome doubling are frequent in human tumors and have been associated with genetic instability favoring tumor progression. How polyploid cells deal with increased centrosome numbers and DNA content remains unknown. Using Drosophila neuroblasts and human cancer cells to study mitotic spindle assembly in polyploid cells, this study found that most polyploid cells divide in a multipolar manner. Even if an initial centrosome clustering step can occur at mitotic entry, the establishment of kinetochore-microtubule attachments leads to spatial chromosome configurations, whereby the final coalescence of supernumerary poles into a bipolar array is inhibited. Using in silico approaches and various spindle and DNA perturbations, this study shows that chromosomes act as a physical barrier blocking spindle pole coalescence and bipolarity. Importantly, microtubule stabilization suppressed multipolarity by improving both centrosome clustering and pole coalescence. This work identifies inhibitors of bipolar division in polyploid cells and provides a rationale to understand chromosome instability typical of polyploid cancer cells (Goupil, 2020).
The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, this study identified a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage (Galletta, 2020).
An essential element of functional flagellated sperm is proper attachment between the head, which contains the genetic material, and the tail, which generates the force for swimming. A failure in this connection can result in decapitated, decaudated, or malformed sperm, ultimately leading to reduced fertility. The most well documented human study investigated 10 infertile males with acephalic sperm. These patients showed a variety of 'abnormal head-neck configurations,' including breaks between the head and tail and sperm with nuclei laterally attached to the midpiece near the centrioles. The head to tail linkage is centered around the centriole, referred to in this context as a basal body (the term centriole is used for simplicity). The 'distal end' of the centriole templates and anchors the axoneme, the core structural element of the tail, while the 'proximal end' of the centriole forms a connection with the nuclear surface. Thus, the proximal and distal ends of centrioles play distinct and critical roles in sperm assembly. Note that the proximal end and distal end of an individual centriole are being discussed; these are distinct from the 'proximal centriole' and 'distal centriole' terms used in mammalian systems to describe the two centrioles within each sperm. Except when specifically discussing mammalian spermiogenesis, the latter terms will not be used or referenced in this study (Galletta, 2020).
A prerequisite for a tight connection between the head and tail of the sperm is the relocation of the centriole to the nuclear envelope during early spermiogenesis in Drosophila and in mammals. In Drosophila, after the exit from meiosis II, the centriole is repositioned against the reformed nuclear envelope and eventually becomes embedded in the nuclear envelope and is surrounded by an electron-dense material suggested to provide a tight connection. Similarly in mammals, the 'proximal centriole'" moves and attaches to the nucleus where an electron-dense material accumulates and the 'connecting piece' assembles around the centriole pair. In both systems, the centriole templating the flagellar axoneme has its proximal end closest to the nucleus and is positioned perpendicular to the nuclear surface (Galletta, 2020).
In mammalian model systems, there are examples of mutations that cause the head-tail connection to fail, including mutations in centriole proteins Centrin 1 and Centrobin, but little is understood at the molecular level of how these centriole proteins are involved in establishing the head-tail connection. Mutant analysis in Drosophila has identified additional players such as Asunder, Lis-1, Spag4, Yuri gagarin, and dynein and dynactin components, indicating that positioning the microtubule (MT) motor dynein at the nuclear envelope is critical. Finally, mutations in gamma tubulin ring complex proteins, which are required for proper MT formation, result in defective centriole-nuclear attachment in older developing spermatids (STs). It is believed these studies in totality suggest a model whereby dynein on the nuclear surface binds and acts on centriolar MTs to reposition the centriole to the nucleus. However, this process has never been documented in live STs, the role of the centriole itself in this process has not been examined, and the molecular mechanism that ensures correct centriole orientation and docking has not been investigated in detail (Galletta, 2020).
The highly stereotypical proximal end-on docking of centrioles to the nucleus suggests that the centriole proximal end is specialized. Many studies have carefully defined proteins uniquely positioned along the proximal-distal centriole axis. This polarized localization can convey local functions. For example, proteins that regulate centriole length such as Klp10A, Cep97, and Cp110 are positioned at the distal end of the centriole where centriole elongation is thought to exclusively occur. When a new centriole forms, proteins such as Ana2/STIL and Sas6, structural elements required for earliest steps daughter centriole formation, accumulate at the proximal end of the mother centriole. Additionally, pericentriolar material (PCM), which is critical for nucleating and organizing MTs, appears to be restricted to the centriole proximal end in mammalian systems and the meiotic centrioles of Drosophila spermatocytes (SC). This suggests that MTs are predominantly produced and anchored at the proximal end of centrioles. However, unlike distal-end protein components, the importance of restricting PCM to the proximal end and the mechanism by which this restriction is achieved are unknown. This study sought to identify the mechanism by which PCM is proximally restricted, and it was hoped, in turn, to gain insight into proximal end nuclear docking during spermatogenesis (Galletta, 2020).
Functional sperm require a stable linkage between sperm head and tail, which is mediated by the centriole. To date, little is known about the molecular mechanisms underlying defective head-tail attachment, except for a few reports identifying mutations in genes such as Spata6, Sun5, BRDT, PMFBP1, and TSGA10. Directly relevant to this study on the role of the centriole in head-tail attachment, is TSGA10 (Cep135 paralog) and two other centriole proteins -- Centrin1 and Centrobin. Thus, the limited patient sequence analysis and mammalian model system mutants point to a critical role for the centriole in head-tail attachment (Galletta, 2020).
Live imaging of centrioles in Drosophila STs revealed that the docking of the centriole to the nucleus is a two-step process. The first is 'Nuclear Search,' where the centriole searches for its docking partner, the nucleus, immediately following meiotic exit. The second is 'Nuclear Attachment,' where a stable connection of the centriole to the nuclear surface is formed. Work in Drosophila has also identified a number of mutations that ultimately result in decapitation, several of which affect the ability of the MT motor dynein to localize to the NE, or to generate pulling forces on MTs. Interestingly, studies from mice have also implicated the dynein adapter Hook1 and the sun protein SPAG4 in ensuring proper head-tail linkage. These proteins have been shown across species to position the centrosomes adjacent to the nucleus in interphase of normally cycling cells. Thus, a simple model emerges where in developing STs a conserved dynein-based system on the surface of the nucleus interacts with MTs emanating specifically from the proximal end of the centriole to draw the proximal centriole end specifically to the nuclear surface. Imaging of MTs in Drosophila suggests that this model is quite plausible, showing MTs specifically emerge from the proximal end of the centriole, which was then shown is key to proper centriole-nuclear docking. One exciting future direction is to reexamine the known Drosophila decapitation mutants to separate players that affect Nuclear Search from those that affect only the later step of Nuclear Attachment (Galletta, 2020).
While this study focuses on the formation of functional sperm, it also provides insight into centrosome architecture and how this architecture relates to function, in this case nuclear docking. In recent years, enormous progress has been made in identifying centrosome proteins and mapping their localization with nanometer precision. The challenge now is to link protein position with function; progress on this front has been most notably made at the centriole. For example Ana2/STIL, Sas6, and Cep135 form a cartwheel structure inside a new centriole at its proximal end, serving as a template for centriole symmetry. Another protein complex, Cep97, CP110, and Klp10A, localizes to the distal end and to control centriole length. Thus, subcentriolar localization and function are intimately linked. Despite PCM being documented at the proximal end of the centriole, a link between the position and function of the PCM at the proximal end has not been established. One possible role for the PCM at the proximal end is to dictate the position of daughter centriole formation. Previous work has shown that new daughter centriole nucleation requires PCM, and overexpression of PCM results in additional daughter centriole formation. However, the position of the ectopic centrioles along the proximal-distal axis was not examined, and the importance of the proximal position could not be inferred. This study provides a clear demonstration of a proximal-specific function for PCM (Galletta, 2020).
Through a series of wild-type, mutant, and misexpression experiments, this study showed that the bridge protein PLP is critical in proper centriole docking in STs. By examining plp mutant testes, it was found that PLP is a major driver of PCM recruitment or retainment at centrioles in round spermatids (RSTs). In the absence of PLP, PCM is disorganized and the centriole is improperly positioned away from the nucleus. This study then investigated how PLP itself is restricted to the proximal end. Using endogenously tagged PLP protein, single-cell RNA-seq and whole mount in situ, this study showed that proximal restriction of PLP is achieved through a reduction in PLP mRNA and protein concentration prior to centriole elongation. It will be important in future studies to determine precisely how PLP concentration is reduced; there is likely a delicate balance between decreased PLP translation, increased PLP degradation, and simple dilution of PLP as spermatocyte size increases (Galletta, 2020).
While numerous studies have shown that PLP is necessary for PCM organization around centrioles and that PLP can interact with PCM components, the precise mechanism of how PLP acts to recruit or anchor γ-tub is not known. In addition to interacting with PCM components, both PLP, and its mammalian ortholog pericentrin, have been found in complexes with γ-tub itself. It remains to be determined precisely how the multiple possible pathways through which PLP can affect the PCM function at the centriole in different contexts. Whether direct or indirect, this study shows that mislocalizing PLP on the centriole is sufficient to dictate PCM position on the centriole. When PCM is present at a more distal position along the centriole in STs, MTs emanate from the entire centriole, which can result in lateral capture to the nucleus. This lateral capture appears unstable and firm Nuclear Attachment does not occur. It is proposed that these failed, or defective, centriole-nuclear attachments do not survive the forces applied as a result of axoneme and mitochondrial derivative elongation, nuclear clustering, and/or ST individualization, ultimately resulting in sperm decapitation. This is consistent with several studies linking a failure in individualization to a failure in centriole-nuclear attachment. Additional studies will be required to determine how the tight attachment between the nucleus and the centriole forms, precisely when the Nuclear Attachment fails in STs with laterally docked centrioles and how this connection relates to the machinery that drives the massive cellular reorganization required to build sperm (Galletta, 2020).
The transcriptional mechanism to restrict protein localization this study identified is one way to achieve sub-centriolar protein compartments. Other mechanisms include specific docking-site recognition, such as the LID (longitudinal tubulin-tubulin interaction domain) domain of Sas4 recognizing the plus end of MTs at the centriole distal end and protein symmetry-breaking and coalescence as seen with Plk4's ability to concentrate into a single spot on the surface of the centriole. While it is unknown if other proteins use a transcriptional mechanism like PLP for centriole position control, single-cell RNA-seq data could help identified such proteins (Galletta, 2020).
One exciting finding from this work is that simply altering the timing of PLP expression can have major deleterious effects. This can be analogous to many human diseases that are frequently reported to correlate with higher levels of protein expression; understanding the underlying cell biology and physiology of the protein overexpression becomes quite critical. For example, overexpression of the master regulator of centriole duplication Plk4 is sufficient to promote tumorigenesis and renal cysts. Another example is seen in the case of having an extra copy of pericentrin (the ortholog of PLP), which in humans is present on chromosome 21. An increase in pericentrin protein levels by 50% can cause defects in ciliogenesis and cilia function. Therefore, understanding the role of the centriole in human disorders will not only require understanding the consequences of loss of protein function but also the consequence of protein misexpression and misregulation (Galletta, 2020).
Apoptosis and compensatory proliferation, two intertwined cellular processes essential for both development and adult homeostasis, are often initiated by the mis-regulation of centrosomal proteins, damaged DNA, and defects in mitosis. Fly Anastral spindle 3 (Ana3) is a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation. This study reports that ana3m19 is a suppressor of lethality induced by the overexpression ofSol narae (Sona), a metalloprotease in a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family. ana3m19 has a nonsense mutation that truncates the highly conserved carboxyl terminal region containing multiple Armadillo repeats. Lethality induced by Sona overexpression was completely rescued by knockdown of Ana3, and the small and malformed wing and hinge phenotype induced by the knockdown of Ana3 was also normalized by Sona overexpression, establishing a mutually positive genetic interaction between ana3 and sona. p35 inhibited apoptosis and rescued the small wing and hinge phenotype induced by knockdown of ana3. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies and reduced the number of dying cells, demonstrating that Ana3 actively promotes cell survival. Knockdown of Ana3 decreased the levels of both intra- and extracellular Sona in wing discs, while overexpression of Ana3 in S2 cells dramatically increased the levels of both cytoplasmic and exosomal Sona due to the stabilization of Sona in the lysosomal degradation pathway. It is proposed that one of the main functions of Ana3 is to stabilize Sona for cell survival and proliferation (Cho, 2021).
The ability to resist and recover from external stresses is important for all living organisms that face stresses such as heat, reactive oxygen species, and irradiation during development and in the adult stage. Damaged cells need to be removed by apoptosis and replaced with newly formed cells by compensatory proliferation. The wing imaginal disc of Drosophila melanogaster is the primordium of the adult wing, and shows a very low level of cell death during normal larval development. In contrast, it shows extensive cell death by environmental stresses, and yet can develop into a normal wing even after 40% to 60% cell death (Cho, 2021).
The centrosome consists of a pair of centrioles and pericentriolar materials (PCMs). DNA damage and mitotic defects cause the overduplication of centrosomes and the formation of multipolar spindles, leading to mitotic failure and cell death. Defects in PCMs interrupt spindle assembly and activate the spindle assembly checkpoint. Fly Anastral spindle 3 (Ana3) is a PCM responsible for the cohesion of centrioles, prevention of premature centriolar segregation, and formation of basal bodies (Stevens, 2009). Ana3 and its mammalian homolog Rotatin (RTTN) contain multiple Armadillo repeats known to interact with Wnt signaling components and potentiate the Wnt pathway (Song, 2003). Wnt has critical roles in growth, development, adult homeostasis, and regeneration. Ana3 and RTTN are also important for the formation of cilia and basal bodies (Kheradmand Kia, 2012; Stevens, 2009). Loss of RTTN causes polymicrogyria (PMG), situs inversus, isomerism, and heterotaxia in humans (Cho, 2021).
From a previous genetic screen, 28 mutants were found to be as responsible for the suppression of lethality caused by the overexpression of Sol narae (Sona) (Kim, 2020). The present study identified one of suppressors as ana3m19. Sona is a member of a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family (Kim, 2016). Most ADAMTSs are secreted proteases that cleave components in the extracellular matrix, and their malfunctions result in multiple diseases including cancer. Sona is positively involved in Wingless (Wg) signaling, and secreted by both the exosomal secretion pathway and Golgi transport (Kim, 2016; Won, 2019). Sona cleaves the linker region of extracellular Wg and generates a new functional form of Wg that is specialized in cell proliferation (Cho, 2021).
Sona is important for cell survival, with the level of Sona correlated with the extent of cell survival (Tsogtbaatar, 2019). Cells expressing a high level of sona are cell autonomously resistant to γ-ray irradiation, while Sona secreted from these cells induces Cyclin D (Cyc D) in the neighboring cells for cell survival and proliferation in a non-cell autonomous manner. Interestingly, Wg-CTD but not full-length Wg induces Cyc D, which demonstrates that Sona is involved in intercellular communication to support the normal development of damaged tissues by regulating Wg signaling. Consistent with this, sona suppressors such as wntless, arrow, pou domain motif 3, and archipelago are related to Wg signaling (Cho, 2021).
This paper reports that Ana3 is also important for cell survival. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies, and the amount of Ana3 correlated with the extent of organism survival under irradiation. The level of Ana3 in wing discs was significantly increased by 1 h after irradiation, indicating that Ana3 may be one of the proteins that respond to irradiation at the front line. Ana3 expressed in S2 cells increased the level of both intracellular and secreted Sona by negatively regulating the lysosomal degradation pathway, which is consistent with the finding of ana3m19 as a sona suppressor. These data demonstrate a new role of Ana3 in the stabilization of Sona (Cho, 2021).
This paper reports that the ana3m19 mutant is a suppressor of Sona-induced lethality. Fly ana3 has a positive genetic interaction with sona that encodes a metalloprotease involved in Wnt signaling, establishing a potential link between Ana3/RTTN and Wnt signaling. Ana3/RTTN is a peripheral member of the centrosome complex whose malfunction leads to embryonic lethality in both ana3 mutant flies and RTTN knockout mice. Both Ana3 and Sona are involved in cell survival and resistance to irradiation. Consistent with their positive genetic interaction and functional similarity, this study found that Ana3 stabilizes Sona and increases the level of Sona in both wing discs and S2 cells. The truncated region in ana3m19 protein is the most conserved region in Ana3/RTTN homologs, suggesting that this region plays a key role in stabilizing Sona. Some PMG mutations have also been identified in the Armadillo repeats in this carboxyl region of RTTN protein (Cho, 2021).
Both lethality and the small wing phenotype induced by Sona overexpression were completely rescued by knockdown of Ana3, suggesting that one of the main functions of Ana3 is to stabilize Sona. It is worth noting that a degradation of Sona occurs in the lysosome but not in the proteasome complex, as well as that another sona suppressor Arr also stabilizes Sona (Han, 2020). Since the original genetic screen was aimed at identifying suppressors that reduce Sona activity, it makes sense that both ana3 and arr mutants are identified as sona suppressors. Interestingly, Ana3 dramatically increased the level of exosomal Sona but not soluble Sona. This suggests that Ana3 stabilizes Sona in the exosomal secretion pathway that is interconnected with the lysosomal degradation pathway and the endosomal pathway but not in Golgi transport (Cho, 2021).
The loss of ana3 induced cell death, which is a common phenotype of centrosome components. Interestingly, overexpression of Ana3 enhanced the survival rate of irradiated flies, with wing discs showing the increased level of Ana3 1 h after irradiating the larvae, indicating that signals initiated by irradiation increase the level of Ana3 to prevent cell death. Since Ana3 stabilizes Sona, and knockdown of ana3 completely rescues the lethality caused by overexpressed Sona, the ability of Ana3 in promoting cell survival may stem from stabilized Sona. Previous work has shown that Sona-expressing cells are resistant to irradiation in a cell autonomous manner, and Sona secreted from these cells enables neighboring cells to survive and proliferate in a non-cell autonomous manner (Tsogtbaatar, 2019). Thus, it is possible that the increased level of Ana3 by irradiation contributes to increasing the level of Sona, which in turn functions to promote cell survival in both cell-autonomous and non-cell autonomous manners (Cho, 2021).
Extracellular Sona cleaves Wg and generates Wg-CTD that increases the level of Cyc D for initiating cell cycles (Won, 2019). Cyc D1 in mammalian cells promotes cell proliferation in response to mitogens, but overexpression of Cyc D1 leads to centrosome amplification, deregulation of the mitotic spindle, and chromosome abnormalities. Cyc D1 is oncogenic in many human cancer cells because it contributes to malignant transformation, with centrosome amplification by ras oncogene depending on Cyc D1. The link between fly Cyc D, Sona, and Wg-CTD, as well as the association of many components in Wnt signaling such as Disheveled, Armadillo/β-catenin, Axin, and Arrow/LRP6 with centrosomes, suggests that Sona may participate in the regulation of centrosomal duplication for the initiation of cell cycles (Cho, 2021).
A functional centrosome is vital for the development and physiology of animals. Among numerous regulatory mechanisms of the centrosome, ubiquitin-mediated proteolysis is known to be critical for the precise regulation of centriole duplication. However, its significance beyond centrosome copy number control remains unclear. Using an in vitro screen for centrosomal substrates of the APC/C ubiquitin ligase in Drosophila, several conserved pericentriolar material (PCM) components were identified, including the inner PCM protein Spd2. Spd2 levels are controlled by the interphase-specific form of APC/C, APC/C (Fzr), in cultured cells and developing brains. Increased Spd2 levels compromise neural stem cell-specific asymmetric PCM recruitment and microtubule nucleation at interphase centrosomes, resulting in partial randomisation of the division axis and segregation patterns of the daughter centrosome in the following mitosis. Evidencse is provided that APC/C(Fzr) -dependent Spd2 degradation restricts the amount and mobility of Spd2 at the daughter centrosome, thereby facilitating the accumulation of Polo-dependent Spd2 phosphorylation for PCM recruitment. This study underpins the critical role of cell cycle-dependent proteolytic regulation of the PCM in stem cells (Meghini, 2023).
Centrosomes are multi-protein organelles that function as microtubule (MT) organizing centers (MTOCs), ensuring spindle formation and chromosome segregation during cell division. Centrosome structure includes core centrioles that recruit pericentriolar material (PCM) that anchors γ-tubulin to nucleate MTs. In Drosophila melanogaster, PCM organization depends on proper regulation of proteins like Spd-2, which dynamically localizes to centrosomes and is required for PCM, γ-tubulin, and MTOC activity in brain neuroblast (NB) mitosis and male spermatocyte (SC) meiosis. Some cells have distinct requirements for MTOC activity due to differences in characteristics like cell size or whether they are mitotic or meiotic. How centrosome proteins achieve cell-type-specific functional differences is poorly understood. Previous work identified alternative splicing and binding partners as contributors to cell-type-specific differences in centrosome function. Gene duplication, which can generate paralogs with specialized functions, is also implicated in centrosome gene evolution, including cell-type-specific centrosome genes. To gain insight into cell-type-specific differences in centrosome protein function and regulation, this study investigated a duplication of Spd-2 in Drosophila willistoni, which has Spd-2A (ancestral) and Spd-2B (derived). Spd-2A functions in NB mitosis, whereas Spd-2B functions in SC meiosis. Ectopically expressed Spd-2B accumulates and functions in mitotic NBs, but ectopically expressed Spd-2A failed to accumulate in meiotic SCs, suggesting cell-type-specific differences in translation or protein stability. This failure to accumulate and function in meiosis was mapped to the C-terminal tail domain of Spd-2A, revealing a novel regulatory mechanism that can potentially achieve differences in PCM function across cell types (O'Neill, 2023).
Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. This study shows that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. Neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation (Panda, 2020).
Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. This study shows that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. These findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).
Centrosomes play an important part in many aspects of cell organisation, and they form when a mother centriole recruits pericentriolar material (PCM) around itself. The PCM contains several hundred proteins, allowing the centrosome to function as a major microtubule (MT) organising centre, and also as an important coordination centre and signalling hub. Centrosome dysfunction has been linked to several human diseases and developmental disorders, including cancer, microcephaly and dwarfism (Alvarez-Rodrigo, 2019).
During interphase, the mother centriole recruits a small amount of PCM that is highly organised. As cells prepare to enter mitosis, however, the PCM expands dramatically around the mother centriole in a process termed centrosome maturation. Electron microscopy (EM) studies suggest that centrioles organise an extensive 'scaffold' structure during mitosis that surrounds the mother centriole and recruits other PCM components such as the γ-tubulin ring complex (γ-TuRC) (Alvarez-Rodrigo, 2019).
In the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, a relatively simple pathway seems to govern the assembly of this mitotic PCM scaffold. The conserved centriole/centrosome protein Spd-2/SPD-2 (fly/worm nomenclature) cooperates with a large, predominantly predicted-coiled-coil, protein (Cnn in flies, SPD-5 in worms) to form a scaffold whose assembly is stimulated by the phosphorylation of Cnn/SPD-5 by the mitotic protein kinase Polo/PLK-1. Mitotic centrosome maturation is abolished in the absence of this pathway, and some aspects of Cnn and SPD-5 scaffold assembly have recently been reconstituted in vitro. Vertebrate homologues of Spd-2 (Cep192), Cnn (Cdk5Rap2/Cep215) and Polo (Plk1) also have important roles in mitotic centrosome assembly, indicating that elements of this pathway are likely to be conserved in higher metazoans. In vertebrate cells another centriole and PCM protein, Pericentrin, also has an important role in mitotic centrosome assembly that is dependent upon its phosphorylation by Plk1. Pericentrin can interact with Cep215/Cnn, but in flies the Pericentrin-like-protein (Plp) has a clear, but relatively minor, role in mitotic PCM assembly when compared to Spd-2 and Cnn (Alvarez-Rodrigo, 2019).
Although most of the main players in mitotic centrosome-scaffold assembly appear to have been identified, several fundamental aspects of the assembly process remain mysterious. Cells entering mitosis, for example, contain two mother centrioles that assemble two mitotic centrosomes of equal size. It is unclear how this is achieved, as even a slight difference in the initial size of the two growing centrosomes would be expected to lead to asymmetric centrosome growth-as the larger centrosome would more efficiently compete for scaffolding subunits. The centrioles in fly embryos appear to overcome this problem by constructing the PCM scaffold from the 'inside-out': Spd-2 and Cnn are only incorporated into the scaffold close to the mother centriole, and they then flux outwards to form an expanded scaffold around the mother centriole. In this way, the growing PCM scaffold could ultimately attain a consistent steady-state size-where incorporation around the mother centriole is balanced by loss of the scaffold at the centrosome periphery-irrespective of any initial size difference in the PCM prior to mitosis (Alvarez-Rodrigo, 2019).
A potential problem with this 'inside-out' mode of assembly is that the rate of centrosome growth is limited by the very small size of the centriole. Mathematical modelling indicates that the incorporation of a crucial PCM scaffolding component only around the mother centriole cannot easily account for the high rates of mitotic centrosome growth observed experimentally. To overcome this problem, it has been proposed that centrosome growth is 'autocatalytic', with the centriole initially recruiting a key scaffolding component that can subsequently promote its own recruitment. It has been proposed that Spd-2 and Cnn could form a positive feedback loop that might serve such an autocatalytic function: Spd-2 helps recruit Cnn into the scaffold, and Cnn then helps to maintain Spd-2 within the scaffold, thus allowing higher levels of Spd-2 to accumulate around the mother centriole, which in turn drives higher rates of Cnn incorporation (Alvarez-Rodrigo, 2019).
In worms and vertebrates, SPD-2/Cep192 can help recruit PLK1/Plk1 to centrosomes and Cep192 also activates Plk1 in vertebrates, in part through recruiting and activating Aurora A, another mitotic protein kinase implicated in centrosome maturation. It is suspected, therefore, that in flies Spd-2 might recruit Polo into the centrosome-scaffold to phosphorylate Cnn and so help to generate a positive feedback loop that drives the expansion of the mitotic PCM. In flies, however, no interaction between Polo and Spd-2 has been reported. Indeed, an extensive Y2H screen for interactions between key centriole and centrosome proteins identified interactions between Spd-2 and the mitotic kinases Aurora A and Nek2, and between Polo and the centriole proteins Sas-4, Ana1 and Ana2, but not between Polo and Spd-2. A possible explanation for this result is that Polo/Plk1 is believed to be largely recruited to its many different locations in the cell, including centrosomes, through its Polo-Box-Domain (PBD), which binds to phosphorylated S-S/T(p) motifs. Perhaps any such Polo binding sites in fly Spd-2 were simply not phosphorylated in the Y2H experiments. In support of this possibility, phosphorylated S-S/T(p) motifs in SPD-2/Cep192 have previously been shown to help recruit PLK1/Plk1 to centrosomes in worms, frogs and humans (Alvarez-Rodrigo, 2019).
This study examined the potential role of Spd-2 in recruiting Polo to centrosomes in Drosophila embryos. Like Cnn, Spd-2 is largely unphosphorylated in the cytosol, but is highly phosphorylated at centrosomes, where Spd-2 and Polo extensively co-localise within the pericentriolar scaffold. A Spd-2 fragment containing 19 S-S/T motifs exhibits enhanced binding to the PBD in vitro when it has been phosphorylated by Plk1, but no enhancement is seen if these S-S/T motifs are mutated to T-S/T-a mutation that strongly perturbs PBD binding. This study expressed forms of Spd-2 in vivo in which either all 34 S-S/T motifs, or the 16 most conserved S-S/T motifs, have been mutated to T-S/T to perturb PBD-binding. These mutant Spd-2 proteins are still recruited to mother centrioles, as are Polo and Cnn, and these proteins assemble a PCM scaffold around the mother centriole. Strikingly, however, this PCM scaffold can no longer expand outwards, and centrosome maturation fails. These observations provide strong support for the hypothesis that Spd-2, Polo and Cnn cooperate to form a positive feedback loop that is required to drive the rapid expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).
It was previously proposed that three proteins -- Spd-2, Polo and Cnn -- together form a scaffold that expands around the mother centriole to recruit other PCM components to the mitotic centrosome. The data presented in this study suggests that these three proteins cooperate to form a positive feedback loop that drives the dramatic expansion of the mitotic PCM scaffold in fly embryos (Alvarez-Rodrigo, 2019).
The following model is proposed (see Polo and Cnn appear to form a positive feedback loop that drives the expansion of the mitotic PCM scaffold). In interphase cells, Spd-2, Polo and Cnn are recruited around the surface of the mother centriole, but Polo is inactive and Spd-2 and Cnn are not phosphorylated-so no scaffold is assembled. As cells prepare to enter mitosis, centrosomal Spd-2 becomes phosphorylated. In vitro data suggests that Polo is involved in this phosphorylation (via a 'self-priming and binding' mechanism), but other mitotic kinases may also be involved. Phosphorylation allows Spd-2 to form a scaffold that fluxes outwards and that can recruit both Polo (via phosphorylated S-S/T(p) motifs) and Cnn . The active Polo phosphorylates Cnn, allowing it to also form a scaffold. The Spd-2 scaffold is inherently unstable, so it can only accumulate around the mother centriole if it is stabilised by the Cnn scaffold. The Cnn scaffold therefore allows the Spd-2 scaffold to expand outward, increasing Spd-2 levels within the PCM scaffold and allowing Spd-2 to recruit more Cnn and more Polo into the scaffold. This is a classical positive feedback loop in which the Output (the PCM scaffold in toto) directly increases the Input (the Spd-2 scaffold) (Alvarez-Rodrigo, 2019).
If Spd-2 cannot efficiently recruit Polo, as appears to be the case with the Spd-2-ALL and Spd-2-CONS mutants, it can still recruit Cnn, and this is, at least initially, phosphorylated by the pool of Polo that is still present around the mother centriole. The data suggests that this centriolar pool of Polo is not recruited by Spd-2 (at least not via the PBD), and it is suspected that S-S/T(p) motifs in other centriole proteins, such as Sas-4, normally recruit Polo to centrioles. As a result, mutant Spd-2 proteins can still support the assembly of a 'mini-scaffold' around the mother centriole, and this can recruit some PCM and organise some MTs. The mutant Spd-2 scaffold that fluxes outwards from the mother centriole, however, cannot recruit Polo. Therefore the Cnn recruited by the expanding Spd-2 network cannot be phosphorylated, and it cannot form a scaffold to support the expanding Spd-2 network. As a result, the expanding mitotic PCM scaffold rapidly dissipates into the cytosol (Alvarez-Rodrigo, 2019).
Although this mechanism is autocatalytic-as the expanding Spd-2 scaffold allows Polo and Cnn to be recruited into the PCM at an increasing rate-crucially, the mother centriole remains the only source of Spd-2. This potentially explains the conundrum of how mitotic PCM growth is autocatalytic, but at the same time requires the mother centriole. This requirement for centrioles can also potentially explain how two spatially separated centrosomes usually grow their mitotic PCM to the same size, as PCM size may ultimately be determined by how much Spd-2 can be provided by the centrioles, rather than how much PCM was present in the centrosome when maturation was initiated (Alvarez-Rodrigo, 2019).
A key feature of this proposed mechanism is that Cnn cannot recruit itself or Spd-2 or Polo into the scaffold (although it helps to maintain the Spd-2 scaffold recruited by the centriole). If it could do so, mitotic PCM growth would no longer be constrained by the centriole as Cnn could catalyse its own recruitment. Interestingly, although Spd-2 and Cnn are of similar size in flies (1146aa and 1148aa, respectively) Spd-2 has >5X more conserved potential PBD-binding S-S/T motifs than Cnn. Moreover, a similar ratio of conserved sites is found when comparing human Cep192 (1941aa) to human Cep215/Cdk5Rap2 (1893aa), even though the human and fly homologues of both proteins share only limited amino acid identity. Perhaps, these two protein families have evolved to ensure that phosphorylated Spd-2/Cep192 can efficiently recruit Polo/Plk1, whereas phosphorylated Cnn/Cep215 cannot (Alvarez-Rodrigo, 2019).
The data indicates that multiple S-S/T(p) motifs in Spd-2 may be involved in Polo recruitment to the PCM. When only the most conserved motifs are mutated, other motifs in Spd-2 appear to be able to help recruit Polo, as evidenced by the additive effect of the Spd-2-ALL mutant compared to the Spd-2-CONS mutant. This mechanism of multi-site phosphorylation and recruitment could help amplify the maturation process (as the additional Polo recruited would allow Cnn to be phosphorylated at a higher rate) and so contribute to the establishment of the positive feedback loop (Alvarez-Rodrigo, 2019).
Another important feature of this proposed mechanism is that Spd-2 is incorporated into the mitotic PCM at the centriole surface and then fluxes outwards. This Spd-2-flux has so far only been observed in Drosophila embryos and mitotic brain cells. In fly embryos, Cnn also fluxes outwards but, unlike Spd-2, this flux requires MTs and is only observed in embryos. In C. elegans embryos, SPD-5 behaves like Cnn in somatic cells: it does not flux outwards and is incorporated isotropically throughout the volume of the PCM. Moreover, a very recent study found no evidence for an outward centrosomal flux of SPD-2 in worm embryos. Clearly, it will be important to determine whether Spd-2/Cep192 homologues flux outwards in other species and, if so, whether this flux provides the primary mechanism by which the mother centriole influences the growth of the expanding mitotic PCM (Alvarez-Rodrigo, 2019).
In vertebrates, Cep192 serves as a scaffold for Plk1 and also Aurora A-another mitotic protein kinase that plays an important part in centrosome maturation in many species. There appears to be a complex interplay between Cep192, Plk-1 and Aurora A in vertebrates, with Cep192 acting as a scaffold that allows these two important regulators of mitosis to influence each other's activity and centrosomal localisation. Spd-2 clearly plays an important part in recruiting Aurora A to centrosomes in fly cells-although it is unclear if this is direct, as fly and worm Spd-2/SPD-2 both lack the N-terminal region in vertebrate Cep192 that recruits Aurora A. How Aurora A might influence the assembly of the Spd-2, Polo/PLK-1 and Cnn/SPD-5 scaffold remains to be determined, although in worms AIR-1 (the Aurora A homologue) is required to initiate centrosome maturation, but is not required for subsequent PCM growth (Alvarez-Rodrigo, 2019).
Finally, there has been great interest recently in the idea that many non-membrane bound organelles like the centrosome may assemble as 'condensates' formed by liquid-liquid phase separation. In support of this possibility for the centrosome, purified recombinant SPD-5 can assemble into condensates in vitro that have transient liquid-like properties, although they rapidly harden into a more viscous gel- or solid-like phase. Moreover, a mathematical model that describes centrosome maturation in the early worm embryo treats the centrosome as a liquid, and it is from this model that the importance of autocatalysis was first recognised. In vivo, however, the Cnn and SPD-5 scaffolds do not appear to be very liquid-like and fragments of Cnn can assemble into micron-scale assemblies in vitro that are clearly solid- or very viscous-gel-like. The current data suggests that the incorporation of Spd-2 into the PCM only at the surface of the centriole, coupled to an amplifying Spd-2/Polo/Cnn positive feedback loop, could provide an 'autocatalytic' mechanism that functions within the conceptual framework of a non-liquid-like scaffold that emanates from the mother centriole (Alvarez-Rodrigo, 2019).
Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and it has been shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. This study shows that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. It is concluded that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly (Alvarez-Rodrigo, 2021).
Polo kinase (PLK1 in mammals) is an important cell cycle regulator. During mitosis, it is recruited to several locations in the cell -- such as centrosomes, kinetochores and the cytokinesis apparatus -- where it performs multiple functions. PLK1 is usually recruited to these locations by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs in target proteins. Mutating the first serine in the PBD-binding motif to threonine strongly reduces PBD binding in vitro and in vivo (Alvarez-Rodrigo, 2021).
PLK1 has several key functions at centrosomes. These organelles are important microtubule (MT) organising centres that form around a pair of centrioles (comprising a mother and daughter centriole) when the mother recruits a matrix of pericentriolar material (PCM) around itself. During interphase, centrosomes organise relatively little PCM, but as cells prepare to enter mitosis the PCM expands dramatically in a process termed centrosome maturation. PLK1 is an essential driver of this process, and several PCM proteins have been identified as PLK1 targets. In vertebrate cells, PLK1 phosphorylates pericentrin, which cooperates with CDK5RAP2 (also known as Cep215) to promote mitotic PCM assembly, whereas in flies and worms Polo/PLK1 kinases phosphorylate Cnn and SPD-5 (functional homologues of CDK5RAP2), respectively, which allows these proteins to assemble into a PCM scaffold around the mother centriole that recruits other PCM proteins (Alvarez-Rodrigo, 2021).
Towards the end of mitosis, the mother and daughter centrioles disengage from each other. PLK1 is essential for disengagement and also for the subsequent maturation of the daughter centriole into a new mother centriole that is itself capable of duplicating and organising PCM. The old mother (OM) and new mother (NM) centrioles then both duplicate during S phase by nucleating the assembly of a daughter centriole on their side. PLK1 is not essential for centriole duplication per se, but it is required for the growth of the centriole MTs that occurs during G2, at least in human cells, and for the subsequent maturation of the daughter centriole into a new mother centriole. After duplication in S phase, the two centrosomes (each now comprising a duplicated centriole pair) are held together by a linker, and PLK1 also helps disassemble this linker to promote centrosome separation as cells prepare to enter mitosis (Alvarez-Rodrigo, 2021).
How PLK1 is recruited to centrosomes to execute its multiple functions is largely unclear, although this recruitment appears to be dependent on the PBD. In vertebrate systems, Cep192 is required for centrosome maturation and it is phosphorylated by Aurora A (also known as AURKA) to create PBD-binding sites that recruit PLK1; this promotes the activation of both kinases at the centrosome. The fly and worm homologues of Cep192, Spd-2 and SPD-2, respectively, are concentrated at centrioles and centrosomes, and their phosphorylation also helps recruit Polo/PLK1 kinases to the mitotic PCM to phosphorylate Cnn in flies and SPD-5 in worms. In fly embryos, Spd-2, Polo and Cnn have been proposed to form a positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. In this scenario, Spd-2 starts to be phosphorylated at centrioles as cells prepare to enter mitosis, and this allows Spd-2 to form a scaffold that can recruit other PCM proteins and that fluxes outwards from the mother centriole. The Spd-2 scaffold itself is weak, but it can recruit Polo and Cnn; the recruited Polo phosphorylates Cnn, which then forms a Cnn scaffold that recruits other PCM components and strengthens the Spd-2 scaffold. This allows more Spd-2 to accumulate around the centriole, which in turn drives the recruitment of more Polo and Cnn - so forming a positive feedback loop. In this way, Spd-2 recruits Polo and Cnn to the PCM to help drive centrosome maturation in flies (Alvarez-Rodrigo, 2021).
If Drosophila Spd-2 cannot efficiently recruit Polo (because all its S-S/T motifs have been mutated to T-S/T) Polo recruitment to the PCM is dramatically reduced, but Polo is still strongly recruited to the mother centriole, indicating that other proteins must help recruit Polo to centrioles. The centriole protein Sas-4 is phosphorylated by Cdk1 during mitosis on threonine 200 (T200), creating a PBD-binding site that recruits Polo to newly formed daughter centrioles. This allows the daughter to recruit Asl (Cep152 in humans), which allows the daughter to mature into a new mother that can duplicate and organise PCM - since Asl is required for both of these processes. Although the single PBD-binding site in Sas-4 recruits Polo to mother centrioles, it is suspected that other proteins must also be required. This study attempted to identify such proteins by mutating all the S-S/T motifs to T-S/T in several candidates. The centriole protein Ana1 (Cep295 in humans) was found to normally help recruit Polo to mother centrioles. Ana1 and Cep295 are required for centriole maturation, and in flies Ana1 helps recruit and/or maintain Asl at new mother centrioles. Thus, flies lacking Ana1 lack centrioles, centrosomes and cilia, presumably because the centrioles cannot duplicate without Ana1 as they cannot recruit Asl. This study shows that centrioles that do not efficiently recruit Polo via Ana1 can still recruit Sas-4, Cep135 and Asl, and can still duplicate, disengage and organise cilia, but they cannot efficiently recruit mitotic PCM or elongate during G2. It is proposed that Ana1 recruits Polo to centrioles specifically to promote centriole elongation in G2 and mitotic PCM assembly (Alvarez-Rodrigo, 2021).
Polo has many important functions at centrioles and centrosomes, and previous work has shown that it is initially recruited to newborn centrioles in flies when Cdk1 phosphorylates the Sas-4 T200 S-T motif during mitosis. This initial recruitment of Polo is important to allow the newborn centrioles to subsequently mature into mothers that can recruit Asl and so duplicate and recruit mitotic PCM. This study showed that the centriole protein Ana1 also plays an important part in recruiting Polo to mother centrioles. The data suggests that Ana1 can recruit Polo directly and that Polo itself can phosphorylate Ana1 at several S-S/T motifs to 'self-prime' its own recruitment. It cannot be excluded that other protein kinases may prime these S-S/T motifs, or that Ana1 could recruit Polo to centrioles indirectly in ways that are disrupted when the S-S/T motifs are mutated to T-S/T. Regardless of mechanism, the Ana1-dependent centriolar pool of Polo appears to be required to drive efficient mitotic PCM expansion and centriole elongation in G2 (Alvarez-Rodrigo, 2021).
Although Ana1 helps recruit and/or maintain Asl at centrioles, and therefore is essential for both mitotic PCM recruitment and centriole duplication, this function of Ana1 does not appear to require the ability to recruit Polo. Thus, Ana1-S34T centrioles recruit and maintain normal levels of Asl (and of Cep135, as well as slightly increased levels of Sas-4) and can duplicate normally. This is in contrast to the situation with Sas-4, where T200 phosphorylation is required for proper Asl recruitment and so for both centriole duplication and mitotic PCM assembly. Presumably, the Polo recruited by Sas-4 is either sufficient for Asl recruitment, or it phosphorylates centriole substrates other than Ana1 to promote Asl recruitment. Interestingly, PLK1 is also essential for efficient centriole disengagement, but neither the Ana1-S34T nor Sas-4-T200 mutations appear to perturb this process, indicating that a separate pathway must recruit Polo to centrioles to drive centriole disengagement. Centrosome separation in G2 is also normally dependent on PLK1, and centrosomes/duplicated centriole pairs were often observed that failed to separate properly in embryos expressing Ana1-S34T. As these centriole pairs almost always organised very little PCM, however, it is suspected that this defect may be an indirect consequence of the failure to properly recruit PCM, rather than a direct consequence of the inability of Ana1 to recruit Polo (Alvarez-Rodrigo, 2021).
These new findings further support the hypothesis that centrioles activate a Spd-2-Polo-Cnn positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. A key feature of this model is that Spd-2 can only be phosphorylated to initiate scaffold assembly at the surface of the mother centriole, and the phosphorylated Spd-2 then fluxes outwards away from the centriole: the Spd-2-Polo-Cnn scaffold itself cannot phosphorylate and/or recruit new Spd-2 into the scaffold. This is important, as it can explain why the mother centriole is required to drive efficient mitotic PCM assembly, why the size of the centriole influences the size of the mitotic PCM and why centrioles are constantly required to drive the growth of the mitotic PCM. All of these findings can be explained if the mother centriole is the only source of the phosphorylated Spd-2 scaffold. The observation that the pool of Polo recruited by Ana1, which unlike Spd-2, is not a PCM component and is restricted to the centriole - is required for the efficient expansion of the PCM demonstrates that the PCM-associated pool of Polo (recruited by Spd-2) is not sufficient to drive efficient PCM expansion on its own. It is important to stress, however, that so far an outward flux of Spd-2 from the centriole has only been observed in fly embryos and cells and has not been detected for SPD-2 in C. elegans embryos\. Clearly it will be important to establish whether such a Spd-2 or Cep192 flux exists in other species (Alvarez-Rodrigo, 2021).
The ability of Ana1 to recruit Polo also appears to be required for centriole elongation during G2. In human cells, PLK1 is required for this process, although a previous study did not report any change in centriole length after long-term Polo-inhibition in fly spermatocytes. Clearly more work is required to establish whether Polo recruitment by Ana1 has a role in G2 centriole elongation in flies, as the current work suggests, and, if so, what Polo's relevant substrates are at the centriole distal end (Alvarez-Rodrigo, 2021).
Finally, it is noted that both the Ana1/Cep295 and Spd-2/Cep192 protein families have a relatively high density of potential PBD-binding sites (S-S/T motifs) when compared to several other centriole and centrosome proteins. This suggests that these proteins might have evolved to function as scaffolds that amplify Polo levels at specific locations within the cell during mitosis. It will be interesting to examine whether other proteins with a high density of potential PBD-binding domains serve a similar function at other locations within the mitotic cell. The strategy of mutating all S-S/T motifs to T-S/T in candidate proteins may be a good way of testing this possibility as, for both Ana1 and Spd-2 at least, the S-to-T substitutions seem to specifically impair Polo-recruitment without more generally perturbing the function of the proteins or centriole/centrosome structure (Alvarez-Rodrigo, 2021).
After centrosome duplication, centrioles elongate before the M phase. To identify genes required for this process and understand the regulatory mechanism, this study investigated the centrioles in Drosophila premeiotic spermatocytes, expressing fluorescently tagged centrioles. An essential microtubule polymerisation factor, Orbit/CLASP, was identified that accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically for determining centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. Loss of centriole integrity was observed, including fragmentation of centrioles and earlier separation of the centriole pairs in Cp110 null mutant cells either overexpressing Orbit or harbouring Klp10A depletion. Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they are unpaired. These findings contribute to understanding the mechanism of centriole integrity, leading to chromosome instability in cancer cells (Shoda, 2021).
The centrosome plays an indispensable role as the major microtubule-organising centre (MTOC) in a cell. During mitosis, centrosomes duplicated in the S phase move apart from each other and reach the opposite poles of the cell. Each centrosome is involved in the assembly of spindle poles, which enables construction of the bipolar spindle microtubule structure. A centrosome consists of two components: a pair of centrioles and the surrounding pericentriolar matrix (PCM). After replication of the centrioles, the longer centriole (mother centriole) engages with the shorter one (daughter procentriole) in a V-shape. When a cell enlarges in the G2 phase, the short daughter procentriole undergoes elongation to a certain length before the subsequent M phase. A single centriole consists of a microtubule doublet or triplet, which is equivalent to the cytoplasmic microtubule. Several factors localised on centrioles have been shown to be involved in the centriole elongation process. The most critical step in centrosome duplication is the duplication of centrioles, which requires stringent regulation. However, the entire mechanism underlying regulation of centriole elongation and the regulatory factors required for the process are not known. Among the centriole-associated proteins, those belonging to the kinesin-13 family are known to act as microtubule-severing kinesins. Klp10A, a Drosophila member of the family, has been shown to play an indispensable role in the regulation of centriole elongation. Based on these observations, it is speculated that some of the factors regulating microtubule length might overlap with those required for centriole elongation. It is possible that the production of centrioles of specific length requires a balance between polymerisation and depolymerisation of the triplet microtubules. However, the main factor(s) counteracting Klp10A and promoting centriole elongation remain to be identified (Shoda, 2021).
Another characteristic complex containing Cp110 is localised at the distal tip of the centriole, where it regulates the accessibility of the distal end to the shrinking and hypothetical lengthening factors, thereby regulating centriole elongation at this end. In the absence of Klp10A, the longer centrioles harbour incomplete ninefold symmetry at their ends in Drosophila cultured cells and tend to undergo fragmentation. Importantly, Cp110 depletion differentially affects centriole elongation in a species- and/or cell type-specific manner. In Drosophila S2 cultured cells, Cp110 depletion results in centriole length diminution. This effect is rescued by simultaneous depletion of Klp10A. In contrast, CP110 (also known as CCP110) depletion results in centriole elongation in mammalian cells. The centriolar microtubules were dramatically elongated in somatic cells, such as wing discs and larval brain cells, in the Drosophila Cp110-null mutant, whereas subtle elongation of the structure was observed in the premeiotic spermatocytes of the mutant (Shoda, 2021).
The premeiotic spermatocyte in Drosophila is a good model for investigating centrosomes and centrioles. Drosophila spermatogenesis involves four mitotic and two meiotic cycles for the formation of haploid spermatids. In the same spermatocyte cyst, each of the 16 cells undergoes synchronous cell growth, which can be divided into the S1 stage, corresponding to S phase, and five subsequent stages, S2 to S6, before initiation of meiosis I. The centrioles, in particular, can be studied more easily in this cell type, since these organelles dramatically elongate until the onset of meiosis and the centriole cylinder is composed of microtubule triplets. In early spermatocytes that possess a pair of centrioles initially, centrioles duplicate at S1 stage. As primary spermatocytes enter in the growth phase, centrioles migrate toward the surface where they assemble the primary cilium at the distal end of basal body. At the beginning of meiotic division I, centrioles move close to the nucleus with their associated 'membrane pocket' on the distal end of the cilium-like region (CLR). Between the CLR and the basal body there is the transition zone (TZ), which plays an important role in elongating the primary cilium of the spermatocyte. Centrioles are no longer duplicated between the two meiotic divisions. Primary spermatocytes hold two pairs of centrioles composed of nine triplet microtubules and engaged by a cartwheel structure at the proximal ends. The centriole pair is disengaged during prophase II, and, consequently, singlet centrioles organise the centrosomes of secondary spermatocytes (Shoda, 2021).
Previous studies have shown that Orbit (the Drosophila CLASP orthologue, encoded by chb) is essential for microtubule polymerisation, as it adds tubulin dimers to the plus end of the microtubules; however, its role in centriole elongation has not been examined. Hence, this study aimed to investigate whether Orbit was involved in centriole elongation in the mature premeiotic spermatocytes before male meiosis. As Orbit antagonises Klp10A, a severing factor determining the length of spindle microtubules in cultured Drosophila cells, whether Orbit was also involved in centriole length regulation was assessed (Shoda, 2021).
In addition, this study highlighted the importance of these regulators of centriole dynamics and the distal end capping proteins in the centriole elongation process using Drosophila spermatocytes. The importance of regulating the elongation of duplicated centrioles to a certain length for proper chromosome inheritance during male meiotic divisions is also discussed (Shoda, 2021).
Centrioles in Drosophila spermatocytes consist of ninefold triplets of microtubules. This study showed that the centrioles elongated to a certain length as the cells grew before male meiosis. Klp10A, which is a Drosophila kinesin-13 orthologue essential for shortening the microtubules, plays an indispensable role in regulation of centriole length. Microtubule length can be determined by the balance between polymerisation and depolymerisation of tubulin heterodimers and protofilaments. Thus, dynamic factors that promote microtubule elongation might also play critical roles in the determination of centriole length. This study has presented evidence suggesting that overexpression of Orbit results in excessively long centrioles in premeiotic spermatocytes. Conversely, shorter centrioles were observed in hypomorphic orbit mutants. These results are consistent with the idea that Orbit is essential for promotion of centriole elongation in spermatocytes. Orbit was initially identified as a microtubule-associated protein that adds tubulin heterodimers at the plus end of microtubules. Hence, the probability that Orbit might also elongate the triplet microtubules of centrioles by adding tubulin dimers, similar to its function in microtubule elongation, is high. In contrast, the kinesin-13 orthologue Klp10A acts as a microtubule depolymerising factor at the plus ends of microtubules in interphase and is an important regulator of centriole elongation. In Drosophila S2 cells, Klp10A antagonises Orbit in bipolar spindle formation and its maintenance. This study has shown that simultaneous overexpression of Orbit and depletion of Klp10A further enhanced centriole elongation. Based on these findings, it is proposed that these two factors act antagonistically to produce centrioles of specific length. Very long GFP-Orbit signals were observed that extended from basal bodies in spermatocytes overexpressing the protein. Accordingly, it is hypothesised that these are overly long axoneme microtubules produced as a consequence of excessively stimulated polymerisation of tubulins by the overexpression. Orbit, like other CLASP proteins, has a microtubule-binding activity to stimulate tubulin polymerisation at the plus end. Alternatively, a possibility cannot be excluded that Orbit polymerises itself to construct microtubule-like structures on the distal tip of the basal body. It would be interesting to investigate whether basal body and axonemal microtubules overly elongate in cells overexpressing Orbit without a fluorescent tag (Shoda, 2021).
In addition to these two factors regulating centriole microtubules, it is hypothesised that Cp110 plays a role as a cap to restrict these factors acting on the distal ends of the microtubules at an earlier stage. After Cp110 releases from the ends at mid-stage, Orbit can access the distal ends more easily and stimulate the centriole microtubules to extend to a certain length. Although the loss of the cap protein resulted in only a subtle extension of centrioles, Orbit overexpression in the absence of Cp110 can change the centriole microtubule dynamics significantly. Consequently, remarkably long centriole microtubules would be produced in the spermatocytes. Consistent with this hypothesis, Cp110Δ1 mutation also significantly enhanced the overly long centriole phenotype in Klp10A-depleted spermatocytes. By contrast, the Cp110-null mutation did not enhance the shorter centriole phenotype caused by Klp10A overexpression. Further experiments need to be performed to verify the model. The fact that Cp110 influences centriole length depending on the cell type and cellular context has been demonstrated in previous studies. It is likely that differential regulation of the conserved core components underlies ciliary basal diversity in different cell types. As argued previously, cellular-specific and tissue-specific regulation in centriole duplication may be indispensable to regenerate diverse centriole structures (Shoda, 2021).
Previous reports have not investigated whether Orbit and Klp10A are localised on centrioles or around the PCM in Drosophila mitotic cells; however, a considerable amount of Orbit accumulates in centrosomes in early embryos, Drosophila cultured cells and germline stem cells during Drosophila oogenesis and spermatogenesis. Whether the protein is localised in the PCM or in the centrioles is not clear. Similarly, studies on the cellular localisation of Orbit/CLASP orthologues in other species have also shown centrosome localisation of these proteins. Anti-human CLASP1 immunostaining in Hela cells has demonstrated that the protein is localised on centrosomes during M phase (Maiato et al., 2003). Furthermore, a CLASP orthologue in Caenorhabditis elegans has been observed in the centrosomes of its embryonic cells. These reports did not mention whether the orthologues were associated with the centrioles. Recently, it has been reported that Klp10A is dominantly localised in the TZ of the ciliary structures in spermatocytes, spermatids and sensory neurons (Persico, 2019). Centrosomal localisation of the protein has also been reported in mitotic cells and germline stem cells in both sexes. Whether Klp10A localises on the cylindrical microtubules of the centrioles or in the PCM of mitotic cells has not been demonstrated. Therefore, whether these antagonistic regulators are localised on centrioles in mitotic cells should be determined at a higher resolution. Furthermore, whether these two regulators are required for centriole length determination in somatic cells warrants further investigation (Shoda, 2021).
In addition to excessively elongated centrioles, this study observed several abnormalities in centriole organisation and structure in spermatocytes overexpressing Orbit and/or harbouring Klp10A depletion. In the absence of Cp110, the loss-of-centriole-integrity phenotypes were also enhanced. Small pieces of centrioles observed may be broken pieces of over-elongated centrioles, as observed in cancer cells. Alternatively, they may have been unpaired centrioles separated precociously from centriole pairs containing daughter procentrioles, which are smaller than normal centrioles. By contrast, the loss-of-centriole-integrity phenotypes were not observed in cells overexpressing a shortening factor, Klp10A. Hence, it is considered that improvised construction of the basal body microtubules may be associated with the loss-of-integrity phenotype; thereby, centriole engagement would be lost. Centriole fragments were found with reduced diameter in the centriole microtubules. The presence of disintegrated centrioles supports this idea, but further investigations are necessary to test the current hypothesis (Shoda, 2021).
Spermatocytes homozygous for loss-of-function mutation of Sas6 and of Ana2 commonly demonstrate premature centriole separation before meiosis. Hence, Sas6 and Ana2 are considered to be required for centriole engagement and/or maintenance of the pairs. Orbit overexpression and/or Klp10A depletion may affect centriole engagement through interfering with Sas6 (or Ana2) function. It is also possible that the premature disengagement can occur independently of Sas6 or Ana2. In addition, it has been reported that APC/C activation and activation of separate, thereby unexpected, cleavage of Scc1 (also known as RAD21) cohesin can take place in mammalian cultured cells. The possibility cannot be excluded that alteration of microtubule dynamics in centrioles by altered expression of Orbit and/or Klp10A led to unexpected APC/C activation. This hypothesis will be tested by several experiments in future work (Shoda, 2021).
Previous studies have also mentioned that Cp110-null mutant spermatocytes or syncytial-stage embryos do not show detectable defects in centrosome behaviour, spindle formation or chromosome segregation. By contrast, this study has show that disintegrated centrioles and multipolar spindle microtubules emanating from the centriolar fragments existed in Cp110-null mutant spermatocytes and cells overexpressing Orbit, as well as in Klp10A-depleted cells less frequently. Cells homozygous for the loss-of-function Klp10A mutation also display multipolar spindle structures. However, it cannot be excluded that the spindle phenotype would result from abnormal microtubule organisation caused by Klp10A mutation, rather than centriole disintegration. Surprisingly, in Cp110 mutants overexpressing Orbit and undergoing meiosis I, it was observed that unpaired single centrioles, even a part of them, could act as the MTOC. Centrosomes must be 'licensed' to function as an MTOC that nucleates microtubules, although the mechanism whereby the 'license' is granted remains unclear. Nevertheless, a recent study has reported that excessive elongation of centrioles in cancer cells is related to the generation of overduplicated, fragmented or hyperactive centrosomes that nucleate considerably more microtubules during cell division. Chromosome segregation is disturbed in cells harbouring these abnormal centrioles. Interestingly, generation of cells harbouring extra centrosomes has been suggested to be able to drive spontaneous tumorigenesis in mice. Additional studies have reported that excessively elongated centrioles in Drosophila spermatocytes affect spermatogenesis via the production of defective flagella. Consistently, immotile sperm production and significant decrease in male fertility were observed in the Cp110-mutant males with spermatocyte-specific Klp10A depletion. These observations suggested that production of excessively elongated centrioles can affect cell division and subsequent spermatogenesis. Once an abnormal spindle microtubule structure is constructed, the extra MTOC results in chromosome mis-segregation and eventually chromosome instability, as observed in cancer cells (Shoda, 2021).
Certain abnormalities such as mis-segregation of chromosomes and the resulting aneuploidy might appear in the subsequent division of cells containing abnormal centrioles. Conversely, excessively short centrioles may be inadequate as templates for the duplication of centrioles in S phase. Hence, regulation of centrosome length via the antagonistic activities of elongation and shrinking factors, such as Orbit and Klp10A, is important. However, the loss of centriole integrity and the resulting aneuploidy may occur in meiotic cells, but not in mitotic cells, which have stricter microtubule assembly checkpoints. Further investigations are required to understand how centrosome length is regulated in mitotic cells (Shoda, 2021).
Current findings suggest the presence of mutually antagonistic regulation to determine centriole length and the significance of the production of centrioles with a certain length for centriole integrity, and for assurance of proper chromosome segregation. It is believed that these findings may enable the identification of a mechanism whereby the loss of centriole integrity causes chromosome mis-segregation in cancer cells (Shoda, 2021).
Centrosomes are essential parts of diverse cellular processes, and precise regulation of the levels of their constituent proteins is critical for their function. One such protein is Pericentrin (PCNT) in humans and Pericentrin-like protein (PLP) in Drosophila. Increased PCNT expression and its protein accumulation are linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remain underexplored. A previous study demonstrated that PLP levels are sharply down-regulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. It was hypothesized that the sharp drop in PLP protein was a result of rapid protein degradation during the male germ line premeiotic G2 phase. This study shows that PLP is subject to ubiquitin-mediated degradation and identified multiple proteins that promote the reduction of PLP levels in spermatocytes, including the UBR box containing E3 ligase Poe (UBR4), which was shown to bind to PLP. Although protein sequences governing posttranslational regulation of PLP are not restricted to a single region of the protein, this study identified a region that is required for Poe-mediated degradation. Experimentally stabilizing PLP, via internal PLP deletions or loss of Poe, leads to PLP accumulation in spermatocytes, its mispositioning along centrioles, and defects in centriole docking in spermatids (Galletta, 2023).
The duplication and 9-fold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. This study used hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its sub-cellular localization. A core stabilization sequence within Gorab's C-terminal coiled-coil domain was identified that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an anti-parallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable hetero-trimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a 16-fold reduced binding affinity for Gorab that can not support centriole duplication. Thus Gorab dimers at the Golgi exist in equilibrium with Sas-6 associated monomers at the centriole to balance Gorab's dual role (Fatalska, 2021).
Centrioles are the ninefold symmetrical microtubule arrays found at the core of centrosomes, the bodies that organize cytoplasmic microtubules in interphase and mitosis. Centrioles also serve as the basal bodies of both non-motile and motile cilia, and flagellae. The core components of centrioles and the molecules that regulate their assembly are highly conserved. The initiation of centriole duplication first requires that the mother and daughter pair of centrioles at each spindle pole disengage at the end of mitosis. Plk4 then phosphorylates Ana2 (Drosophila)/STIL(human) at its N-terminal part, which promotes Ana2 recruitment to the site of procentriole formation, and at its C-terminal part, which enables Ana2 to bind and thereby recruit Sas6. The ensuing assembly of a ninefold symmetrical arrangement of Sas6 dimers provides the structural basis for the ninefold symmetrical cartwheel structure at the procentriole's core. Sas6 interacts with Cep135 and in turn with Sas4 (Drosophila)/CPAP (human), which provides the linkage to centriole microtubules (Fatalska, 2021).
An unexpected requirement has been identified for the protein, Gorab, to establish the ninefold symmetry of centrioles (Kovacs, 2018). Flies lacking Gorab are uncoordinated due to basal body defects in sensory cilia, which lose their ninefold symmetry, and also exhibit maternal effect lethality due to failure of centriole duplication in the syncytial embryo. Gorab is a trans-Golgi-associated protein. Its human counterpart is mutated in the wrinkly skin disease, gerodermia osteodysplastica. By copying a missense mutation in gerodermia patients that disrupts the association of Gorab with the Golgi, this study was able to create mutant Drosophila Gorab, which was also unable to localize to trans-Golgi. However, this mutant form of Gorab was still able to rescue the centriole and cilia defects of gorab null flies. It was also found that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype that can be overcome by a second mutation preventing Golgi targeting. Thus, centriole and Golgi functions of Drosophila Gorab are separable (Fatalska, 2021).
The Golgi apparatus both delivers and receives vesicles to and from multiple cellular destinations and is also responsible for modifying proteins and lipids. Gorab resembles a group of homodimeric rod-like proteins, the golgins, which function in vesicle tethering. The golgins associate through their C-termini with different Golgi domains, and their N-termini both capture vesicles and provide specificity to their tethering. There is known redundancy of golgin function, reflected by the overlapping specificity of the types of vesicles they capture. Gorab is rapidly displaced from the trans-side of the Golgi apparatus by Brefeldin A, suggesting that its peripheral membrane association requires ARF-GTPase activity (Fatalska, 2021).
Previous studies of human Gorab indicated its ability to form a homodimer in complex with Rab6 and identified its putative coiled-coil region as a requirement to localize at the trans-Golgi (Egerer, 2015; Witkos, 2019). Studies on its Drosophila counterpart supported Gorab's ability to interact with itself, potentially through the predicted coiled-coil motif. However, this region was also found to overlap with the region required for Gorab's interaction with Sas6 (Kovacs, 2018). These findings raised the questions of how Gorab's putative coiled-coil region could facilitate interactions with the Golgi, on the one hand, and its Sas6 partner, on the other. To address this, s hydrogen-deuterium exchange (HDX) was employed in conjunction with mass spectrometry (MS). HDX enables the identification of dynamic features of protein by monitoring the exchange of main chain amide protons to deuteria in solution. This study used HDX-MS to monitor the retarded exchange of amide protons localized between interacting regions of Gorab and Sas6 to identify the interacting surfaces within the Gorab-Sas6 complex. Together with other biophysical characterizations, this has revealed that Gorab is able to form a homo-dimer through its coiled-coil region but that it interacts as a monomer with the C-terminal coiled-coil of Sas6. Mutation of a critical amino acid in Sas6's Gorab-binding domain generates a variant of Sas6 with a sixteenfold reduced binding affinity for Gorab that is no longer able to support centriole duplication (Fatalska, 2021).
Together, these findings indicate that Gorab exists at the trans-Golgi network as a homodimer. Dimerization requires its coiled-coil motif (residues 200-315) within which is a core sequence (residues 270-287) that represents the most stable part of this dimerization region. Dimerization enables Gorab to interact with Rab6, and this in turn enables its association with the trans-Golgi. In contrast, Gorab interacts with Sas6 as a monomer. Gorab's binding to Sas6 occurs with a higher affinity than its homodimerization, enabling a Gorab monomer to associate with the Sas6 dimer. Thus, the relatively small number of Sas6 molecules at the centriole would more avidly bind the Gorab monomer, allowing greater excess of Gorab to accumulate as dimers at the trans-Golgi. Sas6 and Gorab interact through short interfaces within their coiled-coil regions. Disruption of this region of Sas6 through mutation of a single conserved leucine residue, L447, results in a failure of Gorab to bind to Sas6 and localize to the centriole. While the possibility cannot be formally excluded that the L447A mutation affects some other aspect of Sas6 function, the finding that expression of this mutant phenocopies a strong gorab hypomorph in its effects upon both co-ordination and centriole duplication suggests that failure to recruit Gorab is responsible for the Sas6-L447A defect. The finding of some residual apparent Gorab-like function in Sas6-L447A-expressing flies may reflect the overexpression of the protein due to the technical requirements of the experiment and the fact that Sas6-L447A still binds Gorab but with a sixteenfold reduced affinity compared to wild-type Sas6. Given that Sas6-L447A greatly diminishes the interaction with Gorab, whereas the mutation, M440A, in the adjoining 'a' position of the 'a-g' coiled-coil heptad repeat does not, leads to the conclusion that Gorab binds to a narrow region near the C-terminus of the coiled coil of Sas6 (Fatalska, 2021).
Gorab shows many of the properties typical of golgins, a family of tentacle-like proteins that protrude from the Golgi membranes to capture a variety of target vesicles. Redundancy between golgins in their ability to bind target vesicles could act as a functional safeguard and might explain why loss-of-function gorab mutants display no obvious Golgi phenotype, contrasting to the Golgi defects shown by the C-terminally tagged Gorab molecule (Kovacs, 2018). Gorab is similar to other golgins, which also associate with the Golgi membranes through their C-terminal parts in interactions that require Rab family member proteins to interact with the C-terminal part of the golgin dimer. The N-terminal parts of the golgins interact with their vesicle targets. Human GORAB's N-terminal part interacts with Scyl1 to promote the formation of COPI vesicles at the trans-Golgi (Witkos, 2019). However, its precise role in the transport of COPI vesicles is not clear, particularly why loss of human GORAB affects Golgi functions in just bone and skin when COPI function is required in multiple tissues. Drosophila Gorab also co-purifies and physically interacts with both Yata, counterpart of Scyl1, and COPI vesicle components, and its importance for transport of COPI vesicles in Drosophila is similarly unclear (Fatalska, 2021).
This study offers a perspective on how Gorab interacts with Sas6 at the centriole and suggests the possibilities for why this interaction is essential to establish the centriole's ninefold symmetry. The heterotrimeric structure formed by a Sas6 dimer and the Gorab monomer will together constitute a single spoke plus central hub unit of the centriole's cartwheel. The C-terminal part of Gorab would be expected to lie in a tight antiparallel association with the C-terminal part of Sas6's coiled-coil region. Gorab's N-terminus might thus be expected to extend towards the centriolar microtubules and their associated proteins. As the microtubules of Drosophila's somatic centrioles exist as doublets of A- and B-tubules, it is tempting to speculate that Gorab interacts with the centriole wall in a region occupied in other cell types by the C-tubule. This could account for the lack of any requirement for Sas6-Gorab interaction in the male germ-line, where centrioles have triplet microtubules and a C-tubule occupies this space. Gorab's partner proteins interacting with its N-terminal region are therefore of great interest at both the Golgi and in the centriole, and it will be key to understand the nature of these interactions in future studies (Fatalska, 2021).
Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. This study established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. At a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, the results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, which other factors modulate de novo centriole assembly was investigate, and proteins of the pericentriolar material (PCM), and in particular γ-tubulin, were found to promote biogenesis, likely by locally concentrating critical components (Nabais, 2021).
Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new 'daughter' centriole is built orthogonally on one side of each radially symmetric 'mother' centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. This study shows that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site (Cunningham, 2022).
Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. This study shows that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process-indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis (Steinacker, 2022).
Two studies have attempted to estimate the levels of one or more of the core centriole duplication proteins in human cells. Fluorescence correlation spectroscopy (FCS) has been used to estimate a Sas-6 cytoplasmic concentration of ~80-360 nM, depending on the cell cycle stage, while another study used quantitative MS to estimate the number of Plk4, Sas-6, CEP152/Asl, and STIL/Ana2 molecules in human cultured cells, which was in the ~2,000-20,000 range, ~10-15X lower than the number of γ-tubulin molecules in the cell. If the volume of a HeLa cell is ~4,000 μm3, then the concentration of these centriole proteins is in the ~1-10 nM range, which seems low, but could reflect that most somatic cells only assemble two tiny daughter centrioles during a cell cycle that can last many hours (Steinacker, 2022).
Given that the early Drosophila embryo assembles several thousand centrioles in <2 h, it was anticipated that centriole assembly proteins would be stored at higher concentrations than in somatic cells, but this does not appear to be the case. It is estimate that Asl, Sas-6, Ana2, and Sas-4 are present in the ~5-20 nM range (note that 20 nM would be the concentration of the Ana2 oligomer), while the cytoplasmic concentration of Plk4 is so low that it cannot be measured by FCS. Interestingly, these concentrations are similar to the MS estimates in human cell lines, suggesting that the early embryo does not store a large surplus of any of these proteins. Why are these key centriole assembly proteins present at such low concentrations? Several of these proteins tend to self-assemble into larger macromolecular structures, so it seems likely that their low cytoplasmic concentration helps to ensure that they normally only start to form a cartwheel at the single kinetically favorable site on the side of the mother centriole. Indeed, the FCS data suggest that the concentration of Sas-6 in the embryo is low enough that it is largely monomeric in the cytoplasm, even though it is almost certainly incorporated into the centriole cartwheel as a dimer. Storing Sas-6 as a monomer would help to ensure that it cannot spontaneously assemble into aberrant structures, and it is wondered whether storing self-assembling proteins that normally function as dimers (or higher-order homo-multimers) in cells as monomers (or lower order homo-multimers) might be a general strategy that helps to prevent their inappropriate self-assembly (Steinacker, 2022).
How cellular structures grow to the correct size is a topic of great interest. In the embryos of C. elegans, mitotic centrosome size appears to be set by a limiting cytoplasmic pool of the centrosome building block SPD-2, although this does not appear to be the case for Spd-2 in early Drosophila embryos. The concept of setting organelle size with a limiting pool of building blocks is attractive, as it allows size to be controlled without the need for a specific mechanism to measure it. The data, however, suggests that although the cytoplasmic concentration of the core duplication proteins is low, none of them act as limiting components to regulate centriole growth in Drosophila embryos. It is concluded that the amount of these proteins sequestered at centrioles may be insignificant compared to the amount in the cytoplasm (a plausible scenario given the large volume of the embryo and small volume of the centriole), and/or that the rate of protein sequestration at centrioles and degradation in the embryo is finely balanced by the rate of new protein synthesis so that a constant cytoplasmic concentration is maintained.
Cdk/Cyclin appears to phosphorylate Ana2 to modulate centriole duplication efficiency (Steinacker, 2022).
In vertebrates, STIL binds and is phosphorylated by CDK1/Cyclin B kinase. The function of this phosphorylation is unclear, but it is thought that binding to (rather than phosphorylation by) CDK1/Cyclin B keeps STIL in an inactive state because Cdk1/Cyclin B binds to the same central coiled-coil (CC) region of STIL that binds PLK4. The current data suggest that in fly embryos Cdk1/Cyclin activity can inhibit daughter centriole growth by phosphorylating, rather than simply binding to, Ana2. Ana2's diffusion rate increases as Cdk/Cyclin activity increases toward the end of S-phase and this increase is abrogated if Ana2 cannot be phosphorylated by Cdk1/Cyclin (due to mutation of all 12 S/T-P motifs). This Ana2(12A) mutant protein can still support centriole duplication, but it is recruited to the duplicating centrioles for an unusually long period of time during S-phase (presumably because its recruitment is not efficiently inhibited by the rising levels of Cdk/Cyclin activity in the embryo), allowing the protein to accumulate at centrioles to abnormally high levels. Mutating these 12 motifs to phosphomimicking D/E motifs has the opposite effect: Ana2(12D/E) is recruited poorly to centrioles and it can no longer support the rapid cycles of centriole duplication in the early embryo. It cannot be ruled out that the 12A and 12D/E mutations alter Ana2 in ways that change its conformation, multimerization, or function in unknown ways. Nevertheless, the ability of both mutants to support centriole duplication in somatic cells and their opposing effects on Ana2's centriole recruitment are consistent with the hypothesis that these mutations prevent or mimic Ana2 phosphorylation, respectively (Steinacker, 2022).
A priori, it is perhaps surprising that the 12A and 12D/E mutants appear to support relatively normal centriole duplication in somatic cells, demonstrating that the phosphorylation of Ana2 by Cdk/Cyclins cannot be essential for duplication-although the 12D/E mutant cannot support centriole duplication in the early embryo. It is speculated that while the Cdk/Cyclin-dependent phosphorylation of Ana2 reduces the efficiency of centriole duplication toward the end of the S-phase, multiple additional regulatory mechanisms-such as the oscillation in centriolar Plk4 levels-help to ensure that daughter centrioles still duplicate at the right time and place even if Ana2 cannot be phosphorylated by Cdk/Cyclins. In embryos, the 12D/E mutant is lethal, as the rapidly dividing centrioles do not have time to compensate for the reduction in duplication efficiency, but this is not the case in somatic cells, where the S-phase is much longer (Steinacker, 2022).
It is not known how the phosphorylation of Ana2 by Cdk1/Cyclins might influence centriole duplication, but it is speculated that it decreases Ana2's affinity for one or more of the other core centriole duplication proteins to which it binds (e.g., Sas-6, Plk4 or Sas-4). Unfortunately, it has not been possible to directly test this in vitro, and it was not possible to detect direct interactions between these endogenous proteins in embryo extracts, probably due to their very low cytoplasmic concentrations. Nevertheless, such a scenario would explain why Ana2's average cytoplasmic diffusion rate normally increases toward the end of the S-phase and why this increase is abrogated in the 12A mutant. FCS analysis also suggests that the average cytoplasmic diffusion rate of all the core duplication proteins analyzed in this study increases slightly as S-phase progresses, perhaps hinting that their cytoplasmic interactions might be generally suppressed by increasing Cdk/Cyclin activity. In embryos expressing Ana2(12A), the failure to efficiently inhibit Ana2's interactions with one or more other duplication proteins toward the end of S-phase could explain why Ana2(12A) and Sas-6 can continue to incorporate into centrioles for an extended period. Such a mechanism could also explain previous observations that inhibiting Cdk1 activity can lead to centriole overduplication in flies (Vidwans et al., 2003) (Steinacker, 2022).
Unexpectedly, expressing Ana2(12A) significantly decreased the amount of Sas-6 recruited to centrioles. This is surprising because Ana2 is thought to help recruit Sas-6 to centrioles, and centriolar Ana2(12A) levels are abnormally high. An intriguing interpretation of this finding is that while the phosphorylation of Ana2 by Cdk/Cyclins in late S-phase helps to inhibit centriole duplication, Cdk/Cyclin-dependent phosphorylation of Ana2 in early S-phase (presumably on different sites) might help promote centriole duplication by increasing the efficiency with which Ana2 interacts with Sas-6 to recruit it to centrioles. The S-phase-initiating CDK2/Cyclin kinase is required for centriole duplication, but its relevant substrate(s) are largely unknown. Perhaps CDK2/Cyclins phosphorylate Ana2 in early S-phase to promote centriole duplication, while CDK1/Cyclins phosphorylate Ana2 from late-S-phase onward to inhibit centriole duplication. Alternatively, the differential phosphorylation of different Cdk/Cyclin targets by different levels of Cdk/Cyclin activity plays an important part in ordering cell cycle events. Perhaps low (early-S-phase-like) levels of Cdk/Cyclin activity phosphorylate Ana2 on certain sites to promote centriole assembly, while higher levels phosphorylate Ana2 at additional sites to inhibit centriole assembly. In either scenario, Ana2 would act as a 'rheostat', responding to global changes in Cdk/Cyclin activity to coordinate centriole duplication with cell cycle progression. Plk4 phosphorylates Ana2 in an ordered fashion at multiple sites to elicit sequential changes in Ana2 behavior, so it seems possible that Cdk/Cyclins might do the same (Steinacker, 2022).
Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. Previous work showed that an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. This study showed that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, in a manner that appears to be entrained by the core Cdk/Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110/Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, changing the levels of CP110/Cep97 appears to alter the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected potential crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which may help to coordinate centriole growth (Aydogan, 2022).
This study shows that fluorescent fusions of CP110 and Cep97 are recruited to the distal-end of daughter centriole MTs in a cyclical manner as they grow during S-phase, with levels peaking, and then starting to decline at about mid-S-phase, which is normally when the centrioles appear to stop growing in these embryos. These recruitment dynamics, however, do not appear to play a major part in determining the period of daughter centriole MT growth, and the findings strongly suggest that centriole MTs do not stop growing when a threshold level of CP110 and Cep97 accumulates at the centriole distal end. Thus, although in many systems the centriole MTs are dramatically elongated in the absence of CP110 or Cep97, it is speculated that they have finished growing, rather than because the centriole MTs grow too quickly as the new daughter centriole is being assembled. It remains a formal, though unlikely, p that this cyclical recruitment of CP110 and Cep97 is an artefact of their fluorescent tagging (Aydogan, 2022).
CP110 and Cep97 levels do not peak at centrioles because the proteins reach saturating levels on the centriole MTs, as the amount of CP110 and Cep97 recruited to centrioles is increased when either protein is overexpressed. It is unclear how these proteins interact specifically with the distal-ends of the centriole MTs, but it is concluded that their binding sites are normally far from saturated, at least in the rapidly cycling Drosophila embryo. Importantly, the phase of CP110 and Cep97 recruitment appears to be influenced by the activity of the core cell-cycle oscillator (CCO). It is suspected, therefore, that the cyclical recruitment dynamics of CP110 and Cep97 in these embryos might simply reflect the ability of these proteins to bind to centrioles when Cdk-Cyclin activity is low, but not when it is high. CP110 was originally identified as a Cdk substrate, and presumably the CCO modifies (perhaps by phosphorylating) CP110 and/or Cep97 and/or their centriolar recruiting factor(s) to inhibit recruitment as cells prepare to enter mitosis. It is presently unclear why it might be important to prevent CP110 and Cep97 binding to centrioles during mitosis (Aydogan, 2022).
Perhaps surprisingly, this study showed that CP110 and Cep97 levels appear to influence the growth of the centriole cartwheel, at least in part, by altering the parameters of the Plk4 oscillation at the base of the growing daughter centrioles. This reveals an unexpected crosstalk between proteins that are usually thought to influence events at the proximal end of the cartwheel (Plk4) and at the distal end of the centriole MTs (CP110 and Cep97). It is currently not understood how CP110 and Cep97 might influence the behaviour of Plk4, but the data suggests they do not alter the abundance of each other in the cytoplasm. Nevertheless, it might be that Plk4 and CP110 and/or Cep97 interact in the cytoplasm, and this interaction influences the amount of Plk4 available for recruitment to the centriole (explaining why less Plk4 is recruited when these proteins are overexpressed and more is recruited with they are absent). Alternatively, perhaps these proteins interact at the centriole during the very early stages of daughter centriole assembly, when they are all present at the nascent site of assembly but have not yet been spatially separated by the growth of the daughter centriole. Clearly it will be important to test whether Plk4 and CP110 and/or Cep97 interact in Drosophila embryos and, if so, how this interaction is regulated in space and time (Aydogan, 2022).
CP110 and Cep97 are not essential for centriole duplication in mice or flies, but CP110 (also known as CCP110 in mammals) is required for Plk4-induced centriole overduplication in cultured human cells, and Plk4 can interact with and phosphorylate CP110 to promote centriole duplication in these cells. Thus, although the physiological significance and molecular mechanism of Plk4 and CP110 and Cep97 crosstalk is currently unclear, this crosstalk might be conserved in other species (Aydogan, 2022).
Finally, it is important to note that changing the levels of CP110 and Cep97 influences the Plk4 oscillation in a surprising way. In the absence of CP110 and Cep97, the cartwheel seems to grow faster and for a shorter period, but the Plk4 oscillation has a higher amplitude and a longer period. Previous observations would suggest that faster centriole growth for a shorter period would be associated with Plk4 oscillation that has a higher amplitude but a shorter period. One way to potentially explain this conundrum is if Plk4 is more active in the absence of CP110 and Cep97 - so the cartwheel would be built faster but for a shorter period, as was observed - but the inactivated Plk4 is not efficiently released from its centriolar receptors (so Plk4 would accumulate at centrioles to a higher level and for a longer period). Clearly further work is required to understand how the Plk4 oscillation drives cartwheel assembly, and how this process is influenced by CP110 and Cep97 (Aydogan, 2022).
Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing 'mother' centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. This study shows that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size (Aydogan, 2018).
How organelles grow to the right size is a fundamental problem in cell biology. For many organelles, however, this question is difficult to address: the number and distribution of an organelle within a cell can vary, and it can also be difficult to determine whether an organelle’s surface area, volume, or perhaps the amount of a limiting component, best defines its size. Centrioles are highly structured organelles that form centrosomes and cilia. Their length can vary by an order of magnitude between different species and tissues but is very consistent within a given cell type. Centrioles are potentially an attractive system with which to study organelle size control, as their numbers are precisely regulated: most cells are born with a single centriole pair that is duplicated once per cell cycle, when a single daughter centriole grows outwards from each mother centriole during S-phase. Moreover, the highly ordered structure of the centriole means that the complex 3D question of organelle size control can be simplified to a 1D question of daughter centriole length control (Aydogan, 2018).
Much progress has been made recently in understanding the molecular mechanisms of centriole duplication. Polo-like kinase 4 (Plk4) initiates duplication and is first recruited in a ring surrounding the mother centriole; this ring ultimately resolves into a single 'dot' that marks the site of daughter centriole assembly. Plk4 recruits and phosphorylates Ana2/STIL, which helps recruit Sas-6 to initiate the assembly of the ninefold-symmetric cartwheel that forms the structural backbone of the growing daughter centriole. How Plk4 is ultimately localized to a single site on the side of the mother is unclear, but Plk4 can dimerize and autophosphorylate itself in trans to trigger its own destruction. In addition, binding to Ana2/STIL activates Plk4’s kinase activity and also appears to stabilize Plk4. Thus, the binding of Plk4 to Ana2/STIL at a single site on the side of the mother could activate and protect the kinase at this site, whereas the remaining Plk4 around the mother centriole is degraded (Aydogan, 2018).
Although studies have provided important insight into how mother centrioles grow only a single daughter, the question of how daughter centrioles subsequently grow to the correct length has been difficult to address. This is in part because centrioles are small structures (usually 100-500 nm in length), making it hard to directly monitor the kinetics of centriole growth. Also, cells usually only assemble two daughter centrioles per cell cycle, and this makes it difficult to measure centriole growth in a quantitative manner. The early Drosophila melanogaster embryo is an established model for studying centriole and centrosome assembly, and it is potentially an attractive system for measuring the kinetics of daughter centriole growth. First, it is a multinucleated single cell (a syncytium) that undergoes 13 rounds of nearly synchronous, rapid nuclear divisions. During nuclear cycles 10-14, the majority of nuclei (and their associated centrioles) form a monolayer at the cortex, allowing the simultaneous observation of many centrioles as they rapidly and synchronously progress through repeated rounds of S-phase and mitoses without intervening gap phases. Second, centrioles in flies are structurally simpler than those in vertebrates. All centrioles start to assemble around the cartwheel in S-phase, but vertebrate centrioles often exhibit a second phase of growth during G2/M, when the centriolar microtubules (MTs) extend past the cartwheel. Fly centrioles usually do not exhibit this second phase of growth, so the centrioles are relatively short, and the cartwheel extends throughout the length of the daughter centriole. It was reasoned, therefore, that the fluorescence incorporation of the cartwheel components Sas-6-GFP or Ana2-GFP could potentially be used as a proxy to measure daughter centriole length in D. melanogaster embryos(Aydogan, 2018).
This study shows that this is the case, and the first quantitative description is provided of the kinetics of daughter centriole growth in a living cell. The findings reveal an unexpected inverse relationship between the centriole growth rate and growth period: in embryos where daughter centrioles tend to grow slowly, they tend to grow for a longer period. Surprisingly, Plk4 influences both the centriole growth rate and growth period and helps coordinate the inverse relationship between them. Thus, Plk4 functions as a homeostatic clock that helps to ensure daughter centrioles grow to the correct size in fly embryos (Aydogan, 2018).
Several models have been proposed to explain how daughter centrioles might grow to the correct size, but none of these have been tested, primarily because of the lack of a quantitative description of centriole growth kinetics. The observations suggest an unexpected, yet relatively simple, model by which centriolar Plk4 might determine daughter centriole length in flies (Aydogan, 2018).
It is proposed that a small fraction of centriolar Plk4, perhaps the fraction bound to both Asl and Ana2, influences both the rate of cartwheel growth (by determining the rate of Sas-6 and Ana2 recruitment to the centriole) and the period of cartwheel growth (by determining the rate of Plk4 recruitment to the centriole, and so how quickly centriolar Plk4 accumulates to trigger its own destruction). This model is consistent with the observation that daughter centrioles grow at a relatively constant rate even as centriolar levels of Plk4 fluctuate (indicating that the majority of Plk4 located at the centriole during S-phase is not directly promoting daughter centriole growth) and that centriolar Plk4 levels appear to influence the rate at which Plk4 is accumulated at centrioles (suggesting that Plk4 can recruit itself, either directly or indirectly, to centrioles) (Aydogan, 2018).
In this model, Plk4 functions as a homeostatic clock (see Schematic illustration of how a Plk4-dependent homeostatic clock might set daughter centriole length in flies), regulating both the rate and period of daughter centriole growth, and ensuring an inverse relationship between them: the more 'active' the Plk4, the faster the daughters grow, but the faster Plk4 is recruited and so inactivated. The activity of this Plk4 fraction is probably a function of both the total amount of Plk4 in this fraction and its kinase activity. It is speculate that this activity is determined before the start of S-phase by a complex web of interactions between Plk4, Ana2, Sas-6, and Asl that influence each other’s recruitment and stability and also, directly or indirectly, Plk4’s kinase activity. These interactions are likely to be regulated by external factors (such as the basic cell cycle machinery), allowing cells to set centriole growth parameters according to their needs. In cells with a G1 period, for example, Plk4 could be activated as cells progress from mitosis into G1, allowing the mother centriole to recruit an appropriate amount of Sas-6 and Ana2/STIL at this stage, which could then be incorporated into the cartwheel when cells enter S-phase. This could explain why in some somatic cells Plk4 levels appear to be higher during mitosis/G1 than in S-phase, and why Plk4 kinase activity appears to be required primarily during G1, rather than S-phase (Aydogan, 2018).
This model can explain why halving the dose of Plk4 leads to a decrease in the growth rate and an increase in the growth period: halving the dose of Plk4 would be predicted to lower both the kinase activity of centriolar Plk4 (so slowing the growth rate) and the amount of centriolar Plk4 (so increasing the growth period). It can also potentially explain why doubling the dose of Plk4 might change the growth period without changing the growth rate: increasing the dose could lead to an increased rate of Plk4 recruitment (because of its increased cytoplasmic concentration), without increasing the amount or kinase activity of the Plk4 fraction bound to Asl or Ana2 (if these were already near saturation). Finally, it could explain why decreasing the kinase activity of Plk4 decreases the rate of growth without changing the growth period: the decrease in Plk4 kinase activity might affect the rate at which it recruits Ana2/Sas-6 without affecting the amount of centriolar Plk4, and so the rate at which Plk4 recruits itself to centrioles (Aydogan, 2018).
Importantly, although the cartwheel extends throughout the entire length of the daughter centriole in worms and flies, this is not the case in vertebrates, where centrioles exhibit a second phase of growth during G2/M and the centriolar MTs grow to extend beyond the cartwheel. It is suspected that the homeostatic clock mechanism described in this study may regulate the initial phase of centriole/cartwheel growth in all species, but the subsequent extension of the daughter centriole beyond the cartwheel that occurs in vertebrates will likely require a separate regulatory network (Aydogan, 2018).
The concept of a homeostatic clock regulating organelle size has not been proposed previously. This mechanism is plausible for Plk4, because it can behave as a 'suicide' kinase: the more active it is, the faster it will trigger its own inactivation. This mechanism relies on delayed negative feedback, a principle that helps set both the circadian clock and the somite segmentation clock. A similar mechanism might operate with other kinases that influence organelle biogenesis and whose activity accelerates their own inactivation, such as PKC, which regulates lysosome biogenesis. It will be interesting to determine whether homeostatic clock mechanisms that rely on delayed negative feedback could regulate organelle size more generally (Aydogan, 2018).
Centrioles are cylindrical assemblies comprised of 9 singlet, doublet, or triplet microtubules, essential for the formation of motile and sensory cilia. While the structure of the cilium is being defined at increasing resolution, centriolar structure remains poorly understood. This study used electron cryo-tomography to determine the structure of mammalian (triplet) and Drosophila (doublet) centrioles. Mammalian centrioles have two distinct domains: a 200 nm proximal core region connected by A-C linkers, and a distal domain where the C-tubule is incomplete and a pair of novel linkages stabilize the assembly producing a geometry more closely resembling the ciliary axoneme. Drosophila centrioles resemble the mammalian core, but with their doublet microtubules linked through the A tubules. The commonality of core-region length, and the abrupt transition in mammalian centrioles, suggests a conserved length-setting mechanism. The unexpected linker diversity suggests how unique centriolar architectures arise in different tissues and organisms (Greenan, 2018).
The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. This study reports a novel role for the multifunctional adaptor protein centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of gamma-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, these results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division (Malerod, 2018).
During cell division, the mitotic spindle (MS) that forms between the two centrosomes ensures faithful segregation of the chromosomes between the two daughter cells, positions the cleavage furrow, and is anchored to the cell cortex to ensure proper spindle orientation. Different subpopulations of microtubules (MTs); the kinetochore, interpolar/astral, and astral MTs, are involved in controlling each process, respectively. Correct orientation of the MS ensures proper segregation of molecules defining cell fate and is important during asymmetric stem cell division to generate one daughter cell which self-renews and one which undergoes differentiation. The orientation of the MS further defines the cleavage plane of the cell and thereby its position within the tissue, exemplified by the planar division of epithelial cells to generate a monolayered epithelium. The precise orientation of the MS can be influenced by internal cues (cell polarity determinants) or external cues (neighboring cells or extracellular matrix) and is cell type-dependent (Malerod, 2018).
Regardless of the molecular mechanisms setting the orientation, the MS is anchored to the cell cortex by the astral MTs radiating from the centrosomes. The centrosome is the major MT-organizing center in most cell types and nucleates astral MTs and the other MT subpopulations of the MS. The centrosome is composed of a centriole pair and the surrounding pericentriolar material (PCM), generated by dynamic assembly of proteins found to stabilize each other via positive feedback loops. During mitosis, the centrosome matures when the PCM expands extensively due to recruitment of scaffold and MT nucleating proteins, which promote MS formation. The γ-tubulin ring complexes (γTuRCs) of the PCM, composed of γ-tubulin and associated proteins (γ-tubulin complex proteins, GCPs), nucleate MT filaments at the centrosome. The ring of γ-tubulin within γTuRC resembles the MT geometry and serves as a template for assembly of α/β-tubulin-dimers, which polymerize into long filaments, MTs. Although the centrosomes represent the major centers for MT nucleation, MTs may alternatively be formed at the Golgi, chromosomes, nuclear envelope, plasma membrane, and pre-existing MTs. Importantly, γ-tubulin seems to be implicated in the nucleation process regardless of the intracellular localization (Malerod, 2018).
Microtubules of the MS, including the astral MTs, are dynamic and their timely assembly and disassembly is tightly controlled by proteins regulating nucleation, severing, and stability of the filaments. MT stability is regulated by MT-associated proteins. These proteins stabilize MTs by binding to the growing plus-end of the filaments to prevent catastrophe, or alternatively, by decorating the MTs to prevent interaction with severing proteins. Furthermore, the γTuRC itself has also been reported to modulate the stability of MTs by interacting with motor proteins such as dynein, kinesin-5, and kinesin-14 as well as the plus-end tracking protein EB1 (Malerod, 2018).
Astral MT regulation occurs at several levels to achieve proper MS orientation: (1) astral MT nucleation at the centrosomes, (2) astral MT dynamics and stability, and (3) astral MT anchoring and behavior at the cell cortex. Aberrant regulation of astral MTs has been shown to correlate with spindle misorientation. For example, centrosomal proteins regulating γTuRC-mediated nucleation of MTs and MAPs controlling MT stability have been shown to regulate spindle orientation in their capacity of modulating MT dynamics. Despite the emerging insight into how astral MT formation is controlled to ensure proper MS orientation, the molecular mechanisms are incompletely understood (Malerod, 2018).
The multifunctional adaptor protein ALG-2-interacting protein X (ALIX) has been shown to localize to centrosomes in interphase and during cell division. However, the biological roles of centrosomal ALIX are not known. Extensive research has implicated ALIX in a diversity of cellular processes, such as apoptosis, endocytosis and endosome biogenesis, cell adhesion, virus release, plasma membrane repair, and cytokinesis. Specifically, ALIX controls cytokinesis by participating in recruiting abscission-promoting proteins of the endosomal sorting complex required for transport (ESCRT) to the midbody. The current study has investigated the role of centrosomal ALIX during cell division. ALIX is shown to localize to the PCM, where it interacts with and stabilizes γTuRC, thus promoting efficient nucleation of astral MTs. In addition, centrosomal ALIX recruits MAP1S, which stabilizes the newly formed MTs radiating from the centrosomes. It is concluded that ALIX facilitates efficient formation of astral MTs by stimulating their nucleation and stabilization, which promotes correct MS orientation during both asymmetric and symmetric cell division (Malerod, 2018).
This study has unraveled a novel role of ALIX located at the centrosomes during cell division in regulation of MS orientation by modulating the formation of astral MTs. ALIX is recruited to the PCM via DSpd-2/Cep192, which recruit PCM components (including Cnn/Cep215, γ-tubulin, and Dgrip91/GCP3), to promote nucleation of astral MTs. Notably, even though DSpd-2/Cep192 appears to be a major recruiter of ALIX to centrosomes, the fact that ALIX was still partially detected at centrosomes in the absence of DSpd-2/Cep192 indicates that additional recruitment mechanisms exist. Centrosomal protein of 55 kDa (CEP55), which localizes to centrosomes during early phases of cell division and moves to and recruits ALIX to the midbody during cytokinesis, represents a possible additional recruiter of ALIX to centrosomes in human cells. However, because CEP55 orthologues lack in lower eukaryotes, such as D. melanogaster (and C. elegans), other proteins could also participate in recruiting ALIX to centrosomes. Interestingly, a direct interaction between DSpd-2/Cep192 and γ-tubulin has not been elucidated. Based on the current results, it is therefore tempting to speculate that ALIX serves a scaffolding role at the interface between DSpd-2/Cep192 and γTuRC, since it was found that ALIX binds DSpd-2, γ-tubulin, and Dgrip91 in vitro. Thus, the results provide mechanistic insight into DSpd-2/Cep192-mediated regulation of astral MT formation and proper orientation of the MS during metaphase in Drosophila and human cells (Malerod, 2018).
The current model shows the MS orientation in cells with or without ALIX. The PCM protein DSpd-2/Cep192 recruits ALIX to the PCM, where ALIX recruits γ-tubulin of the γTuRC at the centrosomes, thus facilitating nucleation of astral MTs. Furthermore, ALIX recruits MAP1S to the centrosomes, in close proximity to the newly formed MTs which are then stabilized by MAP1S. Thus, ALIX facilitates both nucleation of and stabilization of astral MTs emanating from the centrosomes, thus promoting efficient formation of stable astral MTs which mediate anchoring to the cell cortex and thus correct positioning of the MS. By this mechanism, ALIX is one of several molecules controlling MS orientation, providing robustness to correctly orient the MS during cell division (Malerod, 2018).
Astral MTs seem to be equally essential for correct spindle orientation in diverse cell types, in difference to internal polarity cues or external signals provided by neighboring cells. Interestingly, that loss of ALIX induced spindle misorientation in a variety of cell types, including stem cells (NBs and mGSCs) and epithelial cells (SOPs, FECs, and Caco-2 cells), corresponds well with the current data showing that ALIX controls spindle orientation by facilitating the formation of astral MTs and indicates a general role of ALIX in this process. Furthermore, the defective localization of Miranda and aPKC in alix mutant NBs reflects the compromised formation of astral MTs, rather than aberrant cell polarity, since astral MTs have previously been shown to stabilize these determinants at the basal and apical membranes, respectively (Malerod, 2018).
ALIX was shown to maintain the epithelial blood-cerebrospinal fluid barrier by facilitating assembly of tight junctions, which were recently reported to control spindle orientation in Caco-2 cyst cells. In general, cell-cell contacts such as tight junctions seem to control MS orientation in epithelial cells by F-actin, an essential component of the cell cortex facilitating capture of astral MTs. In human epithelial cells, ALIX might thus affect both the formation of astral MTs, as has been shown in this study, and their anchoring to the cell cortex. Also in Drosophila SOPs, septate junctions, resembling tight junctions, regulate the MS orientation. Whether ALIX regulates septate junctions in Drosophila epithelial cells remains to be elucidated, but the current data showing that cold-induced depolymerization of MTs potentiated the spindle misorientation only in wild-type FECs, and not in alix1 FECs, suggest that ALIX regulates the MS orientation by MT-dependent mechanisms (Malerod, 2018).
The current study suggests a dual role for ALIX during astral MT formation: (1) by promoting nucleation via γ-tubulin recruitment and (2) by stabilization of MTs via stabilizing MAP1S at the centrosomes. Although MAP1S is predominantly associated along MTs, it has also been shown to concentrate at the centrosomes. Here, MAP1S has been suggested to stabilize newly formed MT filaments, which likely explains the reduced regrowth of MTs observed at early time points after cold-induced depolymerization in MAP1S-depleted cells. Accordingly, this study found that ectopically expressed MAP1S was unable to rescue the reduced number of astral MTs observed in ALIX-deficient cells, thus arguing against that MAP1S influences the nucleation of MTs as such. Rather, MAP1S significantly increased the length of astral MTs in ALIX knockdown cells, supporting the hypothesis that ALIX facilitates MT stability via MAP1S. It is envisioned that ALIX stabilizes MAP1S adjacent to the PCM, close to the ends of the newly formed MTs emanating from the centrosomes. A simultaneous interaction of MAP1S with both MTs and ALIX seems plausible since the MT-interacting domain is located in the light chain of MAP1S, whereas ALIX seems to bind the heavy chain (Malerod, 2018).
In summary, the current study identifies a novel evolutionarily conserved role of centrosomal ALIX in promoting astral MT formation to orient the MS. The reduced, rather than absent, recruitment of γ-tubulin, MAP1S and consequently appearance of astral MTs in ALIX-deficient cells, clearly suggests that ALIX represents one of several mechanisms to ensure formation of astral MTs. Thus, ALIX provides robustness to correctly orient the MS during asymmetric and planar cell division (Malerod, 2018).
Centrosomes play a critical role in mitotic spindle assembly through their role in microtubule nucleation and bipolar spindle assembly. Loss of centrosomes can impair the ability of some cells to properly conduct mitotic division, leading to chromosomal instability, cell stress, and aneuploidy. Multiple aspects of the cellular response to mitotic error associated with centrosome loss appears to involve activation of JNK signaling. To further characterize the transcriptional effects of centrosome loss, gene expression profiles were compared of wildtype and acentrosomal cells from Drosophila wing imaginal discs. Elevation was found of expression of JNK target genes, which was verified at the protein level. Consistent with this, the upregulated gene set showed significant enrichment for the AP1 consensus DNA binding sequence. Significant elevation was found in expression of genes regulating redox balance. Based on those findings, oxidative stress after centrosome loss was examined, revealing that acentrosomal wing cells have significant increases in reactive oxygen species (ROS). A candidate genetic screen was performed, and one of the genes upregulated in acentrosomal cells, G6PD, was found to play an important role in buffering acentrosomal cells against increased ROS and helps protect those cells from cell death. These data and other recent studies have revealed a complex network of signaling pathways, transcriptional programs, and cellular processes that epithelial cells use to respond to stressors like mitotic errors to help limit cell damage and maintain normal tissue development (Poulton, 2019).
Primary cilia are key sensory organelles that are thought to be disassembled prior to mitosis. Inheritance of the mother centriole, which nucleates the primary cilium, in relation to asymmetric daughter cell behavior has previously been studied. However, the fate of the ciliary membrane (CM) upon cell division is unknown. This study followed the ciliary membrane in dividing embryonic murine neocortical stem cells and cultured cells. Ciliary membrane attached to the mother centriole was endocytosed at mitosis onset, persisted through mitosis at one spindle pole, and was asymmetrically inherited by one daughter cell, which retained stem cell character. This daughter re-established a primary cilium harboring an activated signal transducer earlier than the noninheriting daughter. Centrosomal association of ciliary membrane in dividing neural stem cells decreased at late neurogenesis when these cells differentiate. These data imply that centrosome-associated ciliary membrane acts as a determinant for the temporal-spatial control of ciliogenesis (Paridaen, 2013).
This study has shown that CM typically remains attached
to the basal body/mother centriole through mitosis at one spindle
pole and is asymmetrically inherited by one daughter cell.
Such persistence of centriole-attached CM
in mitotic somatic cells has not been reported previously.
The data challenge the generally accepted model in which the
primary cilium is completely disassembled prior to mitosis. This study found
that, at the G2-M phase transition, the shortening cilium is actually
internalized with the basal body. Because the
basal body/mother centriole maintains its attachment to the remaining
CM, it serves a dual function as a basal body as well
as a part of one mitotic spindle pole. It was reported recently
that Drosophila spermatocytes assemble and retain cilia at all
centrioles through meiosis (Riparbelli, 2012), showing that basal body function in
nucleating cilia is not incompatible with a function as a spindle
pole (Paridaen, 2013).
Previous studies demonstrated that inheritance of the 'old'
mother centriole is linked to asymmetries in cilium reassembly
and ciliary signaling between daughter cells. However,
it was unclear which feature of the 'old' versus 'new'
mother centriole is responsible for inducing these asymmetries.
This study identifies the CM as a key structural component of the
'old' mother centriole in mitotic cells. Furthermore, it was shown
that intracellular ciliary remnant (CR) inheritance by one daughter cell underlies asymmetric
ciliary signaling between daughter cells (Paridaen, 2013).
Classical studies have demonstrated that ciliogenesis is initiated
either by Golgi-derived intracellular CM vesicles docking
to the mother centriole or by direct docking of the mother
centriole to the plasma membrane. The current
data uncover a pathway of ciliogenesis that does not involve
docking of the older mother centriole to a membrane. Specifically,
one of the daughter cells simply inherits the already
CM-bearing centriole from the mother cell and thus can directly
proceed with cilium outgrowth. In
contrast, the other daughter cell inheriting either the new mother
centriole or the old mother centriole without CM can proceed
with cilium outgrowth only after this centriole has docked to
either an intracellular membrane vesicle or the plasma membrane (Paridaen, 2013).
It was reported previously that nascent differentiating daughter
cells (basal progenitors, neurons) re-establish a cilium at their
basolateral plasma membrane prior to their delamination
(Wilsch-Brauninger, 2012). This basolateral ciliogenesis
occurs by docking of a centriole to the lateral plasma membrane,
whereas apical ciliogenesis in APs typically involves an intracellular
CM vesicle. This study shows that the CR is preferentially inherited
by the daughter cell that retains stem cell character (Paridaen, 2013).
The latter finding provides a plausible mechanistic explanation
for the previous observation that elder centrioles versus new
centrioles are preferentially inherited by apical
progenitors (APs) versus differentiating
daughter cells, respectively. It is likely that the
spatial and temporal asymmetries in cilium reformation between
AP daughter cells differentially expose them to signals, such as
proliferative signals from the cerebrospinal fluid (CSF). Therefore, it is proposed that
daughter cells that inherit the CR remain APs.
After division, the membrane of the CR will insert into the apical
plasma membrane either by direct exocytosis or by transcytosis
from the lateral membrane. In contrast, the centrosome containing
the membraneless new mother centriole will directly dock to
the lateral membrane to nucleate a basolateral cilium de novo, and the cell will subsequently delaminate
and differentiate (Paridaen, 2013).
Given that inheritance of the CR is always an asymmetric
event, an important question is whether and how this inheritance
is compatible with symmetric divisions. During neurogenesis,
two types of symmetric AP division exist. Symmetric proliferative
divisions that give rise to two AP daughter cells constitute the
principal type of division prior to neurogenesis and decrease in
frequency after its onset. In contrast, symmetric neurogenic divisions
that give rise to two differentiating daughters cells increase
in frequency toward the end of murine neurogenesis (Paridaen, 2013).
Specifically, the stage when the small GTPase ADP-ribosylation factor-like 13b (Arl13b) was most frequently detected also at the second centrosome of mitotic APs was the
preneurogenic stage when symmetric proliferative divisions prevail.
It is suggested that this Arl13b immunoreactivity reflects newly
synthesized apically destined CM. It is proposed that in symmetric
proliferative divisions, the new mother centriole is able to capture
Golgi-derived de novo CM. This membrane
capturing can occur already during mitosis, in contrast to the
direct docking of the centriole at the plasma membrane that
can occur only after mitosis. Because apical Golgi-derived membrane
trafficking is downregulated at the onset of neurogenesis, it is proposed that APs lose the capacity
of capturing Golgi-derived membrane as neurogenesis progresses.
Therefore, this property of early neural progenitors is
responsible for the earlier ciliogenesis in daughter cells inheriting
the new mother centriole in symmetric proliferative divisions at early stages versus asymmetric and symmetric neurogenic divisions at later stages (Paridaen, 2013).
Inheritance of CM occurs increasingly in noncentrosomal
form as neurogenesis progresses. This leads to a proposal that in symmetric neurogenic divisions, both (now membraneless) centrosomes dock at the lateral membrane,
and de novo basolateral ciliogenesis occurs in both daughter
cells. Subsequently, both daughters delaminate
from the ventricular surface and differentiate.
In conclusion, this study uncovers an additional feature of the
inherent asymmetries that exist between daughter cells due to
the differences in centriole age. Specifically, CR inheritance emerges as an important means of establishing asymmetric behavior between daughter cells (Paridaen, 2013).
Talpid3/KIAA0586 has been identified as a component of a CP110-containing protein complex important for centrosome and cilia function. Talpid3 assembles a ring-like structure at the extreme distal end of centrioles. Ablation of Talpid3 results in an aberrant distribution of centriolar satellites involved in protein trafficking to centrosomes as well as cilia assembly defects, reminiscent of loss of Cep290, another CP110-associated protein. Talpid3 depletion also leads to mislocalization of Rab8a, a small GTPase thought to be essential for ciliary vesicle formation. Expression of activated Rab8a suppresses cilia assembly defects provoked by Talpid3 depletion, suggesting that Talpid3 affects cilia formation through Rab8a recruitment and/or activation. Remarkably, ultrastructural analyses showed that Talpid3 is required for centriolar satellite dispersal, which precedes the formation of mature ciliary vesicles, a process requiring Cep290. These studies suggest that Talpid3 and Cep290 play overlapping and distinct roles in ciliary vesicle formation through regulation of centriolar satellite accretion and Rab8a (Kobayashi, 2014).
Basal bodies are freed from cilia and transition into centrioles to organize centrosomes in dividing cells. A mutually exclusive centriole/basal body existence during cell-cycle progression has become a widely accepted principle. Contrary to this view, this study shows that cilia assemble and persist through two meiotic divisions in Drosophila spermatocytes. Remarkably, all four centrioles assemble primary cilia-centriole complexes that transit from the plasma membrane encased in a packet of membrane, recruit centrosomal material into microtubule-organizing centers, and persist at the spindle poles through division. Thus, spermatocyte centrioles organize centrosomes and cilia simultaneously at cell division. These findings challenge the prevailing view that cilia antagonize cell-cycle progression and raise the possibility that cilium retention at cell division may occur in diverse organisms and cell types (Riparbelli, 2012; see graphical abstract).
The elegant 9-fold radially symmetric design of the centriole is truly one of nature's aesthetic wonders. Centrioles are found at the core of microtubule-organizing centers in most animal cells. Despite their requirement for the spatial organization of the centrosomal material to nucleate cytoplasmic and spindle microtubules, centrioles are dispensable for somatic cell division where acentrosomal pathways of microtubule organization can suffice. Centrioles have an additional and perhaps more critical function: as templates for cilia and flagella, a context in which they are called basal bodies. The ability of centrioles to function both as centrosomal organizers at the spindle poles and also as basal bodies to template primary cilia was originally proposed in the Henneguy-Lenhossek hypothesis in 1898, which predicted the functional equivalence of these organelles (Riparbelli, 2012).
The cilium is an essential antenna-like projection that consists of the microtubule-based axoneme and a surrounding membrane that is continuous but distinct from the cell's plasma membrane. The axoneme is the structural backbone of the cilium and mirrors the 9-fold radial symmetry conveyed by the basal body. Generally, motile cilia contain a pair of axial singlet microtubules at the center of the axoneme surrounded by the nine doublet microtubules of the axoneme: a '9+2' structure. Nonmotile primary cilia lack the central pair ('9+0'). Primary cilia are sensory organelles for intercellular signaling involving hedgehog, Wnt, and PDGF pathways. Defects in cilia have pleiotropic effects on development and physiology, depending on the molecules affected and the alleles involved, and lesions in affected human genes are collectively called ciliopathies (Riparbelli, 2012).
Centrioles have a double life: as centrioles within the centrosomes, and as basal bodies at the cell membrane in differentiated or resting cells. In cycling cells, centrioles are found within the centrosomal material during cell division but then move to the cell periphery in quiescent cells to nucleate a ciliary axoneme after docking at the plasma membrane. Upon cell-cycle entry the centriole is released from the axoneme and/or the cilium resorbs, and then the centriole migrates into the cytoplasm and recruits pericentriolar material to organize the centrosome. Although cell-cycle-dependent cilium assembly and disassembly are typical of vertebrate cells, it remains unclear how this process is executed and regulated. The switch between centriole and basal body is accompanied by morphological and molecular changes. Moreover, there is considerable evidence that the presence of a cilium is incompatible with cell division and that cells will not divide until their centrioles are freed from cilia. It is generally assumed that the centriole cannot function simultaneously to assemble cilia and mitotic centrosomes. The vast majority of studies of cilium dynamics in the cell cycle were done in cell culture (Riparbelli, 2012).
Contrasting evidence suggested that cilia and flagella might be maintained at cell division. It was reported that 'flagellar' structures were maintained during meiotic divisions of some Lepidopteran, Neuropteran, and Dipteran species. Cilia and flagella may also be retained during the division of some protozoa. The seminal electron microscopy (EM) work by Tates showed the detailed changes that centrioles undergo during spermatogenesis and in retrospect revealed the presence of short cilia in spermatocytes during division (Riparbelli, 2012).
This study investigated the timing and ultrastructure of cilium biogenesis during Drosophila spermatogenesis using molecular markers for basal bodies and cilia, coupled with unequivocal ultrastructural imaging using EM. It was shown that cilia assemble in premeiotic prophase and remain intact through two rounds of cell division in vivo in spermatocytes, a cell type with a discrete developmental program involving two rounds of division followed by differentiation (Riparbelli, 2012).
These findings challenge the principle that cilia are inconsistent with progression through the cell cycle and provide a system to study this regulation. Remarkably, rather than dissociating basal bodies from the cilia, spermatocyte basal body-cilia complexes are internalized with an associated 'pocket' of plasma membrane as their centrioles assemble PCM into centrosomes that participate in spindle assembly. The detailed dynamic changes in the centriole-cilium structures from spermatocytes to spermatid will serve as vital features for future analysis of cilium regulation during cell division (Riparbelli, 2012).
The results clearly define two opposite scenarios in cilia biogenesis: (1) that of most known animal cells in which the primary cilium dynamics appear governed by cell cycle and are exclusive of dividing cells, and (2) that of Drosophila spermatocytes in which the primary cilium is stable through meiotic divisions. These observations shift the general point of view that the centriole must be freed from the cilium to organize a functional centrosome for spindle organization and that dividing cells are unable to form primary cilia. Nevertheless, the ability of spermatocytes to assemble cilia and then maintain this structure at the spindle poles during division could be a specialized feature of spermatocytes and perhaps other cell types. On the other hand, this could be a feature of progenitor cells that are programmed to divide a discrete set of rounds before differentiating into a ciliated cell type. There are few such studies performed in vivo to support or counter this possibility at present (Riparbelli, 2012).
Primary cilia in vertebrate cells are antenna-like projections that detect mechanical and chemical cues from the environment to signal differentiation, proliferation, and other responses. Drosophila spermatocytes develop within a cyst that is separated by the environment by a thin cellular envelope. Cytoplasmic bridges that ensure communication and synchrony interconnect the 16 spermatocytes within the cyst. In this condition the function of primary cilia as sensory organs may be redundant or their role unclear. Although centrosomes are essential for accurate division of spermatocytes, the importance or requirement of the cilia for division is unclear. Such a requirement is difficult to test at present because there are no known mutants that completely disrupt spermatocyte cilia that also leave the centrioles intact (Riparbelli, 2012).
A surprising property of spermatocyte cilia is their independence from intraflagellar transport (IFT) for their assembly. This could therefore be a special class of cilia, similar to the sperm flagellum with its independence on IFT for assembly. However, whereas the spermatid axoneme assembles in the cytoplasm, it is actually encased in membrane that invaginates from the plasma membrane to form a 5- to 10-nanometer-long cilium that retains this length at the distal tip of the axoneme as it grows. A requirement of IFT for assembly of this distal cilium has not been examined. On the other hand, the spermatocyte cilium could be an elaborated transition zone, which can assemble independent of IFT (Riparbelli, 2012).
Questions on the function of spermatocyte cilia are also raised by some of their peculiarities. Spermatocyte cilia are nucleated by all four centrioles, whereas in vertebrate cells only the mother centriole is able to do this. Vertebrate cells assemble a single primary cilium during G1 phase, but Drosophila spermatocytes assemble cilia during G2 phase. Moreover, the ciliary axonemes are nucleated by centrioles that continue to grow while the cilium grows, whereas in vertebrate cells the mother centriole has reached its definitive length at maturity before it organizes the primary cilium. How can the centriole and the cilium grow simultaneously? It can be supposed that the cilium elongates by addition of subunits at its distal tip. But how can the centriole elongate if its distal extremity is committed to form the ciliary axoneme? Although a necessary function is not demonstrated, spermatocyte cilia could be precursors for building the flagellar axoneme for the sperm, despite axonemal reorganization in early spermatids (Riparbelli, 2012).
Collectively, these results show that cilia are not incompatible with cell division as a general rule. Although cilium resorption initiates at cell-cycle entry in most cell culture models, it is not clear that disassembly of cilia is necessary for cell division to occur. In spermatocytes, on the other hand, it is possible that a potential cilium block to cell-cycle progression is repressed. Alternatively, the assembly of cilia in these cells in G2 phase might be a unique mechanism to circumvent a G1-specific cell-cycle block imposed by cilia in quiescent cells. Yet another possibility is that spermatocyte cilia have distinct properties from other primary cilia that are permissive for cell-cycle progression. Consistent with this possibility is the finding that these cilia appear to assemble independent of IFT. These intriguing clues provide a framework to further probe the interdependence of cilia with cell-cycle control (Riparbelli, 2012).
In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear. This study reports that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. Cep290 was shown to be essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity; MKS proteins also delimit a centriole-independent compartment in mouse spermatids. These findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly (Basiri, 2014).
Compartmentalization of cellular processes is a critical aspect of cell biology. In many cases-such as in mitochondria and the ER-a semipermeable membrane surrounds an organelle to create a compositionally distinct compartment. In other cases, such as in the nucleus and cilium, the lumen of the organelle is continuous with the cytoplasm and compartmentalization is achieved by a specialized gate at its cytoplasmic interface. The ciliary gate is found in a region known as the transition zone at the base of the cilium. Within the transition zone, axonemal microtubules are crosslinked to the surrounding membrane and components of the ciliary gate mediate attachments between the axoneme, ciliary membrane, and centriole, giving the impression that the transition zone is a static and rigid structure (Basiri, 2014).
Aside from housing the ciliary gate, the transition zone is the site of multiple ciliary activities. As such, the transition zone is compositionally diverse and it is unclear which of its components comprise the ciliary gate. However, recent studies have suggested that genes mutated in the ciliopathies nephronophthisis (NPHP) and Meckel-Gruber syndrome (MKS) encode proteins that comprise two distinct modules essential for ciliary gate function (Basiri, 2014).
Centrioles and basal bodies, as well as cilia and flagella, are synonymous terms in different cellular contexts. For simplicity, this paper maintains the terms centriole and cilium throughout the text. Cilia can be divided into two types based on the location of the axoneme. In compartmentalized cilia, such as Drosophila sensory cilia, the entire axoneme is assembled and maintained within a membrane-bound compartment projecting from the transition zone at the distal end of a centriole. Cilia formed by compartmentalized ciliogenesis require intraflagellar transport (IFT) to move cargo through the transition zone, and IFT machinery directly interacts with transition zone proteins. Thus, the centriole, IFT, and the transition zone are generally essential for compartmentalized ciliogenesis (Basiri, 2014).
In cytoplasmic cilia, such as in mammalian and Drosophila spermatids and microgametes of the malarial parasite Plasmodium, the axoneme is found in the cytoplasm and the centriole is not attached to the cell or ciliary membrane. In Drosophila spermatids, the axoneme is initially composed of bare microtubules and later acquires its mature composition by incorporating proteins in the cytoplasm. Furthermore, IFT is not essential for cytoplasmic axoneme formation in Plasmodium microgametes and Drosophila spermatids. Thus, the separation of the centriole from a membrane-bound compartment and dispensability of IFT appear contradictory for a role of compartmentalization in cytoplasmic ciliogenesis (Basiri, 2014).
Interestingly, electron microscopy of spermatids in various insects including Drosophila identifies a membranous cap-like structure associated with the growing axoneme tip. This, structure, which is referred to as the ciliary cap, is morphologically similar to a typical compartmentalized cilium. Furthermore, mutations in candidate Drosophila transition zone proteins result in defects in spermatogenesis, and many MKS module genes are highly expressed in Drosophila testes. Together, these observations suggest that transition zone components-which are typically implicated in cilium compartmentalization-may also be involved in the formation of cytoplasmic cilia (Basiri, 2014).
This study provides insight into the mechanism by which transition zone proteins function in cytoplasmic ciliogenesis. MKS module proteins comprise a ciliary gate that migrates to compartmentalize the growing axoneme tip in Drosophila spermatids. These findings provide an example whereby the ciliary gate is dynamically associated with the surrounding ciliary architecture to sustain a compartment housing the site of axoneme assembly (Basiri, 2014).
In most species, ciliary gate function is dependent on both the ciliopathies nephronophthisis (NPHP) and Meckel-Gruber syndrome (MKS) modules, which exhibit some redundancy at the transition zone. However, mutation of MKS module genes in humans is associated with more-severe ciliary disease than is mutation of the NPHP module, suggesting that the MKS module plays a more basic role in cilium formation or function. Consistent with this, it was not possible to detect core genes of the NPHP module in Drosophila, implying that the MKS module is sufficient for cilium compartmentalization and that compartmentalization can be achieved by a relatively small subset of proteins (Basiri, 2014).
Classically, the transition zone has been regarded as a characteristic of the ciliary base that is restricted to the distal end of the centriole. Indeed, a mechanism involved in anchoring transition zone proteins to the centriole has been recently described. However, the current work demonstrates that the transition zone can function independently of the centriole and suggests that the transition zone, and not the centriole, is the determining factor in positioning the ciliary compartment along the axoneme. The assembly of the transition zone upon the distal ends of centrioles prior to cytoplasmic axoneme elongation in Drosophila spermatids suggests that initial transition zone assembly requires the centriole. However, as the axoneme elongates, this transition zone becomes spatially separated from the centriole and continuously migrates to compartmentalize the growing axoneme tip. The spatial separation between the centriole and the ciliary cap demonstrates that the transition zone can function free of centriolar association and while constantly migrating relative to the growing axoneme to create a centriole-independent ciliary compartment.
Transition zone migration may also underlie centriole elongation. In normal spermatocytes, the centriole continues to elongate after the assembly of the transition zone upon its distal end. Because centrioles are not known to grow at their proximal ends, centriole elongation likely occurs by transition zone migration along the axoneme, followed by the expansion of centriolar proteins over freshly exposed axonemal microtubules. The excessive length distribution of centriolar proteins observed in cep290mecH spermatocytes may result from an impairment in the transition zone barrier, allowing centriolar proteins to aberrantly expand along the axoneme at the expense of the transition zone. This interpretation is consistent with the observation that transition zone size is decreased in cep290mecH (Basiri, 2014).
The base of the ciliary cap may contain additional proteins besides those of the MKS module. Previously, it was shown that two proteins, Uncoordinated and Chibby, are found simultaneously in both the centriole and at a distal focus, presumably the ciliary cap. Although the role of these proteins at the ciliary cap remains unclear, it is possible that they cooperate with the MKS module in ciliary cap compartmentalization. Furthermore, another structure associated with the base of the ciliary cap, referred to as the ring centriole, has been described in insects by electron microscopy. It was proposed that the ring centriole is homologous to a structure known as the annulus of mammalian sperm. However, unlike the transition zone, the ring centriole/annulus seems to be localized outside of the ciliary cap on the cytoplasmic side of its base and does not appear to contact the axoneme. Considering this, it is speculated that the ring centriole is a distinct structure from the transition zone and that MKS proteins are not part of the ring centriole but are instead components of the ciliary cap transition zone as in compartmentalized cilia. In support of this, this study observed that CEP290 and MKS1 do not precisely colocalize with Sept4 in mouse spermatids (Basiri, 2014).
In compartmentalized cilia of C. elegans, Chlamydomonas, and vertebrates, transition zone ultrastructure is characterized by features known as 'Y-links,' which are thought to mediate attachments between the axoneme and ciliary membrane. These Y-links are also thought to be involved in regulating entry into the ciliary compartment, and Y-link formation seems to be dependent on MKS protein function. Although Y-links have been observed in the transition zone of some insect sensory cilia, they have not been identified in Drosophila. Still, electron microscopy of Drosophila spermatids identifies vague links between the axoneme and the ciliary cap membrane, implying that structural connections between the axoneme and ciliary cap membrane are also present in migrating transition zones. Indeed, similar vague links are observed in analysis of both the Drosophila ciliary cap base and sensory cilium transition zone (Basiri, 2014).
Similar to Drosophila, mammalian spermatid axonemes also contain a segment that is exposed to the cytoplasm. Distal to this segment, the axoneme resembles a compartmentalized cilium and is closely associated with a membrane of distinct composition. However, it was unknown whether a transition zone defines these distinct axoneme segments in mammalian spermatids. The finding that CEP290 and MKS1 localize to the boundary of cytoplasmic and membrane-bound axoneme segments in mouse spermatids suggests that, similar to Drosophila, the distal, growing region of mammalian spermatid axonemes is also compartmentalized (Basiri, 2014).
Although axoneme compartmentalization is a defining characteristic of cilia, it has remained unclear if compartmentalization is essential for cilium formation or if it is instead required to satisfy ciliary functional requirements. The current observations suggest that compartmentalization is a conserved feature of ciliogenesis that is required for the integrity of axoneme assembly. Although the axoneme may be exposed to the cytoplasm after its initial formation, the process of axoneme assembly must occur within a distinct cellular environment. The transition zone may also play a direct role in axoneme assembly, and it has previously been proposed that the base of the ciliary cap confers 9-fold symmetry to the axoneme by organizing growing axonemal microtubules. This idea is supported by the observation that ciliary cap axonemes of cep290mecH are structurally destabilized (Basiri, 2014).
In summary, these findings demonstrate that the transition zone can migrate relative to the axoneme to sustain a compartment housing the site of axoneme assembly. It is proposed that axoneme compartmentalization is conserved as a prerequisite for proper cilium assembly and that the ciliary gate possesses a high degree of independence from the surrounding ciliary architecture to specify the location of the ciliary compartment along the axoneme (Basiri, 2014).
The first cell of an animal (zygote) requires centrosomes that are
assembled from paternally inherited centrioles and maternally inherited
pericentriolar material (PCM). In some animals, sperm centrioles with
typical ultrastructure are the origin of the first centrosomes in the
zygote. In other animals, however, sperm centrioles lose their proteins
and are thought to be degenerated and non-functional during
spermiogenesis. This study shows that the two sperm centrioles
(the giant centriole [GC] and the proximal centriole-like structure [PCL])
in Drosophila melanogaster are remodeled during spermiogenesis
through protein enrichment and ultrastructure modification in parallel to
previously described centrosomal reduction. The ultrastructure of the
matured sperm (spermatozoa) centrioles is modified dramatically and
the PCL does not resemble a typical centriole. Additionally, Poc1
is enriched at the atypical centrioles in the spermatozoa. Using various
mutants, protein expression during spermiogenesis, and RNAi knockdown of
paternal Poc1, it was found that paternal Poc1 enrichment is essential for
the formation of centrioles during spermiogenesis and for the formation of
centrosomes after fertilization in the zygote. Altogether, these findings
demonstrate that the sperm centrioles are remodeled both in their protein
composition and in ultrastructure, yet they are functional and are
essential for normal embryogenesis in Drosophila (Khire, 2017).
The fruit-fly Drosophila melanogaster harbours different types of ciliary structures: ciliary projections associated with neurons of type I and cilium-like regions (CLRs) found during male gametogenesis. The latter deserve particular attention since they are morphologically similar to vertebrate primary cilia and transform into the sperm axonemes during spermiogenesis. Although, all the centrioles are able to organize the CLRs, this study found that the mother centriole docks first to the plasma membrane suggesting a new intrinsic functional asymmetry between the parent centrioles. The CLRs lack the Y-links that connect the axoneme doublets with the plasma membrane in conventional primary cilia. Moreover, the C-tubules, that are lacking in the axoneme of the primary cilia, persisted along the CLRs albeit modified into longitudinal blades. Remarkably, mutant flies in which the CLRs are devoid of the C-tubules or their number is reduced lack sperm axonemes or have incomplete axonemes. Therefore, the C-tubules are dispensable for the assembly of the CLRs but are essential for sperm axoneme elongation and maintenance in Drosophila (Gottardo, 2018).
Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. This study characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. Assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles (Carvalho-Santos, 2012).
Cilia are microtubule (MT)-based organelles involved in a variety of processes, such as cell motility, fluid flow, and sensing mechanical stimuli and signaling molecules. At the base of each cilium there is at least one modified centriole, the basal body (BB), which templates the growth of the axoneme, the MT-based structure of cilia. Centrioles are also essential for the formation of centrosomes, the primary MT organizer of the cell. Cilia can exist as motile or immotile structures. Most motile cilia have a pair of central MTs within the lumen of their axoneme to coordinate motility. Defects in ciliary motility are associated with a variety of human disorders including infertility, respiratory problems, hydrocephalus and situs inversus, commonly found in patients with primary ciliary dyskinesia. Mice mutant for Hydin, a component of the central MT pair apparatus, show motility defects and develop hydrocephalus (Carvalho-Santos, 2012).
Much is known about axonemal components, mostly from work developed in the green algae. In fully assembled axonemes, the central MT pair starts at the most proximal part of the axoneme, a region called the transition zone. MT regulators are likely to control the assembly of the central MT pair: γ-tubulin and heterotrimeric kinesin-2 are required for this process in protozoa and sea urchin, respectively. However, little is known about the molecular mechanisms that govern the switch of centrioles to BBs and, in particular, when and how central MT pair assembly starts and is coordinated with other cellular processes (Carvalho-Santos, 2012).
Drosophila mutants for Bld10, a conserved player in centriole biogenesis, have short centrioles and the majority of their sperm flagella lack the central MT pair. In Chlamydomonas and Paramecium, BLD10/CEP135 localizes close to the centriolar MTs at the spoke tips of the cartwheel, a nine-fold symmetric structure present at the base of centrioles/BBs that enforces their symmetry. Depletion of BLD10/CEP135 in those organisms severely impairs BB assembly. In human cultured cells, BLD10/CEP135 is required for centriole assembly and localizes to the cartwheel, the centriolar walls, and the lumen of the centriole distal region. Altogether, these data strongly indicate that BLD10/CEP135 has a MT-related function that underlies its role in centriole/BB assembly. Moreover, its localization in the centrioles but not in the axoneme in human and Drosophila suggests a role in the initiation of central MT pair assembly (Carvalho-Santos, 2012).
Drosophila spermatogenesis was used to understand how the central MT pair is assembled and the role played by BLD10/CEP135 in that process. One of the MTs of the pair is assembled prior to the meiotic divisions, within the basal body of a small cilium. This MT is maintained through meiosis, after which the flagellum and the second MT of the pair within it are formed. BLD10/CEP135 directly binds and stabilizes MTs and that in Bld10 mutants the singlet MT does not form and consequently the central MT pair is not assembled (Carvalho-Santos, 2012).
The central MT pair complex is an essential and highly specialized structure present in motile cilia that is required for coordinated motility. The morphological and molecular changes that occur during central MT pair assembly are yet to be characterized. Building on influential morphological work that described spermatogenesis in the fruit fly, this study has established Drosophila spermatogenesis as a genetically tractable system to study central MT pair formation. The assembly of this structure initiates prior to the meiotic divisions, much earlier than previously thought. During that stage, a singlet MT forms within the BB of a small cilium in G2 spermatocytes. This MT is likely very stable as it is present throughout meiotic divisions and until axoneme extension. In early spermatids, the stage at which flagellum assembly takes place, a second MT appears close to the singlet, and completes central MT pair formation. Bld10 emerged as an ideal candidate to regulate central MT pair formation as mutants lack this structure and the protein localizes to both the lumen and distal regions of the centriole/BB. Accordingly, this study demonstrates that spermatogenesis in Bld10 mutants is carried out without assembly of the singlet MT, thus impacting on central MT pair biogenesis. Bld10 is a MAP whose overexpression leads to cytoplasmic MT stabilization in culture cells. Finally, this study directly links MT stabilizing activity of Bld10 to central MT pair assembly as (1) its N terminus, which binds and stabilizes MTs, contributes to this process, and (2) exposure to colchicine during central MT pair formation accentuates Bld10 mutant phenotype (Carvalho-Santos, 2012).
Morphological findings of central MT pair assembly during Drosophila spermatogenesis are summarized in Schematic Representation of Cilium and Flagellum Structures in Drosophila melanogaster Spermatogenesis (Figure 7). The identification and analysis of different stages of this process using TEM single and serial sections, and tomography, suggest this assembly is much more dynamic than was anticipated. It is proposed that central MT pair biogenesis starts with the formation of a singlet MT within the lumen of the BB. The detailed analysis of this process raises several important questions, including where the first singlet MT is nucleated from, and whether it actively participates in the assembly of the second MT of the pair. The identification of singlet MT or central MT pair markers that do not localize to other BB and axoneme structures will allow further mechanistic studies of central MT pair assembly in a less laborious fashion than as it is with electron microscopy (Carvalho-Santos, 2012).
The observation that a singlet MT forms within the BB as a precursor for the biogenesis of the central MT pair of the motile axoneme, implies a broader role for the BB in templating cilia than currently thought. This raises an important question on the significance and conservation of this process. Until now, the study of this process using stills of TEM sections has prevented the discovery and characterization of intermediate steps. Drosophila spermatogenesis proved to be a valuable system to study these fast intermediate stages since central pair assembly takes a few days to be completed. It is proposed that the presence of the singlet MT is an intermediate state until the second MT of the pair is nucleated. In mature sperm both MTs have equal length and therefore is not obvious that one MT was assembled first. In model organisms often used to study flagella assembly, such intermediate stages might have not been observed due to their transitory nature. In Nephrotoma suturalis, an insect that is phylogenetically close to Drosophila melanogaster, a singlet MT has also been observed. The presence of stages where a singlet MT is easily found can be a consequence of a slower central MT pair assembly in these insects (Carvalho-Santos, 2012).
It is intriguing that the central MT pair starts to assemble so early within the BB. An extensive literature search was conducted for ultrastructural studies on sperm flagella assembly in other species to address whether this was a conserved phenomenon. Surprisingly, in both vertebrate and invertebrate species, central MT pairs have been observed in cilia/flagella that assemble during spermatocyte stages, before meiosis I or II . Why the central MT pair assembles at that stage, and in particular whether spermatocyte cilia motility is needed for spermatogenesis, is an important question that deserves further study. It is still unclear whether in these different species the central MT pairs found in spermatocyte cilia/flagella are precursors of the central MT pair of sperm flagella, as is describe in this study for Drosophila (Carvalho-Santos, 2012).
Although much is known about the components of the central MT pair machinery, little is understood about the molecular regulation of its nucleation. In light of the current findings, BLD10/CEP135 is proposed to regulate the initiation of central MT pair biogenesis through MT stabilization. Bld10 conserved N-terminal domain is proposed to play an important role in this process; however, given that removal of that domain does not completely abolish Bld10 activity, it cannot be excluded that other Bld10 protein regions also stabilize the central MT pair. It is common for MAPs to have different MT binding domains. Since the M- and C-Bld10 truncations localize independently to centrioles, it is possible they also stabilize MTs associated with the centriole and the central MT pair, a hypothesis that should be investigated in the future (Carvalho-Santos, 2012).
How general is BLD10/CEP135 MT-stabilizing function? It is possible that BLD10/CEP135 ancestral function is stabilizing special sets of MTs including the centriole triplets and the singlet MT during axoneme central MT pair assembly. BLD10/CEP135 is present in the genome of most organisms that assemble centrioles and flagella but absent from those that lack these organelles, such as higher plants. While a role for BLD10/CEP135 in central MT pair assembly has not been investigated in vertebrates, TSGA10, a BLD10/CEP135 paralog only present in vertebrates, localizes to the sperm flagella and has been linked to male sterility, further corroborating a role for this family of proteins in flagella assembly. BLD10/CEP135 loss-of-function in several species generates phenotypes associated with centriolar MT defects, including MT triplet loss, and shorter centrioles. The link between Bld10 and stabilization of these specific MT sets is also supported by its localization to the cartwheel spokes and BB/centriolar MT triplets (in Chlamydomonas, human cells and Drosophila). The complete lack of BBs in Chlamydomonas BLD10 mutants might reflect defects both in cartwheel assembly and in the recruitment and/or stabilization of the BB MTs onto the cartwheel. This is not the case in Drosophila, as centrioles, albeit shorter, are observed in Bld10 mutants. It is possible that, in the fruit fly, other molecules are redundant with Bld10 in its role of stabilizing cartwheel-associated MTs. In the future, it will be important to understand how Bld10 might stabilize MTs, how its function is regulated in different centriole compartments (cartwheel, lumen, walls) and at different time points, such as during centriole or central MT pair assembly (Carvalho-Santos, 2012).
Centrioles and axonemes are very special organelles, being much more stable than any other MT-based structures. They not only withstand complex MT remodeling environments but in the case of centrioles they also perdure for several cell generations. Additionally, the assembly and stabilization of centrioles and axonemes involves particular MT regulators and posttranslational modifications. This study has shown that BLD10/CEP135 is a special MT regulator specifically involved in the formation of highly stable and specialized MTs. The discovery of these specialized MAPs, where SAS4/CPAP could also be included, opens the door to an exciting new biology of MT regulation. Moreover, given the importance of centrioles and cilia in development and homeostasis, this work will allow further contextualization of these cellular structures in human disease (Carvalho-Santos, 2012).
Cilia play critical roles during embryonic development and adult homeostasis. Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the "gates" of cilia. The evolutionarily conserved TZ component centrosomal protein 290 (CEP290) is the most frequently mutated human ciliopathy gene, but its roles in ciliogenesis are not completely understood. This study reports that CEP290 plays an essential role in the initiation of TZ assembly in Drosophila. Mechanistically, the N-terminus of CEP290 directly recruits DAZ interacting zinc finger protein 1 (DZIP1), which then recruits Chibby (CBY) and Rab8 to promote early ciliary membrane formation. Complete deletion of CEP290 blocks ciliogenesis at the initiation stage of TZ assembly, which can be mimicked by DZIP1 deletion mutants. Remarkably, expression of the N-terminus of CEP290 alone restores the TZ localization of DZIP1 and subsequently ameliorates the defects in TZ assembly initiation in cep290 mutants. Our results link CEP290 to DZIP1-CBY/Rab8 module and uncover a previously uncharacterized important function of CEP290 in the coordination of early ciliary membrane formation and TZ assembly (Wu, 2020).
Spermatogenesis is a dynamic process of cellular differentiation that generates the mature spermatozoa required for reproduction. Errors that arise during this process can lead to sterility due to low sperm counts and malformed or immotile sperm. While it is estimated that 1 out of 7 human couples encounter infertility, the underlying cause of male infertility can only be identified in 50% of cases. This study described and examined the genetic requirements for missing minor mitochondria (mmm), sterile affecting ciliogenesis (sac), and testes of unusual size (tous), three previously uncharacterized genes in Drosophila that are predicted to be components of the flagellar axoneme. Using Drosophila, it was demonstrated that these genes are essential for male fertility and that loss of mmm, sac, or tous results in complete immotility of the sperm flagellum. Cytological examination uncovered additional roles for sac and tous during cytokinesis and transmission electron microscopy of developing spermatids in mmm, sac, and tous mutant animals revealed defects associated with mitochondria and the accessory microtubules required for the proper elongation of the mitochondria and flagella during ciliogenesis. This study highlights the complex interactions of cilia-related proteins within the cell body and advances understanding of male infertility by uncovering novel mitochondrial defects during spermatogenesis (Bauerly, 2022).
Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. This study showed that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. Mob4 was shown to be required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. This study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization (Santos, 2023).
Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal structure called the ciliary rootlet links the cilium to the cell body. Previous studies indicated that rootlets support the long-term stability of some cilia. This study reports that Drosophila melanogaster Rootletin (Root), the sole orthologue of the mammalian paralogs Rootletin and C-Nap1, assembles into rootlets of diverse lengths among sensory neuron subtypes. Root mutant neurons lack rootlets and have dramatically impaired sensory function, resulting in behavior defects associated with mechanosensation and chemosensation. Root is required for cohesion of basal bodies, but the cilium structure appears normal in Root mutant neurons. Normal rootlet assembly requires centrioles. The N terminus of Root contains a conserved domain and is essential for Root function in vivo. Ectopically expressed Root resides at the base of mother centrioles in spermatocytes and localizes asymmetrically to mother centrosomes in neuroblasts, both requiring Bld10, a basal body protein with varied functions (Chen, 2015).
As the major microtubule (MT)-organizing center in animal cells, the centrosome consists of a pair of MT-based centrioles that organizes a protein matrix called the pericentriolar material to regulate MT assembly. In specific cell types, the mother centriole can mature into a basal body to organize a cilium, a slender protrusion that contains an MT-based axoneme assembled from the distal tip of the basal body. Cilia generally fall into two classes: motile cilia and primary (nonmotile) cilia. Motile cilia are often present in specialized epithelia, where they beat in coordinated waves, whereas most vertebrate cells can produce a primary cilium to sense diverse extracellular signals and transduce them into important cellular responses. Disruption of cilium assembly or function causes a spectrum of diseases named ciliopathies (Chen, 2015).
In many cell types, a fibrous cytoskeletal structure called the ciliary rootlet links the base of the cilium to the cell body. Across species, the rootlet ultrastructure consists of cross-striations appearing at intervals of 50-70 nm along its length. The size of rootlets varies among cell types, with prominent ones, for example, in mammalian photoreceptors. In mammals, Rootletin (Root, also known as ciliary rootlet coiled-coil protein) is the primary constituent of ciliary rootlets, and endogenous Root is expressed in photoreceptors and all major ciliated epithelia but absent from the spermatozoa. In mammalian cilia, Root resides only in the rootlet and does not extend into the basal body or cilium (Yang, 2002). However, the Caenorhabditis elegans Root orthologue, CHE-10, localizes at the proximal end of the basal body and extends into the transition zone, the most proximal region of the cilium (Mohan, 2013). In proliferating mammalian cells when cilia are not assembled, Root forms fibrous linkers between the centriole pairs and interacts with its paralog C-Nap1 (also known as CEP250) to promote centrosome cohesion in the cell cycle (Chen, 2015).
Over decades, biologists have been intrigued by what the in vivo function of the rootlet may be. In green algae, the rootlet fibers appear to anchor the flagella and to help absorb the mechanical stress generated by flagellar beating. Root mutant mice lack rootlets yet do not show overt defects in development, reproductive performance, or overall health, and Root is not required for normal ciliary functions during development (Yang et al., 2005). However, Root is important for the long-term stability of the cilium, particularly in specialized cells, such as photoreceptors. Studies in C. elegans showed that CHE-10 (Root orthologue) maintains cilium structure through preserving intraflagellar transport and the integrity of the transition zone and the basal body (Mohan, 2013). However, the role of CHE-10 may have diverged somewhat from Root in other organisms as it localizes to the basal body and transition zone of cilia and is required in neurons that lack rootlets (Chen, 2015).
This study has identified Drosophila Root as the sole orthologue of mammalian Root and C-Nap1, and has shown that it localizes to the ciliary rootlet in sensory neurons and, upon ectopic expression, at the proximal end of mother centrioles in spermatocytes. Root is required for neuron sensory perception, affecting various behaviors related to mechanosensation and chemosensation. Root is essential for basal body cohesion and for organizing the ciliary rootlet, and its N terminus containing the evolutionarily conserved Rootletin domain is critical for Root function and rootlet assembly in vivo. This study shows that Drosophila Root organizes rootlets at the base of primary cilia in sensory neurons and is essential for sensory neuron functions, including negative geotaxis, taste, touch response, and hearing (Chen, 2015).
A recent study of Root loss of function using RNAi knockdown in Drosophila also showed the essential role for Root in sensory perception of Ch neurons (Styczynska-Soczka, 2015). This study shows that Root is not required for normal cilium assembly, and it is likely that the required neuronal function of Root is at the rootlets, as rescue constructs that express tagged versions of Root rescued phenotypes completely or partially, and partial rescue coincided with assembly of smaller rootlets. Root was required for cohesion of the basal body pair in ciliated neurons, and centrioles, but not cilia, were required for rootlet assembly. Furthermore, the conserved Root domain is required for rootlet formation and for Root function, but not for localization to basal bodies (Chen, 2015).
Bld10, a presumptive Root partner, was not required for Root assembly into rootlets in sensory neurons but was required for ectopic Root localization to the proximal base of the centriole at the threshold of the lumen. In addition, ectopic Root localized asymmetrically in NBs, accumulating much more at the mother centrosome (Chen, 2015).
How do rootlets affect sensory neuron function? Because rootlets appear to always be associated with cilia, it is likely that rootlets support the structure and/or functions of cilia, enabling their role as sensors of environmental cues. However, Root mutant mice, which lack rootlets, develop normally, and during development Root is not essential for normal cilium functions, including environmental perception and cilium beating (Yang, 2005); however, the long-term stability of cilia requires Root (Yang, 2005; Chen, 2015 and references therein).
One important consideration for the mouse phenotypes is that the paralog, C-Nap1, may have redundant functions with Root. Indeed, in this study, it was found that even very small rootlets, resembling the localization of C-Nap1 at the base of centrioles, could rescue Root66. How can the rootlet, and especially a short rootlet, support mechanosensation? It has been proposed that a cytoskeletal structure (e.g., possibly the rootlet cytoskeleton) links mechanosensation from extracellular forces via the dendrite to the axon or synapse. Because the rootlet does not span across the neuron from the basal body to the axon, perhaps it links to another cytoskeleton like MTs. The conserved Root domain, which this study shows is essential for Root function but not localization to basal bodies, interacts with several kinesin light chains (Yang, 2005), supporting the idea of a possible linkage between the rootlet and the MT cytoskeleton (Chen, 2015).
In C. elegans, che-10 (Root orthologue) mutants show much more severe defects, with cilium, transition zone, and basal body degeneration during development due to severe defects in intraflagellar transport and preciliary membrane disruption that affects delivery of basal body and ciliary components (Mohan, 2013). But these defects may not necessarily be attributed to the rootlet structure because unlike in mammalian cells, CHE-10 also localizes within the basal body and the transition zone (a 'nonfilament form' of CHE-10) in neurons both with and without rootlets (Mohan, 2013). Moreover, in che-10 mutants, cilium degeneration also occurs in neurons without rootlets. Thus, in C. elegans, CHE-10, which is required for sensory neuron function, may have acquired new functions that have deviated from its function in mammals and Drosophila where Root is restricted to the Rootlet and the proximal base of centrioles. This study found that in Drosophila the loss of rootlets impairs sensory neuron functions (Chen, 2015).
Interestingly, the size of rootlets appears to affect neuronal function, particularly in ChOs that normally have long rootlets, because it was observed that shortened rootlets, resulting serendipitously from GFP-Root expression in the Root66 mutant background, only partially restored the JO hearing impairment. The morphologically normal appearance and stability of cilia in Root66 neurons indicate that rootlets may mediate signal transduction from cilia to the cell body, perhaps as a key structural element of the mechanoreceptor. Shorter rootlets may transduce signal less efficiently than longer ones in the JO, explaining why GFP-Root did not completely rescue the Root66 phenotype (Chen, 2015).
Alternatively, rootlets may be important for ciliary protein trafficking at the base of the cilium and between the dendrite and the cilium. In this scenario, long rootlets may support trafficking along the dendrite more efficiently than short ones. If this is the case, defective trafficking must be limited because loss of intraflagellar transport trafficking would result in failure to maintain the cilium structure and produce a more severe uncoordination phenotype (Chen, 2015).
With ectopic Root expression, this study showed that in a Drosophila cell line without cilia or rootlets, Root organized rootlet-like structures extending from the centrioles. However, ectopically expressed Root in cells such as NBs, spermatocytes, and spermatids localized to a smaller focus in the centrioles/centrosomes. In Ch neurons, Root assembles into longer rootlets than in Es neurons. It will be interesting to know what determines the forms of Root protein (centrosomal form vs. rootlet form), and in the case of rootlets, what defines their length. How Root is targeted to basal bodies and how the Root domain regulates rootlet assembly remain important questions (Chen, 2015).
Root, like its mammalian orthologue C-Nap1, specifically associates with mother centrioles upon ectopic expression in testes or NBs. Centriolar localization of Root in NBs and testes was shown to require the proximal centriolar protein Bld10, yet Bld10 is not required for Root localization to rootlets in ciliated neurons. Therefore, different mechanisms may regulate the recruitment of Root to centrioles in proliferating cells versus rootlet assembly at basal bodies in ciliated neurons. Overall, this study shows that Drosophila Root is a key structural component of ciliary rootlets that assembles in a centriole-dependent manner, and ciliary rootlets are necessary for neuronal sensory functions (Chen, 2015).
In vertebrates, rootletin is the major structural component of the ciliary rootlet and is also part of the tether linking the centrioles of the centrosome. Various functions have been ascribed to the rootlet, including maintenance of ciliary integrity through anchoring and facilitation of transport to the cilium or at the base of the cilium. In Drosophila, Rootletin function has not been explored. In the Drosophila embryo, Rootletin is expressed exclusively in cell lineages of type I sensory neurons, the only somatic cells bearing a cilium. Expression is strongest in mechanosensory chordotonal neurons. Knock-down of Rootletin results in loss of ciliary rootlet in these neurons and severe disruption of their sensory function. However, the sensory cilium appears largely normal in structure and in localisation of proteins suggesting no strong defect in ciliogenesis. No evidence was found for a defect in cell division. It is concluded that the role of Rootletin as a component of the ciliary rootlet is conserved in Drosophila. In contrast, lack of a general role in cell division is consistent with lack of centriole tethering during the centrosome cycle in Drosophila. Although the evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of chordotonal (Ch) neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself (Styczynska-Soczka, 2015).
The ciliary rootlet has long been known from transmission electron microscopy studies as the striated fibrous structure extending from the cilium basal body towards the cell nucleus . A rootlet is present at the base of most cilia, but it is particularly robust in cells with large or motile cilia. For instance, mammalian photoreceptors have a large rootlet at the base of a connecting cilium that links to the large photoreceptive outer segment. The rootlet has been speculated to have various functions, including contraction, association with organelles, transport/trafficking and anchoring of the basal body and axoneme (Styczynska-Soczka, 2015).
Knowledge of the rootlet was advanced by the discovery of its major constituent protein, a coiled-coil protein known as rootletin (encoded by the CROCC [ciliary rootlet coiled-coil] gene in humans). Mouse rootletin is a large 2009 amino acid residue protein with a globular head domain and a tail domain consisting of extended coiled-coil structures. The tail domain mediates polymerisation, whilst the head domain interacts with kinesin light chain 3 (KLC-3). It seems likely that rootletin is the only structural constituent of the ciliary rootlet, and its depletion causes loss of the rootlet. Hence, rootletin-deficient mice have been used to assess the function of the rootlet. Interestingly, mice lacking rootletin only exhibit a prominent ciliary phenotype in photoreceptors, which are cells with high rootletin expression levels and a particularly robust rootlet. Rootletin-depleted mouse photoreceptors show signs of degeneration at 18 months, reflected by shortening, disorganisation and loss of the photoreceptor outer segments. The requirement for the rootlet was interpreted as the need for the small connecting cilium to hold in place the large outer segment. It is notable that cells with less prominent rootlets did not show this phenotype (Styczynska-Soczka, 2015).
Thus, the hypothesis has emerged that the rootlet is required in the stability and function of cilia that are subjected to mechanical stress. Other functions for the rootlet in cilium biology, particularly in cells other than photoreceptors, are unclear. An association with KLC-3 led to suggestions that it might be involved in transport to the cilium or in facilitating intraflagellar transport (IFT) at the base of the cilium, but no transport defect was noted in rootletin mutant mice. However, in Caenorhabditis elegans the rootletin orthologue, Che-10, was shown to indirectly influence IFT by modulating the preassembly/localisation of various IFT proteins to the periciliary membrane compartment. Interestingly, in Che-10 mutants, cilia are initially formed normally but start to degenerate in late larvae (Styczynska-Soczka, 2015).
Aside from cilia, rootletin has also been shown to have a role in centriole cohesion during the centrosome duplication cycle. In metaphase cells, the centrosome consists of two tightly associated centrioles. After mitotic (M) phase exit, but before cell division, these become separated but loosely attached via linker proteins, known as a G1-G2 tether. Rootletin has been shown to be one of these linker proteins and so is required for centriole cohesion in G1 and S phases. It associates with the related C-Nap1, which is itself associated with the ends of centrioles, thereby forming filaments that maintain a loose connection between the centrioles. Phosphorylation of C-Nap1/rootletin by Nek2 kinase allows separation of the centrioles in late G2 before proceeding to M phase (Styczynska-Soczka, 2015 and references therein).
This study explores the function of the presumed Drosophila homologue of rootletin, which is encoded by the gene CG6129 (hereafter referred to as Rootletin). Drosophila displays several distinctive features relevant to Rootletin function. First, the centrosome duplication cycle is modified in Drosophila such that there is no G1-G2 tether either in the early embryo or larval neuroblasts. Instead, the centrosomes split immediately after mitosis. It is therefore of interest to ask whether Rootletin is required for Drosophila centrosome duplication (Styczynska-Soczka, 2015).
A second feature of Drosophila is that it has very few ciliated cell types. The only somatic cells bearing cilia are the type I sensory neurons, in which olfactory, gustatory or mechanosensory reception are performed via a specialised terminal cilium. While the cilia in these classes of neuron all have an associated rootlet, the most robust and prominent rootlets are found in the chordotonal (Ch) neurons. Ch neurons are auditory and proprioceptive mechanosensors and may be presumed to be under mechanical stress. Although nothing has been described of Rootletin function, it is highly represented in the transcriptome of Ch neurons. This study investigated the expression and function of Drosophila Rootletin with particular focus on Ch neuron structure and function (Styczynska-Soczka, 2015).
Drosophila Rootletin (CG6129) is required for the formation of the ciliary rootlet of Ch neurons. This strongly supports the conclusion that it performs a conserved function as a structural component of the rootlet. The expression pattern of Rootletin also highlights this function: it is restricted to ciliated cells, i.e., the type I sensory neurons, and among these cells, it is most abundantly and persistently expressed in Ch neurons, whose cilia have very robust rootlets. In contrast, Rootletin does not seem to be required for non-ciliated cells (Styczynska-Soczka, 2015).
In Ch neurons, loss of Rootletin and the rootlet results in functionally defective sensory responses. Despite this, the Ch neuron cilium itself does not appear to be strongly defective structurally. This may suggest a defective mechanotransduction process rather than defective development of the cilium. Based on observations in mouse photoreceptors, it was proposed that a rootlet is required for mechanical stability of large or motile ciliary structures. Clearly, the Ch neurons could be described in this category as these mechanosensory cells must be under mechanical stress in their function. Moreover, the Ch neuron cilium has the molecular machinery for ciliary motility, and there is biophysical evidence that cell or ciliary motility might be important for mechanotransduction. The rootlet can therefore be seen to provide a solid anchor for this in order to maintain dendritic integrity. Age-related decline in fly proprioceptive function may be consistent with the stress/anchor hypothesis. However, at the light microscope level, no sign of collapse or shortening of the cilium with age was found in Rootletin knock-down flies (Styczynska-Soczka, 2015).
An alternative explanation for Ch neuron dysfunction and age-related decline is a requirement for rootlet/Rootletin in transport of components to the base of the cilium or within the cilium, as has been proposed for the orthologue in C. elegans. In Drosophila Ch neurons, it is clear that transport and IFT are not strongly defective as ciliogenesis appears to occur largely normally. Whilst subtle changes in the channel NompC localisation suggest that Rootletin might be indirectly involved in some aspects of IFT, no change in the localisation of IFT protein, RempA, suggests a lack of general disruption of IFT. It is possible, however, that a subtle impairment of IFT disrupts transport necessary for long-term ciliary homeostasis rather than ciliogenesis. Given the severe loss in neuronal function, even in young flies, an alternative explanation is that the rootlet directly participates in mechanotransduction, such as being required to maintain or transmit tension during cilium stimulation (Styczynska-Soczka, 2015).
In other organisms, rootletin is required for centriole cohesion or tethering after centriole duplication). In vertebrates, rootletin forms the tether in association with the related C-Nap1 (CEP250) protein. It seems unlikely that Rootletin is the centrosome linker protein in Drosophila because it is not expressed generally. Moreover, there is no separate C-Nap1 orthologue in Drosophila. Lack of this function would be consistent with observations that Drosophila cells seem to lack centriole tethering. Instead, centrioles separate immediately upon disengagement during the centrosome cycle. In mammalian cells, centrosome separation upon entering mitosis is achieved by Nek2 phosphorylation of rootletin and C-Nap1. Interestingly, Drosophila retains an orthologue of Nek2, and in cultured Drosophila cells Nek2 knockdown causes mitotic spindle defects. In the absence of a role for Rootletin or C-Nap1, the role of Nek2 in this process is unclear (Styczynska-Soczka, 2015).
Despite the lack of a role in centriole tethering in centrosomes, it seems that Rootletin plays a 'tethering-like' role in the basal body of the Ch neuron cilium since the proximal centriole is lost upon Rootletin knock-down. Indeed, on TEM of wild-type neurons, the proximal centriole appears to be held in place by strands of the rootlet that pass around it before joining with the distal centriole (Styczynska-Soczka, 2015).
It is concluded that the role of Rootletin as a component of the ciliary rootlet is conserved in Drosophila. In contrast, lack of a general role in cell division is consistent with lack of centriole tethering during the centrosome cycle in Drosophila. Although the evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of Ch neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself. In contrast, any effect on ciliary transport appears to be subtle (Styczynska-Soczka, 2015).
Cilia are conserved organelles that have important motility, sensory and signalling roles. The transition zone (TZ) at the base of the cilium is critical for cilia function, and defects in several TZ proteins are associated with human congenital ciliopathies such as Nephronophthisis (NPHP) and Meckel Gruber syndrome (MKS: see MKS1, Tectonic, B9D1 and B9D2). In several species, MKS and NPHP proteins form separate complexes that cooperate with Cep290 to assemble the TZ, but flies appear to lack core components of the NPHP module. This study shows that MKS proteins in flies are spatially separated from Cep290 at the TZ, and that flies mutant for individual MKS genes fail to recruit other MKS proteins to the TZ, while Cep290 appears to be recruited normally. Although there are abnormalities in microtubule and membrane organisation in developing MKS mutant cilia, these defects are less apparent in adults, where sensory cilia and sperm flagella appear to function quite normally. Thus, localising MKS proteins to the cilium or flagellum is not essential for viability or fertility in flies (Pratt, 2016).
The Drosophila eye displays peculiar sensory organs of unknown function, the mechanosensory bristles, that are intercalated among the adjacent ommatidia. Like the other Drosophila sensory organs, the mechanosensory bristles consist of a bipolar neuron and two tandemly aligned centrioles, the distal of which nucleates the ciliary axoneme and represents the starting point of the ciliary rootlets. This study reports that the centriole associated protein Sas-4 colocalizes with the short ciliary rootlets of the mechanosensory bristles and with the elongated rootlets of chordotonal and olfactory neurons. This finding suggests an unexpected cytoplasmic localization of Sas-4 protein and points to a new underscored role for this protein. Moreover, it was observed that the sheath cells associated with the sensory neurons also display two tandemly aligned centrioles but lacks ciliary axonemes, suggesting that the dendrites of the sensory neurons are dispensable for the assembly of aligned centrioles and rootlets (Persico, 2021).
The establishment of left-right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. This study shows that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer's vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2; see Drosophila Van Gogh), to shape a productive LRO flow. These findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry (Juan, 2018).
Cilia are highly specialized antennae-like organelles that extend from the cell surface and act as cell signaling hubs. Intraflagellar transport (IFT) is a specialized form of intracellular protein trafficking that is required for the assembly and maintenance of cilia. Because cilia are so important, mutations in several IFT components lead to human disease. Thus, clarifying the molecular functions of the IFT proteins is a high priority in cilia biology. Live imaging in various species and cellular preparations has proven to be an important technique in both the discovery of IFT and the mechanisms by which it functions. Live imaging of Drosophila cilia, however, has not yet been reported. This study has visualized the movement of IFT in Drosophila cilia using time-lapse live imaging for the first time. NOMPB-GFP (IFT88) was found to move according to distinct parameters depending on the ciliary segment. NOMPB-GFP moves at a similar speed in proximal and distal cilia toward the tip (~0.45 mum/s). As it returns to the ciliary base, however, NOMPB-GFP moves at ~0.12 mμm/s in distal cilia, accelerating to ~0.70 mμm/s in proximal cilia. Furthermore, while live imaging NOMPB-GFP, it was observed one of the IFT proteins required for retrograde movement, Oseg4 (WDR35), is also required for anterograde movement in distal cilia. It is anticipated that the time-lapse live imaging analysis technique in Drosophila cilia will be a good starting point for a more sophisticated analysis of IFT and its molecular mechanisms (Lee, 2018).
The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. This study shows that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. These results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells (Vieillard, 2016).
Cilia and flagella are highly conserved organelles involved in various cellular and physiological processes. Defects in cilia assembly are responsible for many human diseases. Cilia are built around a microtubule core, the axoneme, which grows from the basal body (BB), itself derived from the mother centriole. At the interface between the BB and the plasma membrane, a particular compartment, the transition zone (TZ), plays both structural and functional roles by regulating the traffic in and out of the cilium and by forming structural links between microtubules and the membrane. Several ciliopathies, such as the Joubert syndrome and the Meckel syndrome (MKS) or nephronophthisis (NPHP) are caused by defects in TZ components (Vieillard, 2016).
TZ assembly is a complex process starting with the docking of the mother centriole to cytoplasmic vesicles and the timely and spatially ordered assembly of multiple proteins. TZ components have been extensively described in several organisms. Genetic approaches in Caenorhabditis elegans and biochemical approaches in mammalian cells helped to establish a first comprehensive hierarchy of these components. They fall into different modules, namely MKS, NPHP, and CEP290. Proteins of the MKS module colocalize and together contribute to the formation of the TZ in various organisms, from C. elegans to mammals. In C. elegans, MKS, NPHP, and CEP290 cooperate to assemble the TZ. Upstream, MKS5 (mammalian Rpgrip1L or NPHP8) controls the assembly of the MKS and NPHP complexes on centrioles. However, variations exist between model organisms, and all TZ components are not conserved in all ciliated species. For example, MKS5 and several other NPHP proteins are absent from the Drosophila melanogaster genome. Additional components of the TZ have been identified, but their integration in the hierarchy of TZ assembly is not yet clear. For instance, Chibby (Cby) is a component of the TZ in Drosophila and vertebrates, but how Cby contributes to TZ assembly remains to be characterized. Cby is absent from C. elegans and protozoa genomes. Dila (Azi1 in mammals) is also not present in C. elegans but is required for TZ assembly in Drosophila or for ciliogenesis in mammals and is also associated with centriolar satellites in vertebrates. Therefore, key components of TZ assembly in C. elegans or mammals such as MKS5 are missing in Drosophila, and the mechanisms of TZ assembly in this organism are largely unknown (Vieillard, 2016).
Two main ciliated tissues are found in Drosophila: sensory neurons, in which cilia are required for transducing most senses, and sperm germ cells. Sensory cilia assembly relies on intraflagellar transport (IFT) and is hence compartmentalized in Drosophila. In contrast, flagella assembly in male germ cells is IFT independent and said to be cytosolic. Such cytosolic mode of assembly is also proposed in mammals for the formation of the sperm midpiece. In Drosophila, cytosolic flagella extension takes place inside a membrane ciliary cap, which requires Cep290 for proper organization and architecture. This membrane cap is built in G2 Drosophila spermatocytes, when all four replicated centrioles dock to the plasma membrane and grow a primary cilium. During meiosis, the four primary cilia are internalized together with centrioles, maintaining the membrane ciliary cap connected to the plasma membrane (Vieillard, 2016).
This study shows that MKS proteins are involved in Drosophila TZ assembly, but simultaneous removal of several MKS components only leads to very mild TZ and axonemal defects. Instead, deleting both Cby and Dila, two components of the TZ in Drosophila, leads to extremely severe TZ disorganization with complete loss of MKS components and severe reduction of Cep290. In absence of Cby and Dila, BBs fail to dock to the plasma membrane of spermatocytes and assemble the ciliary membrane cap required for sperm flagella elongation. In absence of this ciliary cap, aberrant growth of axonemal microtubules is observed in spermatocytes. This study has demonstrated that Klp59D, a kinesin-13 family member, is present at the ciliary cap and is required for proper and timely regulated growth of the axoneme. It is therefore propose dthat the ciliary cap is necessary to restrict and timely coordinate ciliary component assembly in Drosophila spermatocytes (Vieillard, 2016).
This study shows that TZ assembly in Drosophila involves cooperative actions of Cby and Dila, both in sensory neurons and male germ cells. More importantly, it is revealed that a timely control of the balance between membrane cap assembly by TZ components and microtubule elongation by kinesin-13 microtubule remodeling is required for proper axoneme formation in Drosophila male germ cells.
(Vieillard, 2016).
Flagella assembly in Drosophila male germ cells does not rely on IFT, as null mutations in IFT components have no consequences on flagella formation. It is thus considered to be cytosolic, in contrast to the more widely conserved IFT-dependent ciliogenesis, which is said to be compartmentalized (Avidor-Reiss, 2015). However, proteins of the ring centriole/TZ such as Unc, Cby, or Cep290 are required to compartmentalize the flagellar growing end in Drosophila through the formation of a ciliary cap. The exact function of this ciliary cap is not clear (Avidor-Reiss, 2015). It was proposed that the formation of the ciliary cap is associated with the formation of a diffusion barrier, which protects the ciliary end from cytoplasmic proteins. The current results are in agreement with this hypothesis and suggest that the ciliary cap is required to create a specific environment that restricts axonemal growth, as precluding ciliary cap formation in dila81; cby1 mutants leads to aberrant microtubule extensions and unstable axonemes. On the other hand, the results show that unbalanced microtubule growth prevents ciliary cap formation. In agreement with this conclusion, previous work showed that treatment of spermatocytes with Taxol leads to Unc domain extension, associated with aberrant axoneme elongation and defective ciliary cap formation, when treatment was applied on spermatocytes before BB docking, but not when applied after BB docking (Riparbelli, 2013). Therefore, cytosolic ciliogenesis in Drosophila is regulated by a precise balance between microtubule extension from the centriole distal end and membrane cap formation (Vieillard, 2016).
This mechanism is apparently specific to cytosolic ciliogenesis. In dila81; cby1 sensory cilia, the total absence of the TZ is not associated with aberrant axoneme elongation. This also indicates that centriole docking in this tissue is a prerequisite to elongate the axoneme. Klp59D is not expressed in sensory neurons, and driving Klp59D shRNA expression in sensory neurons did not reveal any sensory defects. These differences could reflect particular properties of centrioles in Drosophila spermatocytes. Indeed, all four centrioles convert to BB in spermatocytes, whereas only the mother centriole does so in sensory neurons. Therefore, different sets of proteins should be involved in centriole maturation in spermatocytes compared with sensory neurons. Further work will be required to understand molecular differences between sperm and sensory basal bodies (Vieillard, 2016).
Another set of observations highlights the particular behavior of spermatocyte centrioles. Mutations in TZ proteins have shown that reducing the length of primary cilia-like/TZ in spermatocytes is associated with an increase in length of the centrioles. This was observed when removing Cep290, and this study made identical observations on B9d2, tctnΔ mutant testes. Thus, reducing ciliary cap extension allows centriole elongation, suggesting a balance in centriolar extension controlled by the TZ. Such a balance was also revealed by removal of Klp10A, which leads to an increase in centriole elongation and in a simultaneous reduction of primary cilia/TZ extension. Hence, the kinesin-13 proteins Klp10A and Klp59D likely play complementary functions at the spermatocyte TZ by restricting, respectively, centriole elongation or TZ elongation (Vieillard, 2016).
The function of kinesin-13 family members in ciliogenesis is still unclear and sometimes contradictory. In protozoa, such as Giardia intestinalis, Leishmania, or Trypanosoma, removal of kinesin-13 leads to longer flagella in agreement with a tubulin-depolymerizing function of these proteins. It has also been proposed that kinesin-13 proteins play a role in maintaining a free tubulin pool to promote axoneme assembly. However, recent studies suggest more complex roles of kinesin-13 members in cilia assembly. In mammals, Kif24 is required to regulate centriole length by interacting with CP110. Such a function is also true for Klp10A in Drosophila. Thus, one possible explanation for the observed phenotype in the dila81; cby1 double mutant would be that CP110 is prematurely removed from centrioles, as Dila and CP110 have strikingly similar localization inside the TZ lumen. However, complete removal or overexpression of CP110 in Drosophila did not lead to measurable ciliary defects and obviously did not lead to aberrant centriolar extensions in male germ cells. Therefore, modulation of CP110 activity cannot explain the aberrant axonemal growth observed in the testes in both dila81; cby1 mutants and Klp59D KD (Vieillard, 2016).
In Tetrahymena, kinesin-13 protein was shown to act as an axoneme assembly factor by regulating the levels of tubulin modification and in particular tubulin acetylation inside the cilia. It is tempting to speculate that Klp59D could play such a function in the ciliary cap of the sperm germ cell. However, it is difficult to test this hypothesis, as quantifications of differences in tubulin modifications inside the ciliary cap by IF are difficult and biochemical quantification of tubulin modifications is limited by the huge amount of nonaxonemal (cytoplasmic) modified microtubules in male germ cells (Vieillard, 2016).
This study reveals both analogies and differences in TZ assembly between Drosophila and other organisms. In mammals, MKS proteins play a critical function on TZ assembly and subsequently cilia function as revealed by human ciliopathies, in which these proteins are mutated. In C. elegans, mutations in the five core members of the MKS complex (Tctn, MKSR-1 [B9d1], MKSR-2 [B9d2], MKS1 [B9d3], and MKS6 [Cc2d2A]) have no effect on cilia architecture and sensory function. However, functional interactions were revealed between them, as their localization at the TZ is interdependent. In addition, whereas no genetic interactions are revealed when looking at sensory functions, combining mutations in several of these genes led to modified lifespan, suggesting that MKS components cooperate in specific ciliary signaling functions in C. elegans. In Drosophila, the current results indicate that, like in nematodes, the MKS complex has only subtle functions on TZ and cilia assembly and that MKS components exhibit functional interactions, as removing B9d2 and Tctn is sufficient to completely alter the recruitment of the other MKS members. In this context, genetic interactions are not expected to be revealed by combining other mutations in MKS components together with the B9d2, tctn mutant. The current observations therefore suggest that the hierarchy and function of MKS components are likely conserved between C. elegans and Drosophila (Vieillard, 2016).
In contrast, strong genetic interactions have been described between MKS and NPHP components in C. elegans. In particular, MKS5 (Rpgrip1L or NPHP8 in mammals) acts upstream of all other components to build the TZ, and NPHP4 and 1 interact with MKS complex proteins to organize the TZ. These NPHP members are not conserved in Drosophila. Therefore, other proteins should be required to organize the ciliary TZ upstream of the MKS complex in Drosophila. Among conserved candidates in other organisms, Cep290 is required to build the TZ in Drosophila, but MKS components are still assembled at the TZ in Cep290 mutants, thus suggesting that other TZ components act upstream of MKS. This study has demonstrated that cby and dila show strong genetic interaction in TZ assembly, whereas mutating each separately only shows moderate effects. These results highlight for the first time the synergistic roles of Cby and Dila in building the TZ (Vieillard, 2016).
Cby and Dila are not present in s but are conserved in mammals. In mouse, Cby plays an important role in motile ciliated epithelia, as demonstrated by the severe airway ciliogenesis defects observed in Cby knockout mice. In mammalian cells, Cby is also required for primary cilia assembly. Interestingly, mammalian Cby is required to dock centrioles to the plasma membrane, a function that is only revealed in Drosophila by removing both Cby and Dila. Mammalian Cby is required for Rab8-mediated ciliary vesicle assembly, and this study shows that Rab8 recruitment at the ciliary cap is also partially affected in cby1 mutant flies and strikingly completely abolished in dila81; cby1 double mutants. This suggests a conserved role of Cby in Rab8-associated membrane cap assembly from Drosophila to mammals and highlights that Dila is also required for this process in Drosophila. In mammals, Azi1 (Dila in Drosophila) is a centriolar satellite component required for centriole duplication, but it is also localized at centrioles and is required for cilia assembly. However, its function in TZ assembly has not been investigated. In Drosophila, Dila is found restricted to the BB and TZ and is required for cilia assembly, but no specific defects of the TZ were detected in mutant flies. Thus, this study in Drosophila reveals that Dila is also involved in building the TZ but that alternative pathways can compensate for Dila deficiency in TZ assembly. Such compensation mechanisms have also been proposed for Azi1 in mammals (Vieillard, 2016).
In conclusion, this study reveals critical genetic interactions between two yet-unrelated TZ proteins, Cby and Dila. Future work will be required to understand if such interactions are also conserved in mammals. This work also demonstrates that the formation of a ciliary cap is essential to coordinate ciliary assembly during cytosolic ciliogenesis by creating a biochemical environment that controls axonemal microtubule growth (Vieillard, 2016).
Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. This study reports a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in TZ maturation in the Drosophila melanogaster male germline. Reduction of cellular PIP2 levels by ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. It is also reported that the onion rings (onr) allele of Drosophila exo84 decouples TZ hyperelongation from loss of cilium-plasma membrane tethering. These results reveal a requirement for PIP2 in supporting ciliogenesis by promoting proper TZ maturation (Gupta, 2018).
Cilia are sensory organelles important for signalling in response to extracellular cues, and for cellular and extracellular fluid motility. Consistent with their importance, defects in cilium formation (i.e. ciliogenesis) are associated with genetic disorders known as ciliopathies, which can display neurological, skeletal and fertility defects, in addition to other phenotypes. Many ciliopathies are associated with mutations in proteins that localize to the transition zone (TZ), the proximal-most region of the cilium that functions as a diffusion barrier and regulates the bidirectional transport of protein cargo at the cilium base. For example, the conserved TZ protein CEP290 is mutated in at least six different ciliopathies and is important for cilium formation and function in humans and Drosophila (Basiri, 2014). Although the protein composition of TZs has been investigated in various studies, the process of TZ maturation, through which it is converted from an immature form to one competent at supporting cilium assembly, is relatively understudied (Gupta, 2018).
Ciliogenesis begins with assembly of a nascent TZ at the tip of the basal body (BB). During TZ maturation, its structure and protein constituents change, allowing for establishment of a compartmentalized space, bounded by the ciliary membrane and the TZ, where assembly of the axoneme, a microtubule-based structure that forms the ciliary core, and signalling can occur. In Drosophila, nascent TZs first assemble on BBs during early G2 phase in primary spermatocytes. This occurs concomitantly with anchoring of cilia to the plasma membrane (PM), microtubule remodelling within the TZ, and establishment of a ciliary membrane that will persist through meiosis (Gupta, 2018).
TZ maturation has been described in Paramecium, Caenorhabditis elegans and Drosophila (Gottardo, 2013), and is most readily observed in the Drosophila male germline by an increase in TZ length. Previous work has shown that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for proper axoneme structure in the Drosophila male germline. PIP2, which is one of seven different phosphoinositides (PIPs) present in eukaryotes, localizes primarily to the PM, where it is required for vesicle trafficking among other processes. PIP2 has recently been linked to cilium function. Although the ciliary membrane contains very little PIP2 due to action of the cilium resident PIP phosphatase INPP5E, the cilium base is enriched in PIP2. Inactivation of INPP5E causes a buildup of intraciliary PIP2, which disrupts transport of Hedgehog signalling proteins in vertebrates and ion channels involved in mechanotransduction in Drosophila (Park, 2015). In light of current understanding of PIP2 as a modulator of cilium function, this study sought to investigate the cause of defects observed in axoneme assembly in Drosophila male germ cells with reduced levels of PIP2 (Gupta, 2018).
To investigate how reduction of cellular PIP2 affects ciliogenesis in the Drosophila male germline, transgenic flies were used expressing the Salmonella PIP phosphatase SigD under control of the spermatocyte-specific β2-tubulin promoter (hereafter β2t-SigD). To examine whether axoneme defects in β2t-SigD were caused by aberrant TZ function, localization of fluorescently-tagged versions of the core centriolar/BB protein Ana1 (CEP295 homolog) and the conserved TZ protein Cep290 was analyzed during early steps of cilium assembly. Cep290 distribution appeared similar in control and β2t-SigD in early G2 phase, when TZs are still immature. In contrast, Cep290-labelled TZs were significantly longer in β2t-SigD compared to controls by late G2, following TZ maturation. Unlike Drosophila cep290 mutants, which contain longer than normal BBs, Ana1 length was not affected in β2t-SigD, and no strong correlation was observed between Cep290 and Ana1 lengths. Consistent with this result, the ultrastructure of BBs in β2t-SigD is normal, and localization of the centriolar marker GFP-PACT is similar in controls and β2t-SigD. In contrast, TZ proteins Chibby (Cby) and Mks1 exhibited hyperelongation in β2t-SigD, indicating that this phenotype is not unique to Cep290. TZ hyperelongation was highly penetrant (>70%, n >200) and showed high correlation (>0.95) within syncytial germ cell cysts, suggesting a dosage-based response to a shared cellular factor, presumably SigD. Despite persistence of hyperelongated TZs through meiosis, axonemes were able to elongate in post-meiotic cells. Nonetheless, the ultrastructure of these axonemes is frequently aberrant, either lacking nine-fold symmetry or containing triplet microtubules in addition to the usual doublets (Gupta, 2018).
Although PIP2 is its major substrate in eukaryotic cells in vivo, SigD can dephosphorylate multiple PIPs in vitro. To address whether TZ hyperelongation observed in β2t-SigD represented a physiologically relevant phenotype due to decreased PIP2, attempts were made to rescue this phenotype by co-expressing β2t-SigD with fluorescently-tagged Skittles (Sktl) under control of the β2- tubulin promoter. Sktl expression was able to suppress TZ hyperelongation to various degrees in a cilium-autonomous manner. Furthermore, the BB/TZ protein Unc-GFP exhibited TZ hyperelongation at a low penetrance in sktl2.3 mutant clones, indicating that Sktl is important for TZ maturation. Vertebrate type I PIP kinase PIPKIγ is important for cilium formation in cultured cells. The Drosophila PIPKIs, Sktl and PIP5K59B, arose from recent duplication of the ancestral PIPKI gene, and are not orthologous to specific vertebrate PIPKI isoforms. Sktl has diverged more than its paralog PIP5K59B and seems to be functionally related to PIPKIγ and the C. elegans PPK-1 in having roles at cilia. However, unlike the human PIPKIγ, which licenses TZ assembly by promoting CP110 removal from BBs, the current results suggest that Sktl functions in regulating TZ length but not TZ assembly. Consistent with this, neither inactivation nor overexpression of cp110 affects cilium formation in Drosophila, and Cp110 is removed from BBs in early primary spermatocytes (Gupta, 2018).
Attempts were made to examine whether TZ hyperelongation due to SigD expression affected TZ function. Following meiosis in the Drosophila male germline, TZs detach from BBs and migrate along growing axonemes, maintaining a ciliary compartment at the distal-most ~2μm, where tubulin is incorporated into the axoneme. As shown by Unc and Cep290 localization, TZs in β2t-SigD were frequently incapable of detaching from BBs and migrating along axonemes despite axoneme and cell elongation. Indeed, the previously reported 'comet-shaped' Unc-GFP localization in β2t-SigD persists during cell elongation after meiosis despite elongation of the axoneme (Gupta, 2018).
In Drosophila and humans, BBs consist of microtubule triplets, whereas axonemes contain microtubule doublets due to termination of C-tubules at the TZ (Gottardo, 2013). Consistent with a defect in this transition and the presence of microtubule triplets in axonemes in β2t-SigD, a subset of cilia (<5%) in β2t-SigD contained puncta of Ana1 at the distal tips of TZs. Treatment of germ cells with the microtubule-stabilizing drug Taxol increased penetrance of this phenotype from <5% in untreated cells to >25% in cells treated with 4 μM Taxolwithout significantly affecting Cep290 length. Taxol-treated controls did not exhibit TZ- distal Ana1 puncta. Fluorescently-tagged Asterless (CEP152 homolog), a pericentriolar protein, did not localize to TZ-distal puncta in β2t-SigD suggesting these TZ-distal sites are not fully centriolar in protein composition. Taxol has been hypothesized to disrupt TZ maturation by inhibiting microtubule remodelling in the Drosophila male germline (Riparbelli, 2013). Indeed, similar to β2t-SigD, Taxol-treated male germ cells assemble long axonemes that contain triplet microtubules (Riparbelli, 2013), further supporting a functional relationship between PIP2 and microtubule reorganization in TZ maturation (Gupta, 2018).
Male flies homozygous for the onion rings (onr) mutant of Drosophila exo84 are sterile and exhibit defects in cell elongation and polarity similar to β2t-SigD. Exo84 is a component of the octameric exocyst complex, which binds PIP2 at the PM. To investigate whether defects in TZ hyperelongation could be explained by defective Exo84 function, TZs were examined in onr mutants. Unlike β2t-SigD, onr did not display hyperelongated TZs, suggesting Exo84 is dispensable for TZ maturation. Due to involvement of the exocyst in membrane trafficking, whether cilium- associated membranes were affected in β2t-SigD or onr mutants in a manner similar to dilatory; cby mutants (Vieillard, 2016) was examined. Dilatory (Dila), a conserved TZ protein, cooperates with Cby to assemble TZs in the Drosophila male germline (Vieillard, 2016). TZs in β2t-SigD and onr cells were able to dock at the PM initially, but were unable to maintain membrane connections, and were rendered cytoplasmic, similar to TZs in dila; cby mutants. In addition, fluorescently-tagged Exo70, a PIP2-binding exocyst
subunit, localized to BBs. The current results suggest that the exocyst, and Exo84 in particular, regulates cilium-PM association, similar to PIP2, and that TZ hyperelongation and loss of cilium-PM association are genetically separable phenotypes (Gupta, 2018).
Maturation of a TZ from a nascent to a fully functional state, leading ultimately to axoneme assembly and ciliary signalling, requires orchestration of various proteins and cellular pathways. The current results indicate that normal execution of this process requires PIP2, and that depletion of PIP2 induces TZs to grow longer than normal. Similar to β2t-SigD, Drosophila dila; cby and cby mutants display hyperelongated TZs, whereas mks1 mutants have shorter TZs. Because both Cby and Mks1 are hyperelongated in β2t-SigD cells, PIP2 regulates TZ length independently of an effect on Cby or Mks1 recruitment. This study shows that hyperelongated TZs are dysfunctional. Similar to dila; cby (Vieillard, 2016) and cep290 (Basiri, 2014) mutants, axonemes can assemble in β2t-SigD, albeit with aberrant ultrastructure, despite lack of functional TZs or membrane association. The presence of TZ-distal Ana1 puncta in β2t-SigD, without the increase in BB length seen in cep290 mutants lacking a functional TZ barrier, suggests that β2t-SigD selectively disrupts the ability of TZs to restrict C-tubules and Ana1 without abolishing the TZ barrier entirely. CEP295, the human Ana1 ortholog, regulates post-translational modification of centriolar microtubules, which might explain the presence of TZ-distal Ana1 along with supernumerary microtubules in β2t-SigD cells. Asterless (Asl), a pericentriolar protein important for centrosome formation and centriole duplication, did not exhibit this TZ-distal localization, possibly due to differences in dynamics of Ana1 and Asl loading onto centrioles or the more peripheral nature of Asl distribution within the centriole (Gupta, 2018).
The majority of PIP2 at the PM is produced by PIPKIs. Mutation of the PIPKI Sktl induced hyperelongated TZs, and expression of Sktl could suppress TZ hyperelongation in β2t-SigD, suggesting Sktl might function in situ to regulate TZ length. In humans, PIPKIC is linked to lethal congenital contractural syndrome type 3 (LCCS3), which has been suggested to represent a ciliopathy. The recent discovery of a role for
another LCCS-associated protein in cilium function corroborates this hypothesis. The current data support the idea that PIPKIs might represent ciliopathy-associated genes or genetic modifiers of disease. Members of the exocyst complex are important for cilium formation in cultured cell lines and zebrafish, but their precise roles in ciliogenesis are not well understood. The subunits Sec3 and Exo70 regulate exocyst targeting to the PM through a direct interaction with PIP2. Previous work has shown that the onr allele of Drosophila exo84 phenocopies defects in cell polarity and elongation observed in β2t-SigD. This study showed that the onr mutation phenocopies loss of cilium-membrane contacts in β2t-SigD but not TZ hyperelongation. Thus, TZ hyperelongation is not a prerequisite for failure of cilium-PM association in male germ cells, and Exo84 uniquely regulates the latter process, potentially by supplying membrane required to maintain cilium-PM tethering. That the TZ is dispensable for this function is supported by the Drosophila cep290 mutant, which lacks a functional TZ but retains cilium-PM association. Notably, EXOC8, which encodes the human Exo84, has been linked to the ciliopathy Joubert syndrome, and a similar defect in ciliogenesis might be present in humans with mutations in EXOC8 (Gupta, 2018).
Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster, individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. This study shows that combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes (Steinhauer, 2019).
Proper differentiation of germline cells into eggs and sperm is essential for the perpetuation of a species. Sperm cells in mammals and Drosophila melanogaster develop from germline stem cells that produce mitotic spermatogonia, which ultimately undergo meiosis and terminal spermatid differentiation. Drosophila has long been a powerful model organism for studying spermiogenesis, which begins after meiosis, when the syncytial spermatids derived from a single gonialblast (the differentiating stem cell daughter) begin morphological changes required for their differentiation, including mitochondrial differentiation, flagellar elongation, nuclear compaction and acrosome formation. In Drosophila, after meiosis is completed, mitochondria aggregate around the basal body on one side of the nucleus. Subsequently, the mitochondria fuse to form the 'Nebenkern'. As differentiation proceeds, spermatids form acrosomes, their nuclei remodel and compact, and sperm tails (flagella) elongate. During spermatid elongation, both axonemal microtubules (MTs) and the mitochondrial Nebenkern extend to form the flagellum, composed of a central axoneme flanked by major and minor mitochondrial derivatives. Drosophila sperm axonemes are structurally similar to other axonemes, containing a central MT pair ringed by nine outer MT doublets (Steinhauer, 2019).
In Drosophila, as in mammals, incomplete cytokinesis during sperm development leads to cytoplasmic sharing between sister spermatids. Following terminal differentiation, the inter-spermatid cytoplasmic bridges are dissolved and the spermatids' cytoplasmic contents are removed. This process in Drosophila, called individualization, is carried out by the actin-rich individualization complex (IC) that first forms adjacent to the needle-shaped spermatid nuclei, which by this point are clustered in the basal testis. The IC is composed of 64 actin cones, one for each spermatid nucleus of the germline cyst, and it shares similarities with actin comets found on endocytic vesicles. As individualization proceeds, the actin cones of the IC move synchronously away from the nuclei toward the apical domain of the testis, traversing the spermatid tails until they reach the end of the flagella. Each moving IC generates a growing cystic bulge that contains the extruded cytoplasmic contents of the individualizing cyst. When the IC and associated cystic bulge reach the end of the flagella, they form a waste bag (WB) full of discarded organelles, degradation of which may occur by an apoptosis-like program. As a result of individualization, each streamlined spermatozoon resides within its own plasma membrane, most of its cytoplasm has been extruded, and it no longer maintains connections to its sister spermatids. Once this process has been completed, mature sperm are coiled in preparation for release into the seminal vesicle, and WBs and abnormal sperm are eliminated within the base of the testis (Steinhauer, 2019).
Elegant prior studies have provided a detailed view of the polarized IC structure. At the leading edge of the actin cones, F-actin filaments form a meshwork under the control of the Arp2/3 actin-nucleating complex, whereas at their rear, F-actin is organized into parallel bundles by the activity of the actin-bundling proteins Quail/Villin, Chickadee/Profilin and Singed/Fascin. IC activities are thought to be segregated, with the front meshwork being responsible for cytoplasmic extrusion and the rear bundles responsible for movement along the sperm tails. During individualization, cones accumulate actin, and actin polymerization, but not myosin motor activity, is essential for IC progression (Steinhauer, 2019).
Previously identified combover (cmb), a gene encoding two major protein isoforms that are predicted to be largely intrinsically disordered and lack any known domains, as a Rho kinase substrate that acts as a planar polarity effector (PPE) to affect Drosophila wing hair (trichome) formation. During pupal development, each cell of the fly wing forms an actin-rich hair, surrounded by membrane, that points towards the distal tip of the wing. Polarized wing hair formation is governed by the non-canonical Wnt-planar cell polarity (PCP) pathway, during which core PCP proteins including Frizzled (Fz), Dishevelled (Dsh), Van Gogh (Vang), Prickle (Pk), Diego (Dgo), and Flamingo (Starry night) localize asymmetrically across proximal-distal wing cell borders. PCP-generated asymmetry leads to proximal enrichment of the PPE proteins Inturned, Fuzzy, Fritz and Multiple wing hairs (Mwh), which prevent ectopic wing hair formation. Additionally, Mwh prevents the formation of multiple prehairs, and it bundles actin to restrict hair formation to a single hair during trichome outgrowth. Although cmb mutants show no wing phenotype, Cmb overexpression causes formation of a multiple hair cell (MHC) phenotype that is enhanced by removal of one gene dose of Rok or the PPE genes, including mwh. Indeed, the two major isoforms of Cmb, PA and PB, physically interact with Mwh, and the MHC phenotype observed in mwh single mutants is partially suppressed in mwh cmb double mutants. It was therefore suggested that Cmb promotes actin-based wing hair formation under control of Rok, a function that is antagonized by Mwh (Steinhauer, 2019).
It was noticed that cmb mutants are male sterile. This study found that this is due to a requirement for cmb in the germline, in the actin-based process of spermatid individualization. Detailed histochemical and electron microscopic analyses of cmb mutant testes show that early cyst formation is normal. Furthermore, ICs form normally and start to move but do not remain properly aligned, and, although they initially recruit actin at normal levels, they fail to maintain and accumulate actin during IC progression. Despite evidence that Cmb affects actin-related processes (wing hair formation in the pupa and individualization of sperm), Cmb does not directly interact with actin in co-immunoprecipitation (CoIP) or co-sedimentation assays. Furthermore, it was shown that the role of cmb during individualization is independent of and distinct from PCP genes. Significantly, Cmb interacts with the axonemal component Radial spoke protein 3 (Rsp3/CG32392), knockdown of which causes individualization phenotypes highly similar to those of cmb mutants, suggesting that Cmb coordinates IC movement with the axoneme during biogenesis of functional sperm (Steinhauer, 2019).
Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. This study shows that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. Cell type specific roles of this functional module were identified in two different tissues. While it is required for TZ assembly in all Drosophila ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. This study therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization (Lapart, 2019).
At the base of the cilium, a specific compartment, the transition zone (TZ) plays a critical role in cilium assembly and function. Many genes responsible for cilia associated diseases such as the Meckel syndrome (MKS), Joubert syndrome or nephronophthisis (NPHP) are caused by defects in proteins of the TZ. The TZ functions as a ciliary gate by sorting selected components in and out of the cilium, thus controlling the specific composition of the ciliary compartment. TZ assembly starts during the first steps of cilia formation, when the mother centriole associates with cytoplasmic vesicles before docking to the plasma membrane. Assembly of TZ proteins is spatiotemporally controlled and requires, in most organisms, at least three different protein modules namely MKS, NPHP and CEP290. Extensive genetic, biochemical studies and super resolution microscopy analysis helped to establish a hierarchy of these components and a structural view of the TZ architecture (Lapart, 2019).
Although largely conserved from worms to mammals, all TZ proteins are not conserved in all ciliated species and variations exist between model organisms. For example, the NPHP module, present in mammals, worms and protozoa, is not conserved in flies, whereas CEP290 and several members of the MKS module are conserved in most organisms. In addition to the core TZ components, several others have been identified but their precise relationships with the core known TZ components are not understood. Among these other proteins, Chibby (Cby), a conserved TZ component in vertebrates and flies, is required for cilia function both in mammals and Drosophila. In vertebrates, CBY has been shown to interact with several basal body (BB) associated proteins, such as ODF2 or CEP164 and more recently DZIP1L, DZIP1 and FAM92a or b proteins. However, the functional integration of CBY and these interactors in TZ assembly is unclear and some of those, such as Cep164 cannot likely sustain Cby function in Drosophila, as Cep164 does not seem to be expressed in Drosophila testes (Lapart, 2019).
This study shows that the unique Drosophila orthologs Dzip1 (CG13617) and Fam92 (CG6405) of respectively, vertebrate DZIP1 or DZIP1L and FAM92a or b, interact and cooperate with Cby in flies. All three proteins form a strictly ordered functional module, and cooperate in building the TZ in the two Drosophila ciliated tissues, with Dzip1 acting upstream of Fam92 and Cby. While these observations establish that Dzip1 and Fam92 localization at the TZ relies on Cep290, they reveal that Dzip1 and Fam92 exert a negative regulatory feedback loop by restraining Cep290 localization to the ciliary base (Lapart, 2019).
Last, this work reveals remarkable differences in the role of Dzip1 and Fam92 in regulating basal bodies (BB) docking between the two Drosophila ciliated tissues. Whereas, loss of Dzip1 or Fam92 does not affect basal body docking in sensory cilia, it impairs BB-membrane growth and attachment in spermatocytes. As a consequence, aberrant and premature elongation of the axoneme was observed before completion of meiosis, highlighting a primary role of the BB-membrane associated compartment for regulating axonemal microtubule elongation in Drosophila spermatocytes. These aberrant elongations mostly affect the daughter centrioles, revealing functional differences of the mother and daughter centrioles in Drosophila spermatocytes (Lapart, 2019).
In vertebrates, two orthologs have been described for Dzip1 and Fam92 and three for Cby. DZIP1, DZIP1L, FAM92a and b also interact with CBY1 in vertebrates indicating a conservation of the interacting capacities of the family members during evolution. The precise hierarchy between members of the complex has not been established in mammals, but depletion of CBY1 prevents the recruitment of FAM92a/b to the centriole and DZIP1L was shown to act upstream of CBY1 (Lapart, 2019).
In vertebrates, mutations in members of this module lead to ciliogenesis defects of various severities and with different phenotypic outcomes Dzip1 or Dzip1L KO mice die during embryogenesis, whereas Fam92a-/- mice show skeletal defects and Cby1 KO mice exhibit differentiation defects of motile ciliated epithelia. This phenotypic variability is not expected if all three proteins only act together in a functional module at the TZ, as demonstrated by work in Drosophila. This suggests that the different mouse paralogs of Cby and Fam92 may have acquired specialized ciliogenic functions in mouse. However, this study also observed that dzip11 and fam921 mutant phenotypes show small differences indicating specific functions of each proteins. For instance, in both ciliated tissues, dzip11 hypomorphic mutant phenotype is more severe than fam921, suggesting that Dzip1 has additional functions that are not solely mediated by Fam92 (Lapart, 2019).
Despite the conserved role of Dzip1, Cby and Fam92 in TZ and cilia assembly from Drosophila to humans, no homologs could be detected in the genomes of C. elegans. This could be linked to the diversification of cilia function, with both motile and immotile cilia being present from Drosophila to humans, in contrast to C. elegans, where only one subtype of cilia that are immotile is found. Another possible explanation could be that DZIP1/FAM92/CBY are associated with specific signaling or developmental functions in animals that still need to be understood (Lapart, 2019).
This work emphasizes the essential role of Dzip1 and Fam92 in building the ciliary transition zone in the two types of ciliated tissues of Drosophila. Strikingly, it also reveals tissue specific function of these proteins in priming basal body/membrane docking in Drosophila testes. This reveals intrinsic differences in the mechanisms that link basal body to membranes in Drosophila ciliated tissues. In mammals, basal body docking requires transition fibers built from the distal appendages on the mother centriole prior to docking. In Drosophila, distal appendages have not been observed on centrioles, but structures similar to transition fibers are described at the base of sensory cilia, whereas only scarce links could be observed in male germ cells. These differences could explain why destabilization of the TZ leads to basal body anchoring defects in spermatocytes, but not in sensory neurons. This structural characteristic of the spermatocyte TZ is likely to be related to its specific functional properties. Indeed, whereas the TZ is stably built at the ciliary base in sensory neurons, it shows a dynamic behavior during sperm flagella extension, separating from the basal body and migrating along the growing end of the axoneme. In addition, in spermatocytes, basal bodies have a dynamic behavior, being first docked at the plasma membrane and next internalized during meiosis. This could induce mechanical constraints on basal bodies that would increase their sensitivity to TZ disruption. In agreement with this hypothesis, when TZ maturation was challenged by modulating membrane phospholipids, BB were released from the plasma membrane during meiosis, but their initial docking was not impaired. However, this study observed by EM, that BB fail to initially dock in significant occurrences (8 among 13) in dzip11 and fam921 mutant spermatocytes, indicating that Dzip1 and Fam92 are at least involved in the initial steps of BB docking. Previous observations of another strong hypomorphic cep290mecH allele showed docked basal bodies in spermatocytes and spermatids (Basiri, 2014). In cep2900153-G4 mutant, this study did not quantify the number of docked versus undocked basal bodies in spermatocytes, but up to 76% of aberrant axonemal growth was observed, suggesting that basal body to membrane attachment is compromised in this mutant (Lapart, 2019).
Differences in the organization of the ciliary base associated with variations in the distribution and function of several centriolar and TZ proteins have been documented in Drosophila ciliated cells (Jana, 2018). However, none of these identified differences help to explain the behavioral properties of BB docking and TZ dynamics that were identified in the two ciliated Drosophila tissues. Hence, additional screens for specific factors of basal-body docking or TZ assembly either in sensory neurons or male germ cells need to be designed (Lapart, 2019).
In all the observations made in this study, there was a striking phenotypic difference between the mother and daughter centrioles in spermatocytes. In all mutants examined (i.e., dzip1, fam92 and cby) a more penetrant defect was observed on the daughter centriole than the mother. Thus, although the 2 centrioles of each pair are able to form cilia, the daughter centriole appears more sensitive to transition zone perturbations. There are no molecular explanations for these intrinsic differences of mother and daughter centrioles in spermatocytes. In Drosophila sensory neurons, Centrobin plays a critical role in maintaining the daughter centriole fate, precluding its capacity to build a cilium. In sperm cells, Centrobin is required for the formation of the C tubule, which also plays a critical role in TZ assembly. However, in spermatocytes Centrobin is equally distributed at the base of both the mother and daughter centrioles and does not show a functional asymmetry. Recently, a transient microtubule based structure that anchors the base of the mother centriole on one of the centriole pair at the onset of meiosis was identified, but its function is unknown. This structure could stabilize the mother centriole and favor its attachment to the membrane, but the function of this microtubule rootlet in Drosophila spermatocytes needs further investigations. The intrinsic difference of the mother versus daughter centrioles could also be related to the timing of centriole docking during spermatogenesis, where the mother centriole was shown to dock first. This timing difference would be sufficient to better stabilize the TZ of the mother centriole, and hence explain the phenotypic differences observed in this study. There are other situations in the animal kingdom were both mother and daughter centrioles build a cilium. Among the best studied are the bi-flagellated Chlamydomonas and the peculiar case of multiple ciliated epithelia, where numerous de novo centrioles are assembled just at the onset of ciliogenesis. Interestingly, in mammals, CBY1 was shown to play a critical role in multiple ciliated cells to allow proper docking of the multiple basal bodies to the plasma membrane. It is tempting to speculate that the increased susceptibility of multiple ciliated cells to CBY1 loss, is related to a particular status of these de novo centrioles, as was observed for daughter centrioles in male germ cells. More work will be needed to understand the molecular determinants of the mother versus daughter centriolar functional asymmetry in Drosophila male germ cells (Lapart, 2019).
Defects of TZ assembly and/or basal body docking were observed that lead to aberrant elongation of axonemal microtubules as revealed by the specific Drosophila axonemal marker CG6652-GFP. These abnormal elongation defects appear only in late G2 phase, just at the onset of meiosis, indicating that specific signals, yet to be identified, enable centrioles to start axonemal elongation at the onset of meiosis. The membrane cap restricts this capacity by inhibiting axonemal growth until the round spermatid stage, where a second signal turns on axonemal elongation and TZ migration. Among the candidate proteins recruited by the membrane cap which may coordinate axonemal assembly are microtubule depolymerizing kinesins. This membrane cap could also be involved in stabilizing centriolar capping proteins such as CP110 or CEP97, which in mammals need to be removed from centrioles to allow ciliary assembly. However, there are no clear evidence in Drosophila that these proteins need to or are specifically removed before axonemal elongation. The current observations indicate that regulation of axonemal assembly and cell cycle regulation are tightly linked in these dividing cells, but the effectors of this control still need to be identified (Lapart, 2019).
In conclusion, this work demonstrates the critical role of the conserved Dzip1/Fam92/Cby module downstream of Cep290 in initiating the assembly of the ciliary transition zone in flies. It also reveals key tissue specific differences in basal body docking pathways in Drosophila (Lapart, 2019).
Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow for the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g., Plasmodium falciparum microgametocytes and human and Drosophila melanogaster sperm). very little is known about cytoplasmic cilia assembly. This study shows that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the incorporation of these axonemal dyneins into the axoneme directly from the cytoplasm, possibly by localizing translation. This RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. It is proposed that cytoplasmic cilia assembly requires the precise localization of mRNAs encoding key axonemal constituents, allowing these proteins to incorporate efficiently into the axoneme (Fingerhut, 2020).
Cilia are important eukaryotic cellular compartments required for diverse biological functions. Recent studies have revealed that protein targeting into the proper ciliary subcompartments is essential for ciliary function. In Drosophila chordotonal cilium, where mechano-electric transduction occurs, two transient receptor potential (TRP) superfamily ion channels, TRPV and TRPN, are restricted to the proximal and distal subcompartments, respectively. To understand the mechanisms underlying the sub-ciliary segregation of the two TRPs, their localization was analyzed under various conditions. In developing chordotonal cilia, TRPN was directly targeted to the ciliary tip from the beginning of its appearance and was retained in the distal subcompartment throughout development, whereas the ciliary localization of TRPV was considerably delayed. Lack of intraflagella transport-related proteins affected TRPV from the initial stage of its pre-ciliary trafficking, whereas it affected TRPN from the ciliary entry stage. The ectopic expression of the two TRP channels in both ciliated and nonciliated cells revealed their intrinsic properties related to their localization. Taken together, these results suggest that subciliary segregation of the two TRP channels relies on their distinct intrinsic properties, and begins at the initial stage of their pre-ciliary trafficking (Kwon, 2020).
Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalized primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. This study describes phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalized from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells (Hodge, 2021).
Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. This study reports the characterisation of the Drosophila orthologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, the only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly (Lage, 2021).
Cilia are microtubule-based, hair-like organelles involved in sensory function or motility, playing critical roles in many physiological processes such as reproduction, organ development, and sensory perception. In insects, cilia are restricted to certain sensory neurons and sperms, being important for chemical and mechanical sensing, and fertility. Although great progress has been made regarding the mechanism of cilia assembly, the formation of insect cilia remains poorly understand, even in the insect model organism Drosophila. Intraflagellar transport (IFT) is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia. This study investigated the role of IFT52, a core component of IFT-B, in cilia/flagellar formation of Drosophila. Drosophila IFT52 is distributed along the sensory neuronal cilia, and is essential for sensory cilia formation. Deletion of Ift52 results in severe defects in cilia-related sensory behaviors. It should be noted that IFT52 is not detected in spermatocyte cilia or sperm flagella of Drosophila. Accordingly, ift52 mutants can produce sperms with normal motility, supporting a dispensable role of IFT in Drosophila sperm flagella formation. Altogether, IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects (Hou, 2022).
Primary cilia are non-motile sensory cell-organelle that are essential for organismal development, differentiation, and postnatal homeostasis. Their biogenesis and function are mediated by the intraflagellar transport (IFT) system. Pathogenic variants in IFT52, a central component of the IFT-B complex is associated with short-rib thoracic dysplasia with or without polydactyly 16 (SRTD16), with major skeletal manifestations, in addition to other features, this study sought to examine the role of IFT52 in osteoblast differentiation. Using lentiviral shRNA interference Ift52 was depleted in C3H10T1/2 mouse mesenchymal stem cells. This led to the disruption of the IFT-B anterograde trafficking machinery that impaired primary ciliogenesis and blocked osteogenic differentiation. In Ift52 silenced cells, Hedgehog (Hh) pathway upregulation during osteogenesis was attenuated and despite Smoothened Agonist (SAG) based Hh activation, osteogenic differentiation was incompletely restored. Further IFT52 activity was investigated in Drosophila, wherein the only ciliated somatic cells are the bipolar sensory neurons of the peripheral nervous system. Knockdown of IFT52 in Drosophila neuronal tissues reduced lifespan with the loss of embryonic chordotonal cilia, and produced severe locomotion, auditory and proprioceptive defects in larva and adults. Together these findings improve knowledge of the role of IFT52 in various physiological contexts and its associated human disorder (Guleria, 2022).
The Drosophila chordotonal neuron cilium is the site of mechanosensory transduction. The cilium has a 9 + 0 axoneme structure and is highly sub-compartmentalised, with proximal and distal zones harbouring different TRP channels and the proximal zone axoneme also being decorated with axonemal dynein motor complexes. The activity of the dynein complexes is essential for mechanotransduction. The localisation of TRP channels and dynein motor complexes during ciliogenesis was investigated. Differences in timing of TRP channel localisation correlate with order of construction of the two ciliary zones. Dynein motor complexes are initially not confined to their target proximal zone, but ectopic complexes beyond the proximal zone are later cleared, perhaps by retrograde transport. Differences in transient distal localisation of outer and inner dynein arm complexes (ODAs and IDAs) are consistent with previous suggestions from unicellular eukaryotes of differences in processivity during intraflagellar transport. Stable localisation depends on the targeting of their docking proteins in the proximal zone. For ODA, this study characterized an ODA docking complex (ODA-DC) that is targeted directly to the proximal zone. Interestingly, the subunit composition of the ODA-DC in chordotonal neuron cilia appears to be different from the predicted ODA-DC in Drosophila sperm (Xiang, 2022).
Cilia are compartmentalised organelles, and their growth (ciliogenesis) requires mechanisms for transport of cilium-resident proteins and protein complexes into the cilium and their incorporation into the ciliary membrane or onto the microtubular axoneme. Ciliogenesis proceeds by growth at the tip of the cilium. In general, at the basal body/transition zone the majority of ciliary proteins are loaded as cargoes onto 'trains' for transport along the axonemal microtubules by a dedicated process called intraflagellar transport (IFT). Anterograde movement of IFT trains from the base to the tip of the flagellum/cilium is powered by kinesin-2 motors interacting with IFT-B proteins; and retrograde movement from the tip to the base by IFT dynein-2 motors with IFT-A proteins. Most cargoes appear to be transported to the growing ciliary tip before being released for incorporation into the ciliary structure, although some cargoes may be released during anterograde transport itself (Xiang, 2022).
In motile cilia, major cargoes of IFT are the specialised axonemal dynein motor complexes that power ciliary movement. These form Inner and Outer Dynein Arms (IDAs and ODAs). The multicomponent motor complexes are very large (1–2 MDa in size), with subunits including the force-generating heavy chains, scaffolding intermediate and light-intermediate chains, as well as light chains that regulate protein interaction and microtubule-anchoring. In current understanding, especially from studies in unicellular organisms such as the biflagellate Chlamydomonas reinhardtii, dynein motor complexes are pre-assembled within the cytoplasm before transport into the cilium and docking on the axoneme. Pre-assembly requires dedicated co-chaperone assembly factors (generally known as DNAAFs). These pre-assembled complexes must move past the transition zone barrier at the base of the cilium and are then transported through the cilium by IFT trains, to which they bind with the help of adaptor proteins. Subsequent events are less clear and may vary between complexes. IFT trains may take motor cargoes to the tip of flagellum, where cargoes undergo 'maturation' and release, or they may be released during anterograde transport. The complexes are then thought to diffuse locally onto their microtubule anchoring sites, where their periodic binding is guided and stabilised by pre-bound 'docking proteins'. For ODAs, this consists of a three-subunit ODA docking complex (ODA-DC); for IDAs and other motility complexes, several proteins participate in docking, including a '96 nm molecular ruler' formed of two coiled-coil proteins (Xiang, 2022).
The dynein assembly pathway is thought to be conserved in humans and other metazoans, including Drosophila. The pathway is medically important because defects in it are a frequent cause of the inherited disease, primary ciliary dyskinesia (PCD), characterised by reduced or absent ciliary motility. However, while such studies have identified conserved components, there are also differences in metazoans. For instance, the ODA-DC seems to have a different composition from Chlamydomonas. Moreover, mechanistic evidence in metazoans for dynein motor transport and docking is sparse (Xiang, 2022).
It is increasingly apparent that ciliary proteins can be located to different regions of a cilium/flagellum for functional reasons. The mechanisms by which such sub-compartmentation is achieved during assembly are poorly known. A striking example is found in Drosophila in the ciliated dendrite of auditory/proprioceptive sensory neurons (chordotonal neurons) of the antennal sensory organ known as Johnston's Organ. These specialised 9 + 0 cilia are notable for two reasons: (1) they have the features of motile cilia, including axonemal dynein motor complexes the function of which is critical to their mechanotransduction mechanism; (2) they have structurally distinct zones with specialised functions: a sensory 'distal zone' closely connected with an external ciliary cap and containing the mechanosensory TRP channel, NompC, and a motile 'proximal zone' containing heteromeric TRPV channels (Inactive/Nanchung or Iav/Nan) as well as the axonemal dynein motors. These zones are separated by a 'ciliary dilation' of unclear function. Chordotonal neuron cilia thus serve as a model to address two questions: how are dynein motors assembled onto axonemes? How are these and other proteins targeted to zones within cilia (Xiang, 2022)?
For the latter question, localisation of the TRP channels has been extensively studied. Based on mutant analyses of IFT-A proteins (rempA and Oseg4) and IFT dynein (beethoven), retrograde IFT plays a role in TRP channel entry into the cilium and correct localisation to distinct zones. Since the ciliary dilation is also defective in these mutants, and IFT-A protein RempA accumulates at the ciliary dilation that separates the zones, it is suggested that the ciliary dilation is required for correct TRP targeting. But an alternative interpretation is that these retrograde transport genes regulate TRP targeting and ciliary dilation structure in parallel. As with other ciliary membrane proteins, localisation of the TRP channels require a Tulp protein (dTulp/king tubby) as an adaptor linking these cargoes to IFT. As in other organisms, release of TRP cargo from dTulp appears to be regulated by PIP levels in the cilium. In contrast to the TRP channels, nothing is known about how dynein motors are transported and then released and localised specifically to the proximal zone of chordotonal cilia (Xiang, 2022).
To investigate protein localisation in chordotonal cilia, this study first characterise TRP channel localisation during the time course of chordotonal ciliogenesis. Using this as a guide, qualitative differences were demonstrated in how ODA and IDA motors become localised over time, which reflect prior differences in localisation of ODA-DC and the molecular ruler complex, respectively. This study showed that ODA is targeted directly to the proximal zone via an ODA-DC containing conserved Ccdc114 and Ccdc151 subunits. In contrast, IDAs appear to be transported to the ciliary tip before stable docking in the proximal zone. This mode is reflected by IDA docking protein, Ccdc39. The data are consistent with a model in which 'outer' axonemal proteins (ODA and ODA-DC) are released from IFT trains during transport for local axonemal binding, while 'inner' axonemal proteins (including IDAs and their docking factors) are released at the ciliary tip to gain access inside the growing axoneme. In addition, the finding that the ODA docking complex appears to be an entirely different structure in chordotonal cilia compared with sperm flagella is discussed (Xiang, 2022).
This study characterised the dynamics of protein localisation to zones within the highly sub-compartmentalised chordotonal neuron cilium. Ciliogenesis in the chordotonal neurons of Johnston's Organ is prolonged, occurring over several days of pupal development. Over this time, TRP channel proteins appear to be targeted directly to their respective zones, albeit at different times. In contrast ODAs and IDAs are not confined to the proximal zone until late into ciliary maturation. Initially, ODA and IDA markers transiently accumulate ectopically at the ciliary dilation and whole distal region, respectively. Below the roles of docking factors in dynein motor targeting and possible implications and mechanisms are discussed (Xiang, 2022).
During transport, ciliary TRP channels are linked to IFT trains by Tulp adaptors, which release their cargoes in response to PIP levels within the cilium. For Drosophila chordotonal cilia, dTulp is required for correct targeting of both Iav and NompC, and this is affected by PIP signalling regulated by dInpp5e. It is not clear, however, how the different localisations of the two TRP channels are achieved. Perhaps in response to PIP levels, Iav is released from IFT trains sooner than is NompC. In this possibility, differences in drop-off from IFT trains (processivity) may provide the initial localisation. These observations suggest instead that Iav enters the cilium early in ciliogenesis and populates the proximal zone as it is being generated, while NompC enters later and populates the distal zone as it is being generated subsequently. The time of localisation in the cilium suggests a simple mechanism for TRP protein localisation to different zones based on timing of expression, but it was found that early overexpression of NompC did not cause substantial mislocalisation to the proximal zone. It remains possible that timing of entry into the cilium could be controlled, rather than timing of expression (Xiang, 2022).
Since the zones of TRP channel localisation are separated by the ciliary dilation, it has been proposed that this structure actively defines the zones and targets proteins to them. Indeed, disruption of the proteins that locate to, and are required for, the ciliary dilation also affect Iav and NompC localisation. However, this study found that ciliary dilation proteins are not localised until quite late, whereas Iav and NompC are largely targeted to their respective zones immediately upon ciliary entry. It seems more likely therefore that the role of the ciliary dilation may be in later refinement and maintenance of this initial separation. It remains unclear, therefore, what determines the initial localisations of the TRP channels. Since the cilium is ensheathed in scolopale and cap structures from an early stage, one plausible mechanism is that the surrounding cap provides an external cue for distal proteins such as NompC, e.g. via contact between the cap and ciliary tip membrane mediated by the linking protein, NompA. Dynamic changes in such contact during maturation might also explain why the extent of NompC localisation becomes more restricted at later stages of development. A second possibility is a temporal change in axonemal modification mechanisms during synthesis of the proximal and then distal zones (Xiang, 2022).
Very little is known about the dynamics of dynein motor transport and localisation in metazoans. This study found that ODA and IDA complexes enter the cilium early in ciliogenesis of chordotonal neurons. In addition to localisation in the proximal zone, both complexes show temporary accumulation beyond their final destination. For ODA, accumulation is confined to a point just beyond the proximal zone, whereas for IDA it is at the whole of the ciliary tip. It is suggested that these represent transient populations of (excess?) motor complexes that are eventually cleared from the cilium, presumably by retrograde IFT (Xiang, 2022).
Why the difference in non-proximal ODA and IDA populations? A possibility is that this reflects differences in how the complexes are released from IFT trains. This is based on comparison with observations obtained in Chlamydomonas from live imaging and dikaryon analysis (observing the assembly of complexes onto pre-existing flagella after dikaryon fusion). In these studies, most IFT cargoes are transported to the flagellar tip before release. Such a mechanism has been described for IDAs, radial spoke complexes, nexin-DRCs, and central pair components. Release at the tip is thought to occur by 'maturation' and remodelling of IFT trains, probably an IFT-A function. Thus, most of these IFT cargoes show a high processivity (low rate of dissociation from IFT trains) during transport. The complexes are then presumed to diffuse locally onto docking sites accessible at the growing tip of the cilium. Thus, it is speculated that the distal zone accumulation of IDA complexes represents continued transport to the tip throughout ciliary growth, but lack of local docking sites in the distal zone means that these complexes do not bind the axoneme stably and are eventually cleared (Xiang, 2022).
It has been suggested that transport to the tip is required for IDAs (and many other components such as radial spokes, nexin-DRC) because their docking sites in the interior of the axonemal shaft are only accessible via the tip during ciliogenesis. In contrast, ODA motor complexes can theoretically access their docking sites (if present) along the entire length of the axoneme. Thus, ODAs may have low processivity (high rate of dissociation) and so may be released from IFT trains along the length of the cilium before diffusing locally onto docking sites. This may explain the lower level of transient accumulation beyond the proximal zone-most ODA complexes are released before the trains reach the distal zone. It is not known how the release might be regulated, but it does not appear to involve the Tulp/PIP mechanism (Xiang, 2022).
Little is known of transport and localisation of the ODA docking complex, and it has not yet been demonstrated that it is an IFT cargo, although several metazoan ODA-DC proteins are known to associate with IFT proteins. In chordotonal cilia, ODA-DC (as marked by Ccdc114) is directly targeted to the proximal zone, independently of ODA complexes. This pattern of localisation suggests that ODA-DC is either a low processivity IFT cargo, like ODA motors, or that it populates the cilium via simple diffusion from the base (Xiang, 2022).
To understand how dynein motors stably dock only in the proximal zone, it will be necessary to understand how their docking sites are eventually restricted to this zone. For the 96-nm molecular ruler subunit, Ccdc39, its early distribution along the whole cilium is consistent with it being released from IFT trains only at the tip. Why it does not bind stably to the distal zone (and thereby guide the docking of IDAs) is not presently clear. It seems highly probable that the axoneme has different properties in the proximal and distal zones that allow differential binding of docking proteins, and thereafter of dynein complexes. One possibility is that there is a difference in tubulin modification in the proximal and distal axonemes. Another is that there are initial differences in so-called 'microtubule internal proteins' (MIPs). These are proteins that line the lumen of the microtubules, and therefore clearly can only incorporate within the microtubules at their growing tips. Their roles are only now being unravelled, but so far they appear to be critical in defining the binding periodicity of proteins to the outside of the microtubules. It is predicted that one or more key MIPs are incorporated exclusively into the proximal zone during axoneme extension (Xiang, 2022).
Overall, very little is known of compartmentation within cilia. Investigating dynein motor docking proteins in chordotonal cilia offers a useful model for exploring this further (Xiang, 2022).
This investigation has begun to characterise the ODA docking complex of chordotonal cilia. The composition of the ODA-DC had not been well characterized outside Chlamydomonas until recently. Evidence indicates that the metazoan complex differs from that of the alga. Of the three Chlamydomonas ODA-DC subunits (DC1, DC2 and DC3), only DC2 is conserved as CCDC114 in vertebrates. Strong evidence is provided that CCDC114 is conserved in Drosophila as CG14905 (which this study named Ccdc114). This gene is essential for ODA docking in chordotonal cilia. Moreover, it is clear that Ccdc114 is transported and localised independently of ODA complexes (Xiang, 2022).
Until recently, the remaining proteins of the metazoan ODA-DC were not fully known. ODA-DC in human respiratory cilia is suggested to include CCDC114, CCDC151, and ARMC4. Mutation of these human genes results in PCD with loss of ODAs on the axoneme. In humans, TTC25 is required for ODA-DC localisation and function, but its axonemal localisation was retained in the absence of CCDC114 function, making it unclear whether it was part of the complex. Recent ultrastructural analysis, however, has shown that mammalian ODA-DC is a pentameric complex comprising the above subunits, TTC25 and a further subunit, Calaxin/EFCAB139 (Xiang, 2022).
The evidence suggests that the subunits of this complex are conserved in Drosophila, but their requirement seems to vary between cell types. It was found that Ccdc114 and Ccdc151 are likely part of ODA-DC in chordotonal cilia, but notably, neither are expressed in sperm and so cannot account for ODA docking in sperm flagella. In sperm, Ccdc114 expression is replaced by a paralogue, wampa (CG17083). One feature of wampa mutant sperm is that their flagella lack ODAs, consistent with an ODA-DC function. Likewise, the Drosophila TTC25 homologue, CG13502 (Ttc25), is restricted to chordotonal neurons, although it may be represented in sperm by a more distantly related gene, CG15128 (Ttc25like). The single Drosophila ARMC4 homologue, gudu, is only expressed in sperm, where it is required for fertility, whereas Calaxin (CG2256) is only expressed in chordotonal neurons. It is therefore suggested that docking complex composition may differ substantially between chordotonal cilia and sperm flagella. Interestingly, this might be a common feature of metazoans: in humans CCDC114 is required for respiratory cilia but PCD patients with CCDC114 mutations appear to be fertile. It is suggested that its docking function may be replaced in sperm by the paralogue, CCDC6340, which is orthologous to wampa. It seems likely that CG14905/CCDC114 and wampa/CCDC63 form functionally equivalent gene pairs in cilia and sperm respectively (Xiang, 2022).
Why are there celltype-specific ODA-DCs in Drosophila, and perhaps in other metazoans? One possibility relates to the fact that motile cilia and sperm flagella contain distinct ODA complexes, with different heavy chains and intermediate chains, as has been shown for Drosophila and humans. Thus, each ODA-DC may be adapted for recognising and binding different ODA subtypes. Consistent with this, human CCDC114 protein directly binds the non-sperm heavy chain, DNAH9. Alternatively, or in addition, ODA-DC differences may relate to differing modes of ciliogenesis. Chordotonal ciliogenesis is compartmentalised and requires IFT, while sperm flagellogenesis occurs within the cytoplasm independently of IFT. Indeed, human TTC25 interacts with the IFT protein machinery. Phylogenetic analysis of CCDC151 homologues revealed a link between CCDC151 genes and IFT37. CCDC151 homologues are absent from organisms in which motile cilia assemble in an IFT-independent process (Xiang, 2022).
Cilia are cellular projections that perform sensory and motile functions in eukaryotic cells. A defining feature of cilia is that they are evolutionarily ancient, yet not universally conserved. In this study, This study used the resulting presence and absence pattern in the genomes of diverse eukaryotes to identify a set of 386 human genes associated with cilium assembly or motility. Comprehensive tissue-specific RNAi in Drosophila and mutant analysis in C. elegans revealed signature ciliary defects for 70-80% of novel genes, a percentage similar to that for known genes within the cluster. Further characterization identified different phenotypic classes, including a set of genes related to the cartwheel component Bld10/CEP135 and two highly conserved regulators of cilium biogenesis. It is proposed that this dataset defines the core set of genes required for cilium assembly and motility across eukaryotes and presents a valuable resource for future studies of cilium biology and associated disorders (Dobbelaere, 2023).
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