Centrosomal/Centriolar Proteins and cilia and flagella

The Interactive Fly

Centrosomal/Centriolar proteins and cilia and flagella


  • Structure and duplication of the centrosome
  • For additional information about cilia and flagella see Antenna, auditory apparatus, and sound sensation and Testis and spermatogenesis
  • Asymmetric inheritance of mother versus daughter centrosome in stem cell division
  • Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells
  • A role for a novel centrosome cycle in asymmetric cell division
  • Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation
  • Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation
  • Centrosome misorientation reduces stem cell division during ageing
  • Asymmetric centrosome inheritance maintains neural progenitors in the mammalian neocortex
  • Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules
  • Drosophila Ana2 is a conserved centriole duplication factor
  • The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor
  • Klp10A, a microtubule-depolymerizing kinesin-13, cooperates with CP110 to control Drosophila centriole length
  • Dynamic interplay of spectrosome and centrosome organelles in asymmetric stem cell divisions
  • Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts
  • Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division
  • Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells
  • Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos
  • The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts
  • A molecular mechanism of mitotic centrosome assembly in Drosophila
  • The Drosophila centriole: conversion of doublets to triplets within the stem cell niche
  • A Par-1-Par-3-centrosome cell polarity pathway and its tuning for isotropic cell adhesion
  • Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling
  • Loss of function of the Drosophila Ninein-related centrosomal protein Bsg25D causes mitotic defects and impairs embryonic development
  • Centrosome and spindle assembly checkpoint loss leads to neural apoptosis and reduced brain size
  • A homeostatic clock sets daughter centriole size in flies

    Cilia and Flagella
  • Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division
  • The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly
  • Assembly and persistence of primary cilia in dividing Drosophila spermatocyte
  • A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids
  • Centriole remodeling during spermiogenesis in Drosophila
  • BLD10/CEP135 is a microtubule-associated protein that controls the formation of the flagellum central microtubule pair
  • Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila
  • The Drosophila homologue of Rootletin is required for mechanosensory function and ciliary rootlet formation in chordotonal sensory neurons
  • Drosophila sensory cilia lacking MKS-proteins exhibit striking defects in development but only subtle defects in adults
  • Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry
  • Time-lapse live-cell imaging reveals dual function of Oseg4, Drosophila WDR35, in ciliary protein trafficking
  • Centriole
  • Pericentriolar material
  • Gamma tubulin ring complex
  • Required for centrosome function
  • Required for formation or function of cilia and flagella

    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).

    A role for a novel centrosome cycle in asymmetric cell division

    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).

    Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation

    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).

    Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation

    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).

    Centrosome misorientation reduces stem cell division during ageing

    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 centrosome inheritance maintains neural progenitors in the mammalian neocortex

    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).

    Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules

    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).

    Drosophila Ana2 is a conserved centriole duplication factor

    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).

    The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor

    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, a microtubule-depolymerizing kinesin-13, cooperates with CP110 to control Drosophila centriole length

    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).

    Dynamic interplay of spectrosome and centrosome organelles in asymmetric stem cell divisions

    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).

    Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts

    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).

    Centrosome-dependent asymmetric inheritance of the midbody ring in Drosophila germline stem cell division

    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).

    Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells

    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

    Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos

    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).

    The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts

    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).

    A molecular mechanism of mitotic centrosome assembly in Drosophila

    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).

    The Drosophila centriole: conversion of doublets to triplets within the stem cell niche

    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).

    A Par-1-Par-3-centrosome cell polarity pathway and its tuning for isotropic cell adhesion

    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).

    Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling

    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).

    Loss of function of the Drosophila Ninein-related centrosomal protein Bsg25D causes mitotic defects and impairs embryonic development

    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).

    Centrosome and spindle assembly checkpoint loss leads to neural apoptosis and reduced brain size

    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).

    A homeostatic clock sets daughter centriole size in flies

    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).

    Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division

    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).

    The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly

    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).

    Assembly and persistence of primary cilia in dividing Drosophila spermatocyte

    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).

    A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids

    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).

    Centriole remodeling during spermiogenesis in Drosophila

    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).

    BLD10/CEP135 is a microtubule-associated protein that controls the formation of the flagellum central microtubule pair

    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).

    Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila

    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).

    The Drosophila homologue of Rootletin is required for mechanosensory function and ciliary rootlet formation in chordotonal sensory neurons

    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).

    Drosophila sensory cilia lacking MKS-proteins exhibit striking defects in development but only subtle defects in adults

    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).

    Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry

    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).

    Time-lapse live-cell imaging reveals dual function of Oseg4, Drosophila WDR35, in ciliary protein trafficking

    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).

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