InteractiveFly: GeneBrief

anastral spindle 3: Biological Overview | References

Gene name - anastral spindle 3

Synonyms -

Cytological map position - 48F11-49A1

Function - biogenesis of centrioles

Keywords - a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation - actively promotes cell survival - one of the main functions of Ana3 is to stabilize Sol narae for cell survival and proliferation - Ana3 may be one of the proteins that respond to irradiation at the front line - During centriole biogenesis, Ana3 and Rcd4 are sequentially loaded on the newly formed centriole and are required for centriole-to-centrosome conversion through recruiting the Cep135-Ana1-Asterless complex

Symbol - ana3

FlyBase ID: FBgn0266111

Genetic map position -

NCBI classification - Rotatin, an armadillo repeat protein, centriole functioning

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Ana3 orthologs: Biolitmine

The centrosome is the main microtubule-organizing center in animal cells. It comprises of two centrioles and the surrounding pericentriolar material. Protein organization at the outer layer of the centriole and outward has been studied extensively; however, an overall picture of the protein architecture at the centriole core has been missing. This paper reports a direct view of Drosophila centriolar proteins at ~50-nm resolution. This reveals a Sas6 ring at the C-terminus, where it overlaps with the C-terminus of Cep135. The ninefold symmetrical pattern of Cep135 is further conveyed through Ana1-Asterless axes that extend past the microtubule wall from between the blades. Ana3 and Rcd4, whose termini are close to Cep135, are arranged in ninefold symmetry that does not match the above axes. During centriole biogenesis, Ana3 and Rcd4 are sequentially loaded on the newly formed centriole and are required for centriole-to-centrosome conversion through recruiting the Cep135-Ana1-Asterless complex. Together, these results provide a spatiotemporal map of the centriole core and implications of how the structure might be built (Tian, 2021).

The centrosome has multiple crucial functions, including the assembly of the mitotic spindle and establishing the axis of cell division. It comprises two principal components: a pair of orthogonally arranged centrioles and the surrounding pericentriolar material (PCM). Centrioles are stable cylindrical structures comprising nine microtubule blades arranged at the end of nine spokes that radiate from a central hub. During each cell cycle, the centriole pair disengages at the mitotic exit, allowing the new centrioles (or daughter centrioles) to gradually assemble next to each preexisting centriole (the mother centriole). A mother centriole serves as a recruitment and assembly scaffold for the PCM proteins to form spindle poles in mitosis; in many cell types, it also provides a template for cilium or flagellum assembly during cell quiescence, forming a crucial organelle for chemical sensation, signal transduction, locomotion, and so forth. Centrosome defects have been related to a wide range of human diseases, including cancer, microcephaly, and a group of disorders collectively known as the 'ciliopathies' (Tian, 2021).

Understanding how the centrosome functions requires knowledge of its protein composition and organization. The centrosome is composed of >100 different proteins. Their architectural arrangement has begun to be systematically examined since the application of superresolution microscopy. Using 3D structured illumination microscopy (3D-SIM), distinct concentric domains within a centrosome have been documented (e.g., zones I-V of the Drosophila centrosome) and that the PCM has a conserved, ordered structure. Protein organization at several compartments of the centrosome, such as the distal and subdistal appendages, the transition zone, the centrosome linker, and the longitudinal axis of the centriole, has also been studied via 3D-SIM, stimulated emission depletion (STED) microscopy, or stochastic optical reconstruction microscopy. Meanwhile, proteins at the core of the centriole remain largely unresolved. This cartwheel region, revealed as zone I by 3D-SIM, contains the central hub of ~22-nm diameter and the nine spokes that determine the ninefold symmetrical feature of the centriole (Tian, 2021).

Drosophila cultured cells present a consistent model for the study of the centriole core because, contrary to the vertebrate centrosome, the cartwheel persists in the mature centriole. The centriole is composed of doublet microtubules arranged in a ninefold symmetrical cylinder, which is ∼200 nm wide and long and has a cartwheel formation along the entire length. This study first determined which proteins known to be required for Drosophila centriole duplication are the components of the centriole core. A direct view of these proteins is presented at ~50-nm resolution, and a timing order of their assembly is presented using several superresolution techniques. These revealed a ninefold radial scaffold comprising Spindle assembly abnormal 6 (Sas6), Centrosomal protein 135kDa (Cep135), Anastral spindle 1 (Ana1), and Asterless (Asl), as well as concentric toroids formed by Anastral spindle 3 (Ana3) and Reduction in Cnn dots 4 (Rcd4), two novel core centriolar components that are also organized in ninefold symmetry. During centriole biogenesis, Ana3 is recruited to the newly formed daughter centriole later than Sas6 but before Rcd4 and Cep135. These findings thus provide a spatiotemporal map of the centriole core and a model of how the proteins might interact to build the structure (Tian, 2021).

These data reveal the spatiotemporal organization of the proteins at the core region of the Drosophila centriole (see Schematics depicting the lateral organization of centriole core). By superimposing the current measurements to the electron cryotomography data of the Trichonympha, Chlamydomonas, and Drosophila centrioles, this study found that Cep135 overlaps with the C-terminus of Sas6 on the spokes via its C-terminus and extends to the pinheads via the N-terminus. Ana1 localizes from the pinheads to the outer edge of the doublet microtubules. Asl slightly overlaps with the doublet microtubules and extends into PCM in a ninefold manner. It is proposed that the core region of the centriole is composed of two dimensions. One is the ninefold radial dimension that is established by elongated molecules overlapping through their adjacent termini: Sas6, Cep135, Ana1, and Asl. They likely constitute the spoke-pinhead axes and further transmit the ninefold symmetrical geometry to the microtubule wall and into the core PCM. The other is a circular dimension established by a group of compact proteins that are also arranged in ninefold symmetry: Ana3, Rcd4, and possibly Ana2. They likely decorate the radial axes and provide the physical support for the ninefold configuration (Tian, 2021).

Previous work has shown that Cep135, Ana1, and Asl form a complex that is responsible for the centriole-to-centrosome conversion), the final stage in the assembly of the daughter centriole that converts it into a mother centriole able to duplicate. With improved spatial resolution, this study shows that the three proteins are each organized in ninefold manner, reinforcing the idea they are the bona fide components of the spoke-pinhead scaffold. The ninefold radial axes then extend past the centriole microtubule wall via the C-terminus of Ana1, which is positioned between the microtubule blades. Recently, an electron cryotomography study showed that, between adjacent microtubule blades, there are ninefold amorphous brushlike structures in the Drosophila S2 centriole. This study suggests that it could contain Ana1 and Asl, both of which exhibit ninefold symmetry at this region (Tian, 2021).

These findings allocate a role to Drosophila Ana3 and Rcd4, previously known from genome-wide RNAi screens to be required for centriole duplication. Ana3 was later reported to be responsible for the structural integrity of centrioles and basal bodies and for centriole cohesion in the Drosophila testes. This study now provides evidence that both Ana3 and Rcd4 are core centriolar components, localizing to the region where Cep135 is. The N-terminus of Ana3 localizes closest to the center of the centriole, followed by the C-termini of Ana3 and Rcd4 and the N-terminus of Rcd4. Both Ana3 and Rcd4 are organized in ninefold symmetry but seem to be positioned in axes that are not in line with the Cep135-Ana1-Asl complex. Spatial overlapping of Ana3 and Rcd4 indicates these two proteins might interact, which has recently been reported (Panda, 2020) and is conserved to their human counterparts, RTTN and PPP1R35. Depletion of either Ana3 or Rcd4 leads to failure in loading the Cep135-Ana1-Asl complex during centriole biogenesis and thus causes defects in centriole-to-centrosome conversion and the reduction of the centrosome number. This pathway is also conserved in human cells, where PPP1R35 was reported to promote centriole-to-centrosome conversion upstream of Cep295 (human homologue of Ana1) and RTTN and PPP1R35 serve as upstream effectors of Cep295 in mediating centriole elongation (Tian, 2021).

Taken together, these data provide an overall picture of the protein architecture at the centriole core and implications of how the ninefold symmetrical structure might be built. Knowing the spatiotemporal restraints of individual centriolar components will guide the immediate study of the molecular interaction partners and understanding of their functions. Meanwhile, it would also provide information for a higher-resolution approach, including cryo-EM, to eventually obtain a 3D map of the centriole (Tian, 2021).

Anastral spindle 3/Rotatin stabilizes Sol narae and promotes cell survival in Drosophila melanogaster

Apoptosis and compensatory proliferation, two intertwined cellular processes essential for both development and adult homeostasis, are often initiated by the mis-regulation of centrosomal proteins, damaged DNA, and defects in mitosis. Fly Anastral spindle 3 (Ana3) is a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation. This study reports that ana3m19 is a suppressor of lethality induced by the overexpression of Sol narae (Sona), a metalloprotease in a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family. ana3m19 has a nonsense mutation that truncates the highly conserved carboxyl terminal region containing multiple Armadillo repeats. Lethality induced by Sona overexpression was completely rescued by knockdown of Ana3, and the small and malformed wing and hinge phenotype induced by the knockdown of Ana3 was also normalized by Sona overexpression, establishing a mutually positive genetic interaction between ana3 and sona. p35 inhibited apoptosis and rescued the small wing and hinge phenotype induced by knockdown of ana3. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies and reduced the number of dying cells, demonstrating that Ana3 actively promotes cell survival. Knockdown of Ana3 decreased the levels of both intra- and extracellular Sona in wing discs, while overexpression of Ana3 in S2 cells dramatically increased the levels of both cytoplasmic and exosomal Sona due to the stabilization of Sona in the lysosomal degradation pathway. It is proposed that one of the main functions of Ana3 is to stabilize Sona for cell survival and proliferation (Cho, 2021).

The ability to resist and recover from external stresses is important for all living organisms that face stresses such as heat, reactive oxygen species, and irradiation during development and in the adult stage. Damaged cells need to be removed by apoptosis and replaced with newly formed cells by compensatory proliferation. The wing imaginal disc of Drosophila melanogaster is the primordium of the adult wing, and shows a very low level of cell death during normal larval development. In contrast, it shows extensive cell death by environmental stresses, and yet can develop into a normal wing even after 40% to 60% cell death (Cho, 2021).

The centrosome consists of a pair of centrioles and pericentriolar materials (PCMs). DNA damage and mitotic defects cause the overduplication of centrosomes and the formation of multipolar spindles, leading to mitotic failure and cell death. Defects in PCMs interrupt spindle assembly and activate the spindle assembly checkpoint. Fly Anastral spindle 3 (Ana3) is a PCM responsible for the cohesion of centrioles, prevention of premature centriolar segregation, and formation of basal bodies (Stevens, 2009). Ana3 and its mammalian homolog Rotatin (RTTN) contain multiple Armadillo repeats known to interact with Wnt signaling components and potentiate the Wnt pathway (Song, 2003). Wnt has critical roles in growth, development, adult homeostasis, and regeneration. Ana3 and RTTN are also important for the formation of cilia and basal bodies (Kia, 2012; Stevens, 2009). Loss of RTTN causes polymicrogyria (PMG), situs inversus, isomerism, and heterotaxia in humans (Cho, 2021).

From a previous genetic screen, 28 mutants were found to be as responsible for the suppression of lethality caused by the overexpression of Sol narae (Sona) (Han, 2020). The present study identified one of suppressors as ana3m19. Sona is a member of a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family (Kim, 2016). Most ADAMTSs are secreted proteases that cleave components in the extracellular matrix, and their malfunctions result in multiple diseases including cancer. Sona is positively involved in Wingless (Wg) signaling, and secreted by both the exosomal secretion pathway and Golgi transport (Kim, 2016; Won, 2019). Sona cleaves the linker region of extracellular Wg and generates a new functional form of Wg that is specialized in cell proliferation (Cho, 2021).

Sona is important for cell survival, with the level of Sona correlated with the extent of cell survival (Tsogtbaatar, 2019). Cells expressing a high level of sona are cell autonomously resistant to γ-ray irradiation, while Sona secreted from these cells induces Cyclin D (Cyc D) in the neighboring cells for cell survival and proliferation in a non-cell autonomous manner. Interestingly, Wg-CTD but not full-length Wg induces Cyc D, which demonstrates that Sona is involved in intercellular communication to support the normal development of damaged tissues by regulating Wg signaling. Consistent with this, sona suppressors such as wntless, arrow, pou domain motif 3, and archipelago are related to Wg signaling (Cho, 2021).

This paper reports that Ana3 is also important for cell survival. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies, and the amount of Ana3 correlated with the extent of organism survival under irradiation. The level of Ana3 in wing discs was significantly increased by 1 h after irradiation, indicating that Ana3 may be one of the proteins that respond to irradiation at the front line. Ana3 expressed in S2 cells increased the level of both intracellular and secreted Sona by negatively regulating the lysosomal degradation pathway, which is consistent with the finding of ana3m19 as a sona suppressor. These data demonstrate a new role of Ana3 in the stabilization of Sona (Cho, 2021).

This paper reports that the ana3m19 mutant is a suppressor of Sona-induced lethality. Fly ana3 has a positive genetic interaction with sona that encodes a metalloprotease involved in Wnt signaling, establishing a potential link between Ana3/RTTN and Wnt signaling. Ana3/RTTN is a peripheral member of the centrosome complex whose malfunction leads to embryonic lethality in both ana3 mutant flies and RTTN knockout mice. Both Ana3 and Sona are involved in cell survival and resistance to irradiation. Consistent with their positive genetic interaction and functional similarity, this study found that Ana3 stabilizes Sona and increases the level of Sona in both wing discs and S2 cells. The truncated region in ana3m19 protein is the most conserved region in Ana3/RTTN homologs, suggesting that this region plays a key role in stabilizing Sona. Some PMG mutations have also been identified in the Armadillo repeats in this carboxyl region of RTTN protein (Cho, 2021).

Both lethality and the small wing phenotype induced by Sona overexpression were completely rescued by knockdown of Ana3, suggesting that one of the main functions of Ana3 is to stabilize Sona. It is worth noting that a degradation of Sona occurs in the lysosome but not in the proteasome complex, as well as that another sona suppressor Arr also stabilizes Sona (Han, 2020). Since the original genetic screen was aimed at identifying suppressors that reduce Sona activity, it makes sense that both ana3 and arr mutants are identified as sona suppressors. Interestingly, Ana3 dramatically increased the level of exosomal Sona but not soluble Sona. This suggests that Ana3 stabilizes Sona in the exosomal secretion pathway that is interconnected with the lysosomal degradation pathway and the endosomal pathway but not in Golgi transport (Cho, 2021).

The loss of ana3 induced cell death, which is a common phenotype of centrosome components. Interestingly, overexpression of Ana3 enhanced the survival rate of irradiated flies, with wing discs showing the increased level of Ana3 1 h after irradiating the larvae, indicating that signals initiated by irradiation increase the level of Ana3 to prevent cell death. Since Ana3 stabilizes Sona, and knockdown of ana3 completely rescues the lethality caused by overexpressed Sona, the ability of Ana3 in promoting cell survival may stem from stabilized Sona. Previous work has shown that Sona-expressing cells are resistant to irradiation in a cell autonomous manner, and Sona secreted from these cells enables neighboring cells to survive and proliferate in a non-cell autonomous manner (Tsogtbaatar, 2019). Thus, it is possible that the increased level of Ana3 by irradiation contributes to increasing the level of Sona, which in turn functions to promote cell survival in both cell-autonomous and non-cell autonomous manners (Cho, 2021).

Extracellular Sona cleaves Wg and generates Wg-CTD that increases the level of Cyc D for initiating cell cycles (Won, 2019). Cyc D1 in mammalian cells promotes cell proliferation in response to mitogens, but overexpression of Cyc D1 leads to centrosome amplification, deregulation of the mitotic spindle, and chromosome abnormalities. Cyc D1 is oncogenic in many human cancer cells because it contributes to malignant transformation, with centrosome amplification by ras oncogene depending on Cyc D1. The link between fly Cyc D, Sona, and Wg-CTD, as well as the association of many components in Wnt signaling such as Disheveled, Armadillo/β-catenin, Axin, and Arrow/LRP6 with centrosomes, suggests that Sona may participate in the regulation of centrosomal duplication for the initiation of cell cycles (Cho, 2021).

Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis

Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. This study shows that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. Neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation (Panda, 2020).

Hypomorphic and amorphic mutations of the Rcd4 gene generated in Drosophila has allowed demonstration of its requirement for centriole duplication and the correct development of cilia in the neurosensory chordotonal organs. Basal bodies and cilia are completely absent in the chordotonal organs of the rcd42-null flies that consequently show extreme loss of coordination. The partial loss of cilia in the rcd41 hypomorph suggests that centriole duplication was not completed during the final rounds of the division cycle in the scolopidium cell lineage of the femoral chordotonal organs. This conclusion gains support from the absence of centrobin staining of the basal bodies of rcd41 mutant scolopdia, indicating failure to produce daughter centrioles in the mitoses preceding differentiation. As a consequence, instead of the pair of cilia present in WT scolopidia, there was usually either a single cilium or none at all in rcd41 scolopidia. When present, the cilia showed defects in symmetry and structure extending along their length and often made abnormal associations with the ciliary membrane. These findings reveal a requirement for Rcd4 to generate basal bodies that are structurally able to template the formation of cilia and associate with cell membranes correctly (Panda, 2020).

In accord with the requirement for centriole duplication apparent from the rcd41 and rcd42 mutant phenotypes, embryos derived from rcd1 mothers fail in development as a result of mitotic defects associated with loss of centrosomes. This could not be assessed directly with rcd42 females as they were too severely uncoordinated to be able to mate. However, following rescue of Rcd4 function in the nervous system, it was possible to generate perfectly coordinated and motile females that were unable to produce viable offspring. Such females generated embryos in which failure of the centriole duplication cycle led to massive mitotic abnormalities in early syncytial embryos, accounting for the maternal effect lethality (Panda, 2020).

rcd41 encodes an N-terminally truncated protein comprising only the 68 C-terminal amino acids of the 199 amino acid protein. The hypomorphic nature of this rcd41 mutant allele indicates that there is some residual function in the approximate C-terminal third of the Rcd4 protein produced by this mutant. It is this C-terminal part of Rcd4 that shares the greatest homology with its human counterpart protein phosphatase 1 regulatory subunit 35 (PPP1R35), with which it shows overall 24% similarity. PPP1R35 is annotated as a protein phosphatase 1 (PP1) regulatory subunit and is reported to bind and inhibit PP1. However, it was not possible to identify any PP1 in proximity to PPP1R35 in BioID assays, and the PP1 interacting motif was found not to be essential for centriole function in human cells. In a similar vein, no PP1 isoform was found to coaffinity-purify with Rcd4. Ana3 was identified as its copurifying partner in extracts of cultured Drosophila cells. This accords with the identification of the Ana3 homologue, Rotatin, in proximity to PPP1R35 by BioID. This, together with the coimmunoprecipitation and colocalization of Rotatin and PPP1R35 by 3D-SIM, led Sydor (2018) to suggest that Rotatin and PPP1R35 form a complex. However, this was not supported by direct evidence of any physical interaction. The current study's demonstration that purified Rcd4 and Ana3 proteins can form complexes in vitro provides direct evidence for a physical interaction between the counterpart proteins in Drosophila. Moreover, Rcd4 specifically binds to the C-terminal half of Ana3, and it appears to interact more strongly with Ana3-C than with full-length Ana3. Although the underlying reason for this is unknown, wit is speculated that full-length Ana3 might adopt an inhibitory conformation that partly masks the Rcd4 binding region (Panda, 2020).

Observations of centrioles in cultured Drosophila cells by 3D-SIM placed the Rcd4 protein as a component of the distal part of zone I. It is localized close to Sas6, which forms the molecular skeleton of the cartwheel that is assembled upon the initiation of procentriole formation. As the centrioles in cultured Drosophila cells are extremely short, very little can be said about the positioning of Rcd4 along the proximo-distal axis of the centriole other than that it lies in a domain between Sas6 at the proximal end and the distal cap formed by Cp110 and its interacting proteins. However, it was not possible to confirm its prominent localization in the distal part of zone I in the elongated centrioles of primary spermatocytes. This is in an analogous position to PPP1R35, which was described by Sydor (2018) to lie in the proximal lumen of the centriole above the cartwheel (Panda, 2020).

Not only are the Sas6:Ana2 and Rcd4:Ana3 complexes localized in distinct parts of zone I, but whereas Sas6 and Ana2 are recruited upon procentriole formation immediately after procentriole disengagement during anaphase/telophase, Rcd4 and Ana3 are recruited at a later stage during interphase. Ana3 is the first to be recruited, and is present on 73% of interphase procentrioles, followed by Rcd4, which is present on 39% of procentrioles. This study indicates not only that Ana3 is recruited to the procentriole ahead of Rcd4, but also that Rcd4 requires Ana3 to be able to load. In this respect, the loading dependency differs from human cells, where loading of the two corresponding proteins appears to be interdependent (Sydor, 2018). Whether this apparent discrepancy is due to functional differences of the homologous proteins between the different model systems remains to be elucidated (Panda, 2020).

The recruitment of Rcd4 takes place around the same time as Cep135, which is present on 30% of interphase procentrioles and which is required for subsequent recruitment of Ana1. The recruitment of Ana1 ahead of mitotic entry marks the onset of the conversion of the daughter centriole to a centrosome during mitotic progression in both Drosophila and human cells. Both Ana3 and Rcd4 are also required for Ana1 recruitment in Drosophila cells. This concurs with findings in human cells where Chen (2017) has shown that loss of Rotatin, the human counterpart of Ana3, completely prevented loading of Ana1's counterpart, Cep295, and consequently all of the centriole proteins that load late in the duplication cycle. Similar results were reported as a consequence of depletion of PPP1R35. Together, this suggests that the Rcd4:Ana3 heterodimeric complex may serve as a platform to recruit or anchor Ana1, through a role in setting up correct centriole structure to enable subsequent centriole to centrosome conversion in the mitoses of somatic cells rather than directly in conversion per se (Panda, 2020).

Whereas one group have emphasized a requirement for PPP1R35 for centriole to centrosome conversion in human cells (Fong, 2018), another group emphasized the requirement for PPP1R35 in concert with Rotatin to regulate centriole length (Sydor, 2018). The short nature of centrioles in somatic Drosophila cells makes it difficult to assess the requirement of Rcd4 for centriole elongation. Therefore, the focus of this study turned to the giant centrioles of the fly primary spermatocytes. While the rcd4-null allele has some effect on the centriole duplication cycle of the male germ line cells, it does not affect the length of the remaining centrioles. This accounts for the fertility of rcd42 males and is a striking indication that, although essential in somatic tissues, the Rcd4 protein is not absolutely required for centriole or basal body formation in the male germ line. To assess whether Ana3 is required for male germ line development, CRISPR/Cas9-mediated mutagenesis was used to generate an ana3 null allele. Such ana3 nulls are male sterile and display extremely few but highly aberrant centrioles in the male germ line. These findings concur with a previous report (Stevens, 2009) and indicate that, in contrast to Rcd4, Ana3 is absolutely required to generate centrioles for spermatogenesis (Panda, 2020).

In conclusion, the combined findings point to a need for the coordinated recruitment of Ana3 and then Rcd4 to enable the later stages of centriole assembly, which are a precondition for centriole to centrosome conversion in somatic cells. Such failures are less extreme in rcd41 mutants that have enough residual function to generate fChOs with single maternal centriole-derived basal bodies sufficient to generate aberrant cilia, in contrast to the rcd42 null, where no axonemal microtubules form in the fChOs. The disparity in requirements for Rcd4 in somatic tissues and in the male germ line is striking, and it will be a topic of future interest to determine why there are similar requirements for Ana3 and Rcd4 for development of procentrioles in somatic cells but different requirements in spermatogenesis (Panda, 2020).

Ana3 is a conserved protein required for the structural integrity of centrioles and basal bodies

Recent studies have identified a conserved "core" of proteins that are required for centriole duplication. A small number of additional proteins have recently been identified as potential duplication factors, but it is unclear whether any of these proteins are components of the core duplication machinery. This study investigated the function of one of these proteins, Drosophila melanogaster Ana3. Ana3 is shown to be present in centrioles and basal bodies, but its behavior is distinct from that of the core duplication proteins. Most importantly, Ana3 was found to be required for the structural integrity of both centrioles and basal bodies and for centriole cohesion, but it is not essential for centriole duplication. This study shows that Ana3 has a mammalian homologue, Rotatin, that also localizes to centrioles and basal bodies and appears to be essential for cilia function. Thus, Ana3 defines a conserved family of centriolar proteins and plays an important part in ensuring the structural integrity of centrioles and basal bodies (Stevens, 2009).

Functions of Ana3 orthologs in other species

PPP1R35 is a novel centrosomal protein that regulates centriole length in concert with the microcephaly protein RTTN

Centrosome structure, function, and number are finely regulated at the cellular level to ensure normal mammalian development. This study characterize PPP1R35 as a novel bona fide centrosomal protein and demonstrate that it is critical for centriole elongation. Using quantitative super-resolution microscopy mapping and live-cell imaging this study shows that PPP1R35 is a resident centrosomal protein located in the proximal lumen above the cartwheel, a region of the centriole that has eluded detailed characterization. Loss of PPP1R35 function results in decreased centrosome number and shortened centrioles that lack centriolar distal and microtubule wall associated proteins required for centriole elongation. This study further demonstrated that PPP1R35 acts downstream of, and forms a complex with, RTTN, a microcephaly protein required for distal centriole elongation. Altogether, this study identifies a novel step in the centriole elongation pathway centered on PPP1R35 and elucidates downstream partners of the microcephaly protein RTTN (Sydor, 2018).

Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, this study found that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly. Mutations in many centriolar protein-encoding genes cause primary microcephaly. This study shows that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles (Chen, 2017).

RTTN mutations link primary cilia function to organization of the human cerebral cortex

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. This study has identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. This study shows that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, this study has shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals (Kia, 2012).


Search PubMed for articles about Drosophila Anastral spindle 3

Chen, H. Y., Wu, C. T., Tang, C. C., Lin, Y. N., Wang, W. J. and Tang, T. K. (2017). Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles. Nat Commun 8(1): 247. PubMed ID: 28811500

Cho, D. G., Lee, S. S. and Cho, K. O. (2021). Anastral spindle 3/Rotatin stabilizes Sol narae and promotes cell survival in Drosophila melanogaster. Mol Cells 44(1): 13-25. PubMed ID: 33510049

Fong, C. S., Ozaki, K. and Tsou, M. B. (2018). PPP1R35 ensures centriole homeostasis by promoting centriole-to-centrosome conversion. Mol Biol Cell 29(23): 2801-2808. PubMed ID: 30230954

Han, J. H., Kim, Y. and Cho, K. O. (2020). Exosomal arrow (Arr)/lipoprotein receptor protein 6 (LRP6) in Drosophila melanogaster increases the extracellular level of Sol narae (Sona) in a Wnt-independent manner. Cell Death Dis 11(11): 944. PubMed ID: 33139721

Kia, S. K., Verbeek, E., Engelen, E., Schot, R., Poot, R. A., de Coo, I. F., Lequin, M. H., Poulton, C. J., Pourfarzad, F., Grosveld, F. G., Brehm, A., de Wit, M. C., Oegema, R., Dobyns, W. B., Verheijen, F. W. and Mancini, G. M. (2012). RTTN mutations link primary cilia function to organization of the human cerebral cortex. Am J Hum Genet 91(3): 533-540. PubMed ID: 22939636

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Biological Overview

date revised: 18 February 2022

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.