InteractiveFly: GeneBrief

anastral spindle 1: Biological Overview | References

Gene name - anastral spindle 1

Synonyms -

Cytological map position - 96A6-96A6

Function - centriole-to-centrosome conversion

Keywords - centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 and Asterless onto daughter centrioles during mitotic progression in both Drosophila and human - Ana1 helps to recruit Polo to mother centrioles in Drosophila. If recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2.

Symbol - ana1

FlyBase ID: FBgn0262167

Genetic map position - chr3R:24,530,576-24,536,395

NCBI classification - 442620921

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

GENE orthologs: Biolitmine
Recent literature
Wong, S. S., Wilmott, Z. M., Saurya, S., Alvarez-Rodrigo, I., Zhou, F. Y., Chau, K. Y., Goriely, A. and Raff, J. W. (2022). Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly. Embo j 41(11): e110891. PubMed ID: 35505659
Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. This study shows that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process-peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. It is proposed that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.

Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and it has been shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. This study shows that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. It is concluded that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly (Alvarez-Rodrigo, 2021).

Polo kinase (PLK1 in mammals) is an important cell cycle regulator. During mitosis, it is recruited to several locations in the cell -- such as centrosomes, kinetochores and the cytokinesis apparatus -- where it performs multiple functions. PLK1 is usually recruited to these locations by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs in target proteins. Mutating the first serine in the PBD-binding motif to threonine strongly reduces PBD binding in vitro and in vivo (Alvarez-Rodrigo, 2021).

PLK1 has several key functions at centrosomes. These organelles are important microtubule (MT) organising centres that form around a pair of centrioles (comprising a mother and daughter centriole) when the mother recruits a matrix of pericentriolar material (PCM) around itself. During interphase, centrosomes organise relatively little PCM, but as cells prepare to enter mitosis the PCM expands dramatically in a process termed centrosome maturation. PLK1 is an essential driver of this process, and several PCM proteins have been identified as PLK1 targets. In vertebrate cells, PLK1 phosphorylates pericentrin, which cooperates with CDK5RAP2 (also known as Cep215) to promote mitotic PCM assembly, whereas in flies and worms Polo/PLK1 kinases phosphorylate Cnn and SPD-5 (functional homologues of CDK5RAP2), respectively, which allows these proteins to assemble into a PCM scaffold around the mother centriole that recruits other PCM proteins (Alvarez-Rodrigo, 2021).

Towards the end of mitosis, the mother and daughter centrioles disengage from each other. PLK1 is essential for disengagement and also for the subsequent maturation of the daughter centriole into a new mother centriole that is itself capable of duplicating and organising PCM. The old mother (OM) and new mother (NM) centrioles then both duplicate during S phase by nucleating the assembly of a daughter centriole on their side. PLK1 is not essential for centriole duplication per se, but it is required for the growth of the centriole MTs that occurs during G2, at least in human cells, and for the subsequent maturation of the daughter centriole into a new mother centriole. After duplication in S phase, the two centrosomes (each now comprising a duplicated centriole pair) are held together by a linker, and PLK1 also helps disassemble this linker to promote centrosome separation as cells prepare to enter mitosis (Alvarez-Rodrigo, 2021).

How PLK1 is recruited to centrosomes to execute its multiple functions is largely unclear, although this recruitment appears to be dependent on the PBD. In vertebrate systems, Cep192 is required for centrosome maturation and it is phosphorylated by Aurora A (also known as AURKA) to create PBD-binding sites that recruit PLK1; this promotes the activation of both kinases at the centrosome. The fly and worm homologues of Cep192, Spd-2 and SPD-2, respectively, are concentrated at centrioles and centrosomes, and their phosphorylation also helps recruit Polo/PLK1 kinases to the mitotic PCM to phosphorylate Cnn in flies and SPD-5 in worms. In fly embryos, Spd-2, Polo and Cnn have been proposed to form a positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. In this scenario, Spd-2 starts to be phosphorylated at centrioles as cells prepare to enter mitosis, and this allows Spd-2 to form a scaffold that can recruit other PCM proteins and that fluxes outwards from the mother centriole. The Spd-2 scaffold itself is weak, but it can recruit Polo and Cnn; the recruited Polo phosphorylates Cnn, which then forms a Cnn scaffold that recruits other PCM components and strengthens the Spd-2 scaffold. This allows more Spd-2 to accumulate around the centriole, which in turn drives the recruitment of more Polo and Cnn - so forming a positive feedback loop. In this way, Spd-2 recruits Polo and Cnn to the PCM to help drive centrosome maturation in flies (Alvarez-Rodrigo, 2021).

If Drosophila Spd-2 cannot efficiently recruit Polo (because all its S-S/T motifs have been mutated to T-S/T) Polo recruitment to the PCM is dramatically reduced, but Polo is still strongly recruited to the mother centriole, indicating that other proteins must help recruit Polo to centrioles. The centriole protein Sas-4 is phosphorylated by Cdk1 during mitosis on threonine 200 (T200), creating a PBD-binding site that recruits Polo to newly formed daughter centrioles. This allows the daughter to recruit Asl (Cep152 in humans), which allows the daughter to mature into a new mother that can duplicate and organise PCM - since Asl is required for both of these processes. Although the single PBD-binding site in Sas-4 recruits Polo to mother centrioles, it is suspected that other proteins must also be required. This study attempted to identify such proteins by mutating all the S-S/T motifs to T-S/T in several candidates. The centriole protein Ana1 (Cep295 in humans) was found to normally help recruit Polo to mother centrioles. Ana1 and Cep295 are required for centriole maturation, and in flies Ana1 helps recruit and/or maintain Asl at new mother centrioles. Thus, flies lacking Ana1 lack centrioles, centrosomes and cilia, presumably because the centrioles cannot duplicate without Ana1 as they cannot recruit Asl. This study shows that centrioles that do not efficiently recruit Polo via Ana1 can still recruit Sas-4, Cep135 and Asl, and can still duplicate, disengage and organise cilia, but they cannot efficiently recruit mitotic PCM or elongate during G2. It is proposed that Ana1 recruits Polo to centrioles specifically to promote centriole elongation in G2 and mitotic PCM assembly (Alvarez-Rodrigo, 2021).

Polo has many important functions at centrioles and centrosomes, and previous work has shown that it is initially recruited to newborn centrioles in flies when Cdk1 phosphorylates the Sas-4 T200 S-T motif during mitosis. This initial recruitment of Polo is important to allow the newborn centrioles to subsequently mature into mothers that can recruit Asl and so duplicate and recruit mitotic PCM. This study showed that the centriole protein Ana1 also plays an important part in recruiting Polo to mother centrioles. The data suggests that Ana1 can recruit Polo directly and that Polo itself can phosphorylate Ana1 at several S-S/T motifs to 'self-prime' its own recruitment. It cannot be excluded that other protein kinases may prime these S-S/T motifs, or that Ana1 could recruit Polo to centrioles indirectly in ways that are disrupted when the S-S/T motifs are mutated to T-S/T. Regardless of mechanism, the Ana1-dependent centriolar pool of Polo appears to be required to drive efficient mitotic PCM expansion and centriole elongation in G2 (Alvarez-Rodrigo, 2021).

Although Ana1 helps recruit and/or maintain Asl at centrioles, and therefore is essential for both mitotic PCM recruitment and centriole duplication, this function of Ana1 does not appear to require the ability to recruit Polo. Thus, Ana1-S34T centrioles recruit and maintain normal levels of Asl (and of Cep135, as well as slightly increased levels of Sas-4) and can duplicate normally. This is in contrast to the situation with Sas-4, where T200 phosphorylation is required for proper Asl recruitment and so for both centriole duplication and mitotic PCM assembly. Presumably, the Polo recruited by Sas-4 is either sufficient for Asl recruitment, or it phosphorylates centriole substrates other than Ana1 to promote Asl recruitment. Interestingly, PLK1 is also essential for efficient centriole disengagement, but neither the Ana1-S34T nor Sas-4-T200 mutations appear to perturb this process, indicating that a separate pathway must recruit Polo to centrioles to drive centriole disengagement. Centrosome separation in G2 is also normally dependent on PLK1, and centrosomes/duplicated centriole pairs were often observed that failed to separate properly in embryos expressing Ana1-S34T. As these centriole pairs almost always organised very little PCM, however, it is suspected that this defect may be an indirect consequence of the failure to properly recruit PCM, rather than a direct consequence of the inability of Ana1 to recruit Polo (Alvarez-Rodrigo, 2021).

These new findings further support the hypothesis that centrioles activate a Spd-2-Polo-Cnn positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. A key feature of this model is that Spd-2 can only be phosphorylated to initiate scaffold assembly at the surface of the mother centriole, and the phosphorylated Spd-2 then fluxes outwards away from the centriole: the Spd-2-Polo-Cnn scaffold itself cannot phosphorylate and/or recruit new Spd-2 into the scaffold. This is important, as it can explain why the mother centriole is required to drive efficient mitotic PCM assembly, why the size of the centriole influences the size of the mitotic PCM and why centrioles are constantly required to drive the growth of the mitotic PCM. All of these findings can be explained if the mother centriole is the only source of the phosphorylated Spd-2 scaffold. The observation that the pool of Polo recruited by Ana1, which unlike Spd-2, is not a PCM component and is restricted to the centriole - is required for the efficient expansion of the PCM demonstrates that the PCM-associated pool of Polo (recruited by Spd-2) is not sufficient to drive efficient PCM expansion on its own. It is important to stress, however, that so far an outward flux of Spd-2 from the centriole has only been observed in fly embryos and cells and has not been detected for SPD-2 in C. elegans embryos\. Clearly it will be important to establish whether such a Spd-2 or Cep192 flux exists in other species (Alvarez-Rodrigo, 2021).

The ability of Ana1 to recruit Polo also appears to be required for centriole elongation during G2. In human cells, PLK1 is required for this process, although a previous study did not report any change in centriole length after long-term Polo-inhibition in fly spermatocytes. Clearly more work is required to establish whether Polo recruitment by Ana1 has a role in G2 centriole elongation in flies, as the current work suggests, and, if so, what Polo's relevant substrates are at the centriole distal end (Alvarez-Rodrigo, 2021).

Finally, it is noted that both the Ana1/Cep295 and Spd-2/Cep192 protein families have a relatively high density of potential PBD-binding sites (S-S/T motifs) when compared to several other centriole and centrosome proteins. This suggests that these proteins might have evolved to function as scaffolds that amplify Polo levels at specific locations within the cell during mitosis. It will be interesting to examine whether other proteins with a high density of potential PBD-binding domains serve a similar function at other locations within the mitotic cell. The strategy of mutating all S-S/T motifs to T-S/T in candidate proteins may be a good way of testing this possibility as, for both Ana1 and Spd-2 at least, the S-to-T substitutions seem to specifically impair Polo-recruitment without more generally perturbing the function of the proteins or centriole/centrosome structure (Alvarez-Rodrigo, 2021).

Superresolution characterization of core centriole architecture

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 timed 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).

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).

Conserved molecular interactions in centriole-to-centrosome conversion

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. This study shows that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. This study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood (Fu, 2016).

These results are consistent with recent reports that Ana1/Cep295 is required for centriole-to-centrosome conversion in flies and humans, and it was shown that ana1 mutant cells have very few centrosomes in vivo. Studies on fly cells in culture suggested a simple model of centriole-to-centrosome conversion whereby Cep135 is initially recruited to centrioles and this subsequently recruits Ana1 to centrioles in late interphase. In mitosis, Ana1 then recruits Asl to new centrioles; Asl recruitment is crucial for centriole-to-centrosome conversion, as Asl is required to allow newly formed centrioles to recruit PCM during mitosisand to duplicate during the next S-phase. A similar molecular mechanism appears to operate in human cultured cells (Fu, 2016).

These findings suggest, however, that the recruitment of Ana1 to centrioles might be more complicated. The N-terminal region of Ana1 (amino acids 1–935) interacts with Cep135 in vitro and is recruited to centrioles in cultured cells, whereas an N-terminally deleted fragment of Ana1 (amino acids 756–1729) cannot be recruited to centrioles in cultured cells, even in the presence of the endogenous WT Ana1 protein. In contrast, this study found that the C-terminal region of Ana1 can be recruited to centrosomes (although quite weakly). Moreover, GFP–Ana1ΔNT (comprising amino acids 640–1729) can rescue the centrosome assembly defect in ana1 mutant cells and the uncoordinated phenotype of ana1 mutant flies, although this protein is clearly not fully functional because it is only weakly recruited to centrosomes, and the rescued mutant flies are both male- and female-sterile. It is not known why this deletion construct appears to behave differently in embryos and cultured cells, but the findings suggest that several mechanisms help to localise Ana1 to centrioles in embryos. In particular, the middle region of Ana1 contains multiple predicted coiled-coil regions that could allow Ana1 to interact with itself (potentially complicating the analysis of protein localisation experiments performed in the presence of the WT endogenous protein) and also with other proteins such as Cep135 (Fu, 2016).

Surprisingly, it was also found that in the presence of GFP–Ana1ΔCT (a C-terminal Ana1 deletion that cannot interact with Asl) the recruitment of Asl is more strongly reduced at old mother centrioles than at new mother centrioles. This is unexpected, and suggests that the process of recruiting Asl to mother centrioles might also be more complicated than originally thought. Indeed, mother centrioles appear to contain at least two pools of Asl: ~50% of the Asl protein is stably incorporated into centrioles, whereas ~50% is in exchange with the cytoplasmic pool. Interestingly, Asl interacts directly with another centriole protein, Sas-4/CPAP, in both fly and human cells, and this study has shown that Sas-4 plays an important part in recruiting Asl to new mother centrioles, but is not required to maintain Asl at old mother centrioles. Perhaps Sas-4 plays a more important role in initially recruiting Asl to new mother centrioles, whereas Ana1 plays a more important role in maintaining Asl at mother centrioles (Fu, 2016).

In human cells, centrioles are destabilised in the absence of Cep295. This appears to be because the failure in centriole-to-centrosome conversion blocks the recruitment of the PCM, and the PCM is required to stabilise the centrioles after they lose their central cartwheels during the conversion process (as normally occurs in vertebrates). In flies, however, the centrioles do not usually lose their cartwheels during the conversion process, so it is unclear why centrioles appear to be destabilised in the absence of Ana1 (as the permanent central cartwheel structure would presumably stabilise centrioles even when they cannot recruit PCM). This study observed that centriole-like structures can be seen at the electron microscopy level in ana1 mutant cells overexpressing Sas-6 and Ana2, and some Sas-4-containing structures are visible in immunofluorescence images from early ana1 mutant spermatocytes, suggesting that some residual centriole-like structures can persist in ana1 mutant cells. If some residual centriole structures do persist in ana1 mutant tissues, however, they are clearly not capable of supporting centrosome assembly or cilium function (as ana1 mutant flies are severely uncoordinated) (Fu, 2016).

This study found that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are slightly longer when Ana1 is overexpressed, and slightly shorter when ana1 gene dosage is halved. This finding is in contrast to the observation that Ana1 depletion does not lead to a decrease in centriole length in S2 cells, perhaps because Ana1 depletion does not alter the length of centrioles that had already formed in the cell population prior to Ana1 depletion (and dynamic analysis of Ana1 behaviour suggests that Ana1 is irreversibly incorporated into centrioles). Importantly, this function of Ana1 appears to have different molecular requirements to the function of Ana1 in centriole stabilisation, as the N-terminal 639 amino acids of Ana1 are not essential for centrosome assembly or cilium function, but are required to allow Ana1 to promote centriole elongation in spermatocytes and to promote centriole over-elongation when overexpressed (Fu, 2016).

Several centriole proteins can influence centriole length. Positive regulators such as Sas-4/CPAP, Cep135, Cep120 and SPICE can increase centriole length when overexpressed in human cells, and negative regulators such as CP110, Cep97 and Klp10A appear to act to suppress centriole elongation. Although it is unclear how Ana1 influences centriole length, it is intriguing that its N-terminal region, which is required to promote centriole elongation, can interact with Cep135 as centrioles are shorter than normal in Cep135 mutant spermatocytes. The data suggests, however, that Ana1 does not promote centriole elongation simply by recruiting extra Cep135 or Asl to the centrioles. Interestingly, Cep295, the human homologue of Ana1, also promotes centriole elongation in human cells (suggesting that this function is conserved), and it can interact with tubulin –potentially providing a molecular mechanism that can explain how Cep295 promotes centriole elongation. Ana1 is the first protein shown to reduce centriole length when its gene dosage is halved, suggesting that Ana1 is a limiting factor that ensures proper centriole elongation in flies (Fu, 2016).

Functions of Ana1 orthologs in other species

CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion

Centrosomes are essential organelles with functions in microtubule organization that duplicate once per cell cycle. The first step of centrosome duplication is the daughter centriole formation followed by the pericentriolar material recruitment to this centriole. This maturation step was termed centriole-to-centrosome conversion. It was proposed that CEP295-dependent recruitment of pericentriolar proteins drives centriole conversion. This study shows, based on the analysis of proteins that promote centriole biogenesis, that the developing centriole structure helps drive centriole conversion. Depletion of the luminal centriole protein CEP44 that binds to the A-microtubules and interacts with POC1B affecting centriole structure and centriole conversion, despite CEP295 binding to centrioles. Impairment of POC1B, TUBE1 or TUBD1, which disturbs integrity of centriole microtubules, also prevents centriole-to-centrosome conversion. It is proposed that the CEP295, CEP44, POC1B, TUBE1 and TUBD1 centriole biogenesis pathway that functions in the centriole lumen and on the cytoplasmic side is essential for the centriole-to-centrosome conversion (Atorino, 2020).

Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

Centrioles surrounded by pericentriolar material (PCM) serve as the core structure of the centrosome. A newly formed daughter centriole grows into a functional mother centriole. However, the underlying mechanisms remain poorly understood. This study shows that Cep295, an evolutionarily conserved protein, is required for generation of a bona fide mother centriole organizing a functional centrosome. Cep295 is recruited to the proximal centriole wall in the early stages of procentriole assembly. Cep295 then acts as a scaffold for the proper assembly of the daughter centriole. It was also found that Cep295 binds directly to and recruits Cep192 onto the daughter centriole wall, which presumably endows the function of the new mother centriole for PCM assembly, microtubule-organizing centre activity and the ability for centriole formation. These findings led to a proposal that Cep295 acts upstream of the conserved pathway for centriole formation and promotes the daughter-to-mother centriole conversion (Tsuchiya, 2016).

Stabilization of cartwheel-less centrioles for duplication requires CEP295-mediated centriole-to-centrosome conversion

Vertebrate centrioles lose their geometric scaffold, the cartwheel, during mitosis, concurrently with gaining the ability to recruit the pericentriolar material (PCM) and thereby function as the centrosome. Cartwheel removal has recently been implicated in centriole duplication, but whether "cartwheel-less" centrioles are intrinsically stable or must be maintained through other modifications remains unclear. This study identified a newborn centriole-enriched protein, KIAA1731/CEP295, specifically mediating centriole-to-centrosome conversion but dispensable for cartwheel removal. In the absence of CEP295, centrioles form in the S/G2 phase and lose their associated cartwheel in mitosis but cannot be converted to centrosomes, uncoupling the two events. Strikingly, centrioles devoid of both the PCM and the cartwheel progressively lose centriolar components, whereas centrioles associating with either the cartwheel or PCM alone can exist stably. Thus, cartwheel removal can have grave repercussions to centriole stability, and centriole-to-centrosome conversion mediated by CEP295 must occur in parallel to maintain cartwheel-less centrioles for duplication (Izquierdo, 2014).


Search PubMed for articles about Drosophila Ana1

Alvarez-Rodrigo, I., Wainman, A., Saurya, S. and Raff, J. W. (2021). Ana1 helps recruit Polo to centrioles to promote mitotic PCM assembly and centriole elongation. J Cell Sci 134(14). PubMed ID: 34156068

Atorino, E. S., Hata, S., Funaya, C., Neuner, A. and Schiebel, E. (2020). CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion. Nat Commun 11(1): 903. PubMed ID: 32060285

Fu, J., Lipinszki, Z., Rangone, H., Min, M., Mykura, C., Chao-Chu, J., Schneider, S., Dzhindzhev, N. S., Gottardo, M., Riparbelli, M. G., Callaini, G. and Glover, D. M. (2016). Conserved molecular interactions in centriole-to-centrosome conversion. Nat Cell Biol 18: 87-99. PubMed ID: 26595382

Izquierdo, D., Wang, W. J., Uryu, K. and Tsou, M. F. (2014). Stabilization of cartwheel-less centrioles for duplication requires CEP295-mediated centriole-to-centrosome conversion. Cell Rep 8(4): 957-965. PubMed ID: 25131205 x

Panda, P., Kovacs, L., Dzhindzhev, N., Fatalska, A., Persico, V., Geymonat, M., Riparbelli, M. G., Callaini, G. and Glover, D. M. (2020). Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis. J Cell Biol 219(8). PubMed ID: 32543652

Tian, Y., Wei, C., He, J., Yan, Y., Pang, N., Fang, X., Liang, X. and Fu, J. (2021). Superresolution characterization of core centriole architecture. J Cell Biol 220(4). PubMed ID: 33533934

Tsuchiya, Y., Yoshiba, S., Gupta, A., Watanabe, K. and Kitagawa, D. (2016). Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole. Nat Commun 7: 12567. PubMed ID: 27562453

Biological Overview

date revised: 2 January 2023

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