InteractiveFly: GeneBrief

anastral spindle 2: Biological Overview | References

Gene name - anastral spindle 2

Synonyms -

Cytological map position - 44F3-44F3

Function - centriole duplication factor

Keywords - centrosomal protein, spindle orientation, tumor suppressor, neuroblast asymmetical division

Symbol - ana2

FlyBase ID: FBgn0027513

Genetic map position - chr2R:4786228-4787706

Classification - STAN motif and coiled-coil domain protein

Cellular location - centriolar protein

NCBI link: EntrezGene

ana2 orthologs: Biolitmine
Recent literature
Cottee, M.A., Muschalik, N., Johnson, S., Leveson, J., Raff, J.W. and Lea, S.M. (2015). The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies. Elife [Epub ahead of print]. PubMed ID: 26002084
Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the nine-fold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). This study shows that the Drosophila Ana2 CCCD forms a tetramer, and solves its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. The structure of the Drosophila Sas-6 N-terminal domain was also solved to 2.9 Å revealing that it formed higher-order oligomers through canonical interactions. Point mutations that perturbed Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturbed centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.
Dzhindzhev, N. S., Tzolovsky, G., Lipinszki, Z., Abdelaziz, M., Debski, J., Dadlez, M. and Glover, D. M. (2017). Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation. Open Biol 7(12). PubMed ID: 29263250
The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL. Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. This study shows that in Drosophila, Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When the auto-destruction of Plk4 is overriden, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. This study shows that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region termed the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6.
Jana, S. C., Mendonca, S., Machado, P., Werner, S., Rocha, J., Pereira, A., Maiato, H. and Bettencourt-Dias, M. (2018). Differential regulation of transition zone and centriole proteins contributes to ciliary base diversity. Nat Cell Biol 20(8): 928-941. PubMed ID: 30013109
Cilia are evolutionarily conserved structures with many sensory and motility-related functions. The ciliary base, composed of the basal body and the transition zone, is critical for cilia assembly and function, but its contribution to cilia diversity remains unknown. This study has generated a high-resolution structural and biochemical atlas of the ciliary base of four functionally distinct neuronal and sperm cilia types within an organism, Drosophila melanogaster. A common scaffold was uncovered and diverse structures associated with different localization of 15 evolutionarily conserved components. Furthermore, CEP290 (also known as NPHP6) is involved in the formation of highly diverse transition zone links. In addition, the cartwheel components SAS6 and ANA2 (also known as STIL) have an underappreciated role in basal body elongation, which depends on BLD10 (also known as CEP135). The differential expression of these cartwheel components contributes to diversity in basal body length. These results offer a plausible explanation to how mutations in conserved ciliary base components lead to tissue-specific diseases.


Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. This study identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. Two ana2 alleles were isolated that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. These findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division (Wang, 2011).

The Drosophila larval brain neural stem cell, or neuroblast (NB), has recently emerged as a new model for studying stem cell self-renewal and tumorigenesis. NBs divide asymmetrically to generate a self-renewing daughter NB and a ganglion mother cell (GMC) that is committed to differentiation. Asymmetric localization/segregation machinery ensures the polarized distribution of 'proliferation factors,' including atypical protein kinase C (aPKC), and 'differentiation factors,' including basal proteins such as Numb, Miranda (Mira), Brain tumor (Brat), and Prospero, to the daughter NB and GMC, respectively. The failure of asymmetric division of NBs can result in their hyperproliferation and the induction of tumors (Wang, 2011).

To ensure the correct asymmetric segregation of cell fate determinants, the mitotic spindle has to be properly oriented with respect to the polarized proteins on the cell cortex. Inscuteable (Insc) and the heterotrimeric G proteins Gαi and Gβγ and their regulators Partner of Insc (Pins) and Ric-8 control mitotic spindle orientation (Wang, 2011).

Recent work has also implicated centrosome-associated proteins in the regulation of spindle orientation and tumorigenesis (Gonzalez, 2007). Centrosomes function as major microtubule-organizing centers in most animal cells. A centrosome is composed of a pair of centrioles surrounded by an amorphous matrix of pericentriolar material (PCM). Centriole duplication is regulated by centriolar components, such as Asterless (Asl), Sas6, Sas4, and anastral spindle 2 (Ana2). ana2 was identified from genome-wide RNA interference (RNAi) screens, where ana2 RNAi-treated S2 cells exhibited an anastral spindle phenotype (Dobbelaere, 2008; Goshima, 2007). The Ana2 overexpression phenotype and its interaction with Sas6 have suggested a role for Ana2 in centriole duplication (Stevens, 2010). However, no ana2 mutants were previously available for further functional studies (Wang, 2011).

This study has isolated two ana2 alleles that are defective in apical/basal spindle orientation during NB asymmetric division. Ana2 is demonstrated to be a tumor suppressor that suppresses NB overproliferation. The centriolar protein Ana2 directly interacts with Ctp, a dynein light chain that also localizes to the centrioles, and Mud, leading to their localization to the centrosomes. This finding suggests that the tumor suppressor Ana2 ultimately regulates Mud function to direct asymmetric division and prevent tumor formation (Wang, 2011).

This study investigated the role of Drosophila Ana2 during NB asymmetric cell division, focusing on mitotic spindle orientation. Two ana2 alleles were isolated from a genetic screen that produced supernumerary NBs in larval brains and failed to properly align the mitotic spindle with asymmetrically localized proteins. It was demonstrated that Drosophila Ana2 functioned as a tumor suppressor in a transplantation experiment. Using ana2 mutants, it was shown that Ana2 is important for centriole function. Ana2 interacts with Sas-6 through the C-terminal region of Ana2 (201-420 aa), which contains the conserved STAN motif and coiled-coil domain (Stevens, 2010). The data suggest that the N terminus of Ana2 (1-274 aa), which interacted with Ctp, a Ddlc1 (Drosophila Dynein light chain), is sufficient for its function in centriole assembly and spindle orientation. This is not in direct contradiction with the interaction between Ana2 and Sas6 because the C-terminal region of Ana2 (201-420 aa), which interacts with Sas-6, partially overlaps with the Ana2 N1 (1-274 aa). However, this result suggests surprisingly that the STAN motif may be dispensable for Ana2's function during centriole formation. The mammalian Ana2-related protein STIL, which also contains the STAN motif, has been implicated in primary microcephaly, a neurodevelopmental disorder characterized by a reduced brain size (Kumar, 2009). The apparently disparate phenotypes reported for mammalian STIL and fly Ana2 during brain development are likely due to different developmental contexts (Wang, 2011).

The reason that NB overproliferation occurs in ana2 mutants, but not in asterless or sas4 mutants with spindle or centriole defects, may be due to the different behaviors of these mutants in 'telophase rescue,' a phenomenon whereby proteins delocalized from the cortex during early mitosis are restored at anaphase/telophase by a poorly understood compensatory mechanism. The spindle misorientation phenotype in ana2 mutants is much more severe than sas4 or asterless mutants. Likely as a consequence of a relatively weak spindle misorientation phenotype, 'telophase rescue' still occurred in 100% of the asterless and sas4 mutant telophase NBs, and all asymmetrically localized proteins were correctly segregated into different daughter cells. In contrast, in ana2 mutants or mud mutants, which have NB overgrowth in larval brains, asymmetrically localized proteins sometimes mis-segregate into different daughters at telophase (Wang, 2011).

The RNAi screen identified Ctp as an important player in mitotic spindle orientation because ctp mutants displayed spindle misorientation during NB asymmetric division. ctp null mutants display spindle misorientation in NBs similar to that seen in ctp RNAi. It is noted that Ctp localizes to centrioles in Drosophila. Ana2 directly binds and anchors Ctp to the centrioles during NB division. The centriole localization is important for Ctp function during spindle orientation because membrane-targeted CtpCAAX fails to rescue the spindle misorientation phenotype in the ctp null mutant. The interaction between Ctp and Ana2 on the centrioles may be critical for dynein to organize astral microtubules and move its cargo proteins along the microtubules (Wang, 2011).

A dynein component, Ctp, can also bind directly to Mud, a protein downstream of heterotrimeric G protein signaling, that regulates spindle orientation. This interaction is conserved in vertebrates; Xenopus NuMA, a Mud-related protein, also forms a complex with dynein. Ana2 and Ctp are important for spindle pole localization of Mud during spindle orientation in NBs, whereas Mud is not required for centriolar localization of Ana2 or Ctp. Ana2 also directly interacts with Mud. These data suggested that Mud may be an important downstream target of Ana2 and Ctp during spindle orientation. Ana2, Ctp, and Mud are also found in the same protein complex in vivo and in vitro. Mud is involved in spindle pole/centrosome engagement, which has not been reported in previous analyses of Mud function. Ana2 and Ctp also played a similar role during spindle pole/centrosome attachment. Together, these data indicate that the Ana2, Ctp, and Mud complex functioned to regulate spindle pole assembly and spindle orientation during asymmetric division of NBs (Wang, 2011).

Apical/basal spindle orientation is controlled by a two-step mechanism: an early, centrosome-dependent alignment and a later spindle-cortex interaction. The data indicate that Ana2 is not only critical for the early, centrosome-dependent step, but also for the later spindle-cortex interaction. Although the loss of Ana2 or Ctp function does not affect Pins asymmetric localization in NBs, Ana2 and Ctp appear to be important for the interaction between Pins and Mud in larval brains because the Pins-Mud interaction is diminished in ana2 or insc-CtpCAAX, ctp mutant larval brains. These findings suggested that the Dynein-Dynactin complex cooperate with the centriolar protein Ana2 to mediate the spindle-cortex interaction. The spindle-cortex interaction may require the 'search and capture' mechanism, driven by the plus-end microtubule-binding protein EB1 and Dynein-Dynactin complex). It is speculated that Ana2 and Ctp may be involved in such a 'search and capture' mechanism during apicobasal spindle orientation (Wang, 2011).

These data suggested that a multiprotein complex composed of Ana2, Ctp, and Mud is critical during the regulation of spindle orientation. Ana2 and Ctp regulated Mud localization on centrosome/spindle poles as well as on the cell cortex, whereas the heterotrimeric G protein pathway is only important for cortical Mud localization. Thus, the centrosomal Ana2/Ctp/Mud complex converges with the heterotrimeric G protein pathway during spindle orientation. Very little is known about the molecular mechanisms by which centrosomal proteins regulate spindle orientation. Aur-A, a PCM protein, has been shown to phosphorylate Pins on S436 of the Pins Linker domain, which is required for accurate spindle orientation. The current findings suggest important functional links among the centriolar protein Ana2, the dynein complex, and Mud during asymmetric division of NBs. This raises the possibility that a similar mechanism whereby centrosomal proteins interact with dynein complexes to mediate cortical protein localization may exist during asymmetric division and stem cell self-renewal in mammals (Wang, 2011).

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, 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 Anastral spindle 2 (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 (Goshima, 2007; Dobbelaere, 2008). This list includes Sak, DSas-6, and DSas-4, as well as eight other proteins that specifically localize to centrosomes (Ana1, Ana2, 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).

Aydogan, M. G., Wainman, A., Saurya, S., Steinacker, T. L., Caballe, A., Novak, Z. A., Baumbach, J., Muschalik, N. and Raff, J. W. (2018). A homeostatic clock sets daughter centriole size in flies. J Cell Biol. PubMed ID: 29500190

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).

Centriole growth is limited by the Cdk/Cyclin-dependent phosphorylation of Ana2/STIL

Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. This study shows that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process-indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis (Steinacker, 2022).

Two studies have attempted to estimate the levels of one or more of the core centriole duplication proteins in human cells. Fluorescence correlation spectroscopy (FCS) has been used to estimate a Sas-6 cytoplasmic concentration of ~80-360 nM, depending on the cell cycle stage, while another study used quantitative MS to estimate the number of Plk4, Sas-6, CEP152/Asl, and STIL/Ana2 molecules in human cultured cells, which was in the ~2,000-20,000 range, ~10-15X lower than the number of γ-tubulin molecules in the cell. If the volume of a HeLa cell is ~4,000 μm3, then the concentration of these centriole proteins is in the ~1-10 nM range, which seems low, but could reflect that most somatic cells only assemble two tiny daughter centrioles during a cell cycle that can last many hours (Steinacker, 2022).

Given that the early Drosophila embryo assembles several thousand centrioles in <2 h, it was anticipated that centriole assembly proteins would be stored at higher concentrations than in somatic cells, but this does not appear to be the case. It is estimate that Asl, Sas-6, Ana2, and Sas-4 are present in the ~5-20 nM range (note that 20 nM would be the concentration of the Ana2 oligomer), while the cytoplasmic concentration of Plk4 is so low that it cannot be measured by FCS. Interestingly, these concentrations are similar to the MS estimates in human cell lines, suggesting that the early embryo does not store a large surplus of any of these proteins. Why are these key centriole assembly proteins present at such low concentrations? Several of these proteins tend to self-assemble into larger macromolecular structures, so it seems likely that their low cytoplasmic concentration helps to ensure that they normally only start to form a cartwheel at the single kinetically favorable site on the side of the mother centriole. Indeed, the FCS data suggest that the concentration of Sas-6 in the embryo is low enough that it is largely monomeric in the cytoplasm, even though it is almost certainly incorporated into the centriole cartwheel as a dimer. Storing Sas-6 as a monomer would help to ensure that it cannot spontaneously assemble into aberrant structures, and it is wondered whether storing self-assembling proteins that normally function as dimers (or higher-order homo-multimers) in cells as monomers (or lower order homo-multimers) might be a general strategy that helps to prevent their inappropriate self-assembly (Steinacker, 2022).

How cellular structures grow to the correct size is a topic of great interest. In the embryos of C. elegans, mitotic centrosome size appears to be set by a limiting cytoplasmic pool of the centrosome building block SPD-2, although this does not appear to be the case for Spd-2 in early Drosophila embryos. The concept of setting organelle size with a limiting pool of building blocks is attractive, as it allows size to be controlled without the need for a specific mechanism to measure it. The data, however, suggests that although the cytoplasmic concentration of the core duplication proteins is low, none of them act as limiting components to regulate centriole growth in Drosophila embryos. It is concluded that the amount of these proteins sequestered at centrioles may be insignificant compared to the amount in the cytoplasm (a plausible scenario given the large volume of the embryo and small volume of the centriole), and/or that the rate of protein sequestration at centrioles and degradation in the embryo is finely balanced by the rate of new protein synthesis so that a constant cytoplasmic concentration is maintained. Cdk/Cyclin appears to phosphorylate Ana2 to modulate centriole duplication efficiency (Steinacker, 2022).

In vertebrates, STIL binds and is phosphorylated by CDK1/Cyclin B kinase. The function of this phosphorylation is unclear, but it is thought that binding to (rather than phosphorylation by) CDK1/Cyclin B keeps STIL in an inactive state because Cdk1/Cyclin B binds to the same central coiled-coil (CC) region of STIL that binds PLK4. The current data suggest that in fly embryos Cdk1/Cyclin activity can inhibit daughter centriole growth by phosphorylating, rather than simply binding to, Ana2. Ana2's diffusion rate increases as Cdk/Cyclin activity increases toward the end of S-phase and this increase is abrogated if Ana2 cannot be phosphorylated by Cdk1/Cyclin (due to mutation of all 12 S/T-P motifs). This Ana2(12A) mutant protein can still support centriole duplication, but it is recruited to the duplicating centrioles for an unusually long period of time during S-phase (presumably because its recruitment is not efficiently inhibited by the rising levels of Cdk/Cyclin activity in the embryo), allowing the protein to accumulate at centrioles to abnormally high levels. Mutating these 12 motifs to phosphomimicking D/E motifs has the opposite effect: Ana2(12D/E) is recruited poorly to centrioles and it can no longer support the rapid cycles of centriole duplication in the early embryo. It cannot be ruled out that the 12A and 12D/E mutations alter Ana2 in ways that change its conformation, multimerization, or function in unknown ways. Nevertheless, the ability of both mutants to support centriole duplication in somatic cells and their opposing effects on Ana2's centriole recruitment are consistent with the hypothesis that these mutations prevent or mimic Ana2 phosphorylation, respectively (Steinacker, 2022).

A priori, it is perhaps surprising that the 12A and 12D/E mutants appear to support relatively normal centriole duplication in somatic cells, demonstrating that the phosphorylation of Ana2 by Cdk/Cyclins cannot be essential for duplication-although the 12D/E mutant cannot support centriole duplication in the early embryo. It is speculated that while the Cdk/Cyclin-dependent phosphorylation of Ana2 reduces the efficiency of centriole duplication toward the end of the S-phase, multiple additional regulatory mechanisms-such as the oscillation in centriolar Plk4 levels-help to ensure that daughter centrioles still duplicate at the right time and place even if Ana2 cannot be phosphorylated by Cdk/Cyclins. In embryos, the 12D/E mutant is lethal, as the rapidly dividing centrioles do not have time to compensate for the reduction in duplication efficiency, but this is not the case in somatic cells, where the S-phase is much longer (Steinacker, 2022).

It is not known how the phosphorylation of Ana2 by Cdk1/Cyclins might influence centriole duplication, but it is speculated that it decreases Ana2's affinity for one or more of the other core centriole duplication proteins to which it binds (e.g., Sas-6, Plk4 or Sas-4). Unfortunately, it has not been possible to directly test this in vitro, and it was not possible to detect direct interactions between these endogenous proteins in embryo extracts, probably due to their very low cytoplasmic concentrations. Nevertheless, such a scenario would explain why Ana2's average cytoplasmic diffusion rate normally increases toward the end of the S-phase and why this increase is abrogated in the 12A mutant. FCS analysis also suggests that the average cytoplasmic diffusion rate of all the core duplication proteins analyzed in this study increases slightly as S-phase progresses, perhaps hinting that their cytoplasmic interactions might be generally suppressed by increasing Cdk/Cyclin activity. In embryos expressing Ana2(12A), the failure to efficiently inhibit Ana2's interactions with one or more other duplication proteins toward the end of S-phase could explain why Ana2(12A) and Sas-6 can continue to incorporate into centrioles for an extended period. Such a mechanism could also explain previous observations that inhibiting Cdk1 activity can lead to centriole overduplication in flies (Vidwans et al., 2003) (Steinacker, 2022).

Unexpectedly, expressing Ana2(12A) significantly decreased the amount of Sas-6 recruited to centrioles. This is surprising because Ana2 is thought to help recruit Sas-6 to centrioles, and centriolar Ana2(12A) levels are abnormally high. An intriguing interpretation of this finding is that while the phosphorylation of Ana2 by Cdk/Cyclins in late S-phase helps to inhibit centriole duplication, Cdk/Cyclin-dependent phosphorylation of Ana2 in early S-phase (presumably on different sites) might help promote centriole duplication by increasing the efficiency with which Ana2 interacts with Sas-6 to recruit it to centrioles. The S-phase-initiating CDK2/Cyclin kinase is required for centriole duplication, but its relevant substrate(s) are largely unknown. Perhaps CDK2/Cyclins phosphorylate Ana2 in early S-phase to promote centriole duplication, while CDK1/Cyclins phosphorylate Ana2 from late-S-phase onward to inhibit centriole duplication. Alternatively, the differential phosphorylation of different Cdk/Cyclin targets by different levels of Cdk/Cyclin activity plays an important part in ordering cell cycle events. Perhaps low (early-S-phase-like) levels of Cdk/Cyclin activity phosphorylate Ana2 on certain sites to promote centriole assembly, while higher levels phosphorylate Ana2 at additional sites to inhibit centriole assembly. In either scenario, Ana2 would act as a 'rheostat', responding to global changes in Cdk/Cyclin activity to coordinate centriole duplication with cell cycle progression. Plk4 phosphorylates Ana2 in an ordered fashion at multiple sites to elicit sequential changes in Ana2 behavior, so it seems possible that Cdk/Cyclins might do the same (Steinacker, 2022).


Search PubMed for articles about Drosophila Ana2

Blachon. S., et al. (2009). A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication. Genetics 182: 133-144. PubMed ID: 19293139

Dobbelaere, J., et al. (2008). A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biol. 6: e224. PubMed ID: 18798690

Dzhindzhev, N. S., Tzolovsky, G., Lipinszki, Z., Schneider, S., Lattao, R., Fu, J., Debski, J., Dadlez, M. and Glover, D. M. (2014). Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation. Curr Biol 24(21):2526-32. PubMed ID: 25264260

Gonzalez, C. (2007). Spindle orientation, asymmetric division and tumour suppression in Drosophila stem cells. Nat Rev Genet 8: 462-472. PubMed ID: 17510666

Goshima G., et al. (2007). Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417-421. PubMed ID: 17412918

Izraeli, S., et al. (1999). The SIL gene is required for mouse embryonic axial development and left-right specification. Nature 399: 691-694. PubMed ID: 10385121

Kumar, A., Girimaji, S. C., Duvvari, M. R. and Blanton, S. H. (2009). Mutations in STIL, encoding a pericentriolar and centrosomal protein, cause primary microcephaly. Am. J. Hum. Genet. 84: 286-290. PubMed ID: 19215732

Peel, N., Stevens, N. R., Basto, R. and Raff, J. W. (2007). Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation. Curr. Biol. 17: 834-843. PubMed ID: 17475495

Pfaff, K. L., Straub, C. T., Chiang, K., Bear, D. M., Zhou, Y. and Zon, L. I. (2007). The zebra fish cassiopeia mutant reveals that SIL is required for mitotic spindle organization. Mol Cell Biol 27: 5887-5897. PubMed ID: 17576815

Rodrigues-Martins, A., Riparbelli, M., Callaini, G., Glover, D. M., and Bettencourt-Dias, M. (2007a). Revisiting the role of the mother centriole in centriole biogenesis. Science 316: 1046-1050. PubMed ID: 17463247

Rodrigues-Martins, A., et al. (2007b). DSAS-6 organizes a tube-like centriole precursor, and its absence suggests modularity in centriole assembly. Curr. Biol. 17(17): 1465-72. PubMed ID: 17689959

Steinacker, T. L., Wong, S. S., Novak, Z. A., Saurya, S., Gartenmann, L., van Houtum, E. J. H., Sayers, J. R., Lagerholm, B. C. and Raff, J. W. (2022). Centriole growth is limited by the Cdk/Cyclin-dependent phosphorylation of Ana2/STIL. J Cell Biol 221(9). PubMed ID: 35861803

Slevin, L. K., Romes, E. M., Dandulakis, M. G. and Slep, K. C. (2014). The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor. J Biol Chem [Epub ahead of print]. PubMed ID: 24920673

Stevens, N. R., Dobbelaere, J., Brunk, K., Franz, A. and Raff, J. W. (2010). Drosophila Ana2 is a conserved centriole duplication factor. J Cell Biol 188: 313-323. PubMed ID: 20123993

Wang, C., Li, S., Januschke, J., Rossi, F., Izumi, Y., Garcia-Alvarez, G., Gwee, S. S., Soon, S. B., Sidhu, H. K., Yu, F., Matsuzaki, F., Gonzalez, C. and Wang, H. (2011). An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts. Dev Cell 21: 520-533. PubMed ID: 21920316

Biological Overview

date revised: 22 April 2023

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