anastral spindle 2: Biological Overview | References
Gene name - anastral spindle 2
Cytological map position - 44F3-44F3
Function - centriole duplication factor
Symbol - ana2
FlyBase ID: FBgn0027513
Genetic map position - chr2R:4786228-4787706
Classification - STAN motif and coiled-coil domain protein
Cellular location - centriolar protein
|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.
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).
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, Ana3, Asl, DCP110, DCep135/Bld10, DCep97, and Rcd4): these eight are therefore good candidates to play a direct role in centriole duplication (Stevens, 2010).
GFP-Sak, GFP-DSas-6, and DSas-4-GFP share the unique ability to drive de novo formation of centriole-like structures in unfertilized eggs when highly overexpressed from the upstream activation sequence (UAS) promoter. UAS-GFP-Sak and UAS-GFP-DSas-6 induce these structures in ~95% of unfertilized eggs, whereas UAS-DSas-4-GFP does so in ~60% of unfertilized eggs. It was asked if this assay could be used to identify other components likely to function upstream in the centriole duplication pathway. Transgenic lines were generated carrying GFP fusions to all eight potential duplication factors under the control of the UAS promoter, which allowed overexpression in unfertilized eggs. Strikingly, only Ana2 (in 97% of eggs) and Asl (in 33% of eggs) were able to drive de novo formation of centriole-like structures (Stevens, 2010).
Asl has recently been shown to be essential for centriole duplication in flies (Blachon, 2008), whereas, of the six proteins unable to induce de novo centriole formation, two, DCep135/Bld10 and Ana3, are now known not to be essential for centriole duplication in flies. These findings indicate that the overexpression assay can identify those proteins likely to be most intimately involved in centriole duplication. Since Asl has already been shown to be required for centriole duplication (Blachon, 2008), focus was placed on investigating the function of Ana2 (Stevens, 2010).
Ana2 can drive de novo formation of centriole-like structures as efficiently as DSas-6 and Sak (Peel, 2007; Rodrigues-Martins, 2007b). It was important to verify, however, that Ana2 also has a role in canonical centriole duplication. Overexpressing GFP-Sak or GFP-DSas-6 from the ubiquitin (Ubq) promoter induces centriole overduplication in brains and embryos, respectively (Peel, 2007). Surprisingly, however, overexpression of Sak, DSas-6, or DSas-4 cannot drive centriole overduplication in primary spermatocytes (Peel, 2007), which suggests that another duplication protein is limiting. To test if Ana2 might be this limiting factor, Ubq-GFP-Ana2 transgenic lines were generated. Strikingly, it was found that in spermatocytes expressing Ubq-GFP-Ana2, in addition to the normal centriole pairs (doublets), centriole triplets, quadruplets, and even quintets were observed. The extra centrioles in these clusters appeared to be fully functional; they separated from one another by the end of meiosis I (as centriole doublets normally do), and the extra centrioles inherited by secondary spermatocytes recruited PCM and nucleated MT asters, and so formed multipolar spindles during meiosis II (Stevens, 2010).
It was important to compare the localization of Ana2 with that of the other Drosophila centriole duplication factors. DSas-4-GFP, GFP-DSas-6, and GFP-Sak are all enriched at the proximal and distal ends of the large spermatocyte centrioles (Peel, 2007). It was found that, likewise, Ana2-GFP localized preferentially to the proximal and distal centriole tips. Strikingly, however, Ana2-GFP (and GFP-Ana2) also exhibited a unique asymmetric distribution, consistently localizing preferentially along one centriole barrel (Stevens, 2010).
In primary spermatocytes, it is possible to distinguish mother and daughter centrioles, as the daughter can often be observed associating end-on with the side of the mother (Blachon, 2008). In 25 centriole pairs where mother and daughter centrioles could unambiguously distinguished, Ana2-GFP was always enriched on the daughter. Mother and daughter centrioles can show important differences in their behavior in vertebrate cells and during asymmetric stem cell divisions in Drosophila. Although mother and daughter centrioles are morphologically and molecularly distinguishable in vertebrates, this is not the case in Drosophila. Ana2-GFP is the first fly protein shown to localize asymmetrically to mother and daughter centrioles in this manner (Stevens, 2010).
Interestingly, as spermatocytes progressed through meiosis I, this centriolar asymmetry became less pronounced, and this appeared to reflect the selective loss of GFP-Ana2 from the daughter centriole, bringing its levels down to that of the mother. Since overexpression of Ana2 can lead to centriole overduplication, Ana2 levels presumably must normally be tightly regulated to prevent the formation of extra centrioles (Stevens, 2010).
After exit from meiosis II, each spermatid inherits a single centriole, which acts as a basal body to nucleate the flagellar axoneme. Structural components of the centriole, like Ana3 (Stevens, 2009) and Drosophila pericentrin-like protein (D-PLP), continue to localize along the basal body. In contrast, Ana2, like the conserved duplication proteins (Blachon, 2009), was undetectable along the basal body. Ana2 did, however, colocalize with GFP-DSas-6 at the proximal centriole-like structure, a small nodule adjacent to the basal body that has been proposed to be an early intermediate in centriole formation (Stevens, 2010).
Intriguingly, Drosophila homologues have been identified for all the C. elegans centriole duplication factors except SAS-5, which has no clear homologues outside worms. Ana2 and SAS-5 are similar in size and have a single central coiled-coil domain, leading Goshima (2007) to suggest that Ana2 could be the Drosophila equivalent of SAS-5. As SAS-5 interacts with SAS-6 in worms, genetic interaction between Ana2 and DSas-6 was tested in flies (Stevens, 2010).
A small percentage of eggs laid by mothers carrying two copies of a Ubq-GFP-DSas-6 transgene assemble centriole-like structures (Peel, 2007). To see if this effect could be enhanced, flies were generated carrying one copy of Ubq-GFP-DSas-6 and one copy of Ubq-Ana2-GFP, neither of which alone (as a single copy) induces the assembly of centriole-like structures. Strikingly, almost all the unfertilized eggs laid by these females contained hundreds of large structures that stained for centriole markers, recruited PCM, and nucleated asters. Importantly this interaction was specific to Ana2 and DSas-6. In eggs from mothers carrying one copy of either Ubq-Ana2-GFP or Ubq-GFP-DSas-6 together with one copy of either Ubq-GFP-Sak, Ubq-Asl-GFP, or Ubq-DSas-4-GFP, at most a very small number of asters were observed in very few eggs (Stevens, 2010).
Interestingly, the centriole-like structures produced by overexpressing UASp-GFP-DSas-6 differ significantly from those resulting from the overexpression of GFP-Sak, DSas-4-GFP, Asl-GFP, or Ana2-GFP in that they are much larger and often appear ring-shaped, and that only one structure is contained within each aster (Peel, 2007; Rodrigues-Martins, 2007a). The structures in the eggs from females expressing both Ubq-GFP-DSas-6 and Ubq-Ana2-GFP were similar to this DSas-6 type. This suggests that Ana2-GFP acts to promote the assembly of GFP-DSas-6 into these structures (Stevens, 2010).
Having shown that Ana2 functionally interacts with DSas-6, physical interaction was sought. Using a yeast two-hybrid (Y2H) assay, it was found that Ana2 and DSas-6 interact and that the N-terminal region of DSas-6 and the C-terminal region of Ana2 are necessary and sufficient for this interaction. Moreover, like SAS-5, Ana2 also interacts with itself. Attempts to test whether Ana2 and DSas-6 associate in vivo were hindered by their low abundance. However, it was found that DSas-6 antibodies coimmunoprecipitated Ana2-GFP from S2 cells overexpressing Ana2-GFP. Collectively, the evidence of a specific functional and physical interaction between Ana2 and DSas-6 indicates that Ana2 likely represents the Drosophila functional orthologue of SAS-5 (Stevens, 2010).
Having shown that Ana2 is the likely SAS-5 functional orthologue in Drosophila, Ana2/SAS-5 orthologues were sought in other species. Using an iterative BLAST search, significant homology was found between Ana2 and the STIL or SIL protein family. Moreover, the reciprocal iterative BLAST search starting with zebrafish STIL identified Ana2 as the most similar Drosophila protein. Although vertebrate STIL family members are larger than Ana2 or SAS-5, all of these proteins share a short, central, coiled-coil domain. In addition, a particularly conserved region of ~90 aa toward the C terminus of Ana2 and STIL was identified, that was called the STil/ANa2 (STAN) motif. The STAN motif of Ana2 is 31% identical (48% similar) to that of zebrafish STIL. A divergent STAN motif can be detected in SAS-5, which is 12% identical (26% similar) to that of zebrafish STIL. Importantly, the STAN motif is within the regions of SAS-5 and Ana2 that interact with SAS-6 and DSas-6, respectively (Stevens, 2010).
Data from studies of STIL in mice, zebrafish, and humans are consistent with a function in centriole duplication, although this was not appreciated at the time of these studies. First, mitotic spindles often lack centrosomes in stil mutant zebrafish (Pfaff, 2007). Second, STIL mutant mice show defects characteristic of aberrant cilia function, such as randomized left-right asymmetry and neural tube abnormalities (Izraeli, 1999). Most importantly, it has recently been shown that mutations in human STIL cause primary microcephaly (MCPH), a congenital disorder characterized by reduced brain size (Kumar, 2009). Mutations in four other genes, MCPH1, CDK5RAP2, ASPM, and CPAP/CENPJ, are known to cause MCPH, and all are centrosomal proteins, strongly suggesting that STIL is required for efficient centrosome function in humans (Stevens, 2010).
This study has show that of eight centrosomal proteins identified as potential duplication factors in Drosophila tissue culture cells, only two, Asl and Ana2, appear to be able to induce de novo formation of centriole-like structures in unfertilized eggs. Asl has recently been shown to be essential for centriole duplication (Blachon, 2009), and this study provides evidence that Ana2 is also a key centriole duplication factor. Thus, Ana2 and Asl join Sak, DSas-6, and DSas-4 to make up a module of just five proteins known to drive centriole duplication in flies (Stevens, 2010).
These data strongly suggest that the Ana2/STIL family of centrosomal proteins are the long-sought functional orthologues of SAS-5. Thus, four of these five components (Sak/ZYG-1, DSas-6/SAS-6, Ana2/SAS-5, and DSas-4/SAS-4) are functionally conserved between flies and worms. Moreover, three of these proteins are required for centriole duplication in humans, whereas the fourth, SAS-5/Ana2/STIL, also appears likely to be required for this process in vertebrates (Stevens, 2010).
Both flies and worms have an additional protein (SPD-2 in worms, Asl in flies) that appears to be essential for centriole duplication. Intriguingly, both SPD-2 are not only required for centriole duplication, but also for PCM recruitment. There is evidence that the PCM promotes centriole duplication, so SPD-2 and Asl could play a more indirect role in centriole duplication via their ability to recruit PCM. Alternatively, both proteins may act directly in centriole duplication, with the function of SPD-2 in worms perhaps being performed by Asl in flies (Stevens, 2010).
In summary, this study shows that Ana2 acts as a centriole duplication factor in Drosophila and is likely to have a conserved role in other species. Overall, centriole duplication appears to be a highly conserved process, at the heart of which is a small number of key proteins. The challenge will now be to tease apart how these components cooperate to build a centriole of the right size, in the right place, and at the right time (Stevens, 2010).
Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila Anastral spindle-2 (Ana2) is a conserved centriole duplication factor. While recent work demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. This study examined the Ana2-LC8 interaction and mapped two LC8-binding sites within Ana2's central region, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 mμM) while site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 mμM). Both LC8-binding sites flank a predicted ~34-residue alpha-helix. Two independent atomic structures are presented of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form beta-strands that extend a central composite LC8 beta-sandwich. LC8 recognizes the signature TQT motif in Ana2's first LC8 binding site, forming extensive van der Waals contacts and hydrogen bonding with the peptide, while the Ana2 site 2 TQC motif forms a uniquely extended beta-strand, not observed in other dynein light chain-target complexes. Size-exclusion chromatography coupled with multi-angle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization likely enhances Ana2's avidity for centriole binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis (Slevin, 2014).
Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4; see Drosophila SAK) counterpart in other organisms. Although Plk4's centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. This study shows that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent (Dzhindzhev, 2014).
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
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
date revised: 5 February 2015
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