Most animal cells contain structures known as centrioles. Typically, a cell that is not dividing contains a pair of centrioles. But when a cell prepares to divide, the centrioles are duplicated. The two pairs of centrioles then organize the scaffolding that shares the genetic material equally between the newly formed cells at cell division.

Centriole assembly is tightly regulated and abnormalities in this process can lead to developmental defects and cancer. Centrioles likely contain several hundred proteins, but only a few of these are strictly needed for centriole assembly. New centrioles usually assemble from a cartwheel-like arrangement of proteins, which includes a protein called SAS-6. Previous work has suggested that in the fruit fly Drosophila melanogaster, Sas-6 can only form this cartwheel when another protein called Ana2 is also present, but the details of this process are unclear.

Now, Cottee, Muschalik et al. have investigated potential features in the Ana2 protein that might be important for centriole assembly. These experiments revealed that a region in the Ana2 protein, called the ‘central coiled-coil domain’, is required to target Ana2 to centrioles. Furthermore, purified coiled-coil domains were found to bind together in groups of four (called tetramers). Cottee, Muschalik et al. then used a technique called X-ray crystallography to work out the three-dimensional structure of one of these tetramers and part of the Sas-6 protein with a high level of detail. These structures confirmed that Sas-6 proteins also associate with each other.

When fruit flies were engineered to produce either Ana2 or Sas-6 proteins that cannot self-associate, the flies' cells were unable to efficiently make centrioles. Furthermore, an independent study by Rogala et al. found similar results for a protein that is related to Ana2: a protein called SAS-5 from the microscopic worm Caenorhabditis elegans.

Further work is needed to understand how Sas-6 and Ana2 work with each other to form the cartwheel-like arrangement at the core of centrioles.

Centrioles are complex microtubule (MT) based structures that are required for the formation of centrosomes and cilia/flagella. These organelles have many important functions in cells, and their dysfunction has been linked to a plethora of human pathologies, ranging from cancer to microcephaly to obesity (Nigg and Raff, 2009; Bettencourt-Dias et al., 2011). Thus, understanding how these organelles assemble and function is an important goal of both basic and biomedical research.

Although several hundred proteins are thought to be concentrated at centrioles, only a small number appear to form a conserved ‘core’ pathway that is essential for centriole assembly (Delattre et al., 2006; Pelletier et al., 2006; Gönczy, 2012). During canonical centriole duplication, the protein kinase Plk4/Sak/ZYG-1 is recruited to the mother centriole by SPD-2 in worms (Delattre et al., 2006; Pelletier et al., 2006; Shimanovskaya et al., 2014), by Asterless (Asl) in flies (Blachon et al., 2008; Dzhindzhev et al., 2010), or by a combination of the two (Cep192 and Cep152, respectively) in humans (Cizmecioglu et al., 2010; Hatch et al., 2010; Kim et al., 2013; Sonnen et al., 2013). The protein kinase recruits STIL/Ana2/SAS-5 and Sas-6 to a single site on the side of the mother centriole where they assemble with CPAP/Sas-4 into a cartwheel structure that helps to establish the ninefold symmetry of the centriole (Dammermann et al., 2004; Delattre et al., 2004; Leidel et al., 2005; Nakazawa et al., 2007; Peel et al., 2007; Strnad et al., 2007; Stevens et al., 2010a; Tang et al., 2011; Arquint et al., 2012). CPAP/Sas-4 can interact with tubulin (Hung et al., 2004) and is required to recruit the centriole MTs to the outer region of the cartwheel (Pelletier et al., 2006), possibly working together with Cep135/Bld10 (Hiraki et al., 2007; Lin et al., 2013)—although no homologue of this protein has been identified in worms, and it does not appear to be essential for centriole duplication in flies (Carvalho-Santos et al., 2012; Mottier-Pavie and Megraw, 2009; Roque et al., 2012).

Great progress has been made recently in understanding how these proteins interact and how these interactions are regulated to ensure that a new centriole is only formed at the right place and at the right time. In particular, the crystal structure of Sas-6 from several species has revealed how this protein forms a dimer through its C-terminal coiled-coil domain (C–C) that can then further homo-oligomerise through an N-terminal headgroup interaction (N–N) to form a ring structure from which the C–C domains emanate as spokes (Kitagawa et al., 2011; van Breugel et al., 2011, 2014; Hilbert et al., 2013). This Sas-6 ring structure can be modelled into EM tomographic reconstructions of the cartwheel from Trichonympha centrioles (Guichard et al., 2012, 2013), strongly suggesting that these Sas-6 rings form the basic building blocks of the cartwheel. In support of this hypothesis, mutant forms of Sas-6 that cannot homo-oligomerise through the N–N interaction are unable to support efficient centriole duplication (Kitagawa et al., 2011; van Breugel et al., 2011), although they can still target to centrioles, a function that seems to rely on the C–C domain (Fong et al., 2014; Keller et al., 2014).

A crystal structure of the interface between Ana2/STIL and Sas-4/CPAP has also recently been solved (Cottee et al., 2013; Hatzopoulos et al., 2013), as has the interaction interface between Plk4 and both Cep192/SPD-2 and Cep152/Asl (Park et al., 2014); mutations that perturb these interactions in vitro perturb centriole duplication in vivo, indicating that these interactions are also essential for centriole duplication. More recently, it has been shown that Plk4 can recruit STIL to centrioles in human cells (Ohta et al., 2014; Kratz et al., 2015) and that Plk4/Sak can phosphorylate the conserved STIL/Ana2 (STAN) domain in STIL/Ana2 proteins in humans and flies, thereby promoting the interaction of the STAN domain with Sas-6 (Dzhindzhev et al., 2014; Ohta et al., 2014; Kratz et al., 2015). Mutant forms of STIL/Ana2 that could not be phosphorylated strongly perturbed Sas-6 recruitment to centrioles and centriole duplication. Together, these studies have shed important light on the molecular mechanisms of centriole assembly, but many important questions remain.

In particular, it has been proposed that the homo-oligomerisation properties of Sas-6 establish the ninefold symmetry of the centriole (Kitagawa et al., 2011), and, remarkably, a ninefold symmetric ring structure is formed in crystallo by Leishmania major Sas-6 (van Breugel et al., 2014). However, although Sas-6 oligomers appear to have a propensity towards ninefold symmetry, Sas-6 proteins spontaneously assemble into oligomers of varying stoichiometry in vitro (Kitagawa et al., 2011; van Breugel et al., 2011), suggesting that the homo-oligomerisation properties of Sas-6 alone may be insufficient to enforce the rigorous ninefold symmetry that is observed in centrioles from virtually all species (Cottee et al., 2011). Additionally, recent Cryo-EM analysis suggests that the basic building block of the cartwheel stack is not a single ring and spoke structure, but rather a pair of rings that sit on top of one another: these rings do not make direct contact with each other, but are joined in the more peripheral regions through their spokes (Guichard et al., 2012, 2013). Our current knowledge of Sas-6 self-association cannot explain this important feature of the cartwheel structure.

We previously showed that overexpressed Sas-6 can form higher-order aggregates in Drosophila spermatocytes, but these aggregates only adopt a cartwheel-like structure when Ana2 is also overexpressed (Stevens et al., 2010b), and the STIL/Ana2 protein family is essential for the proper recruitment of Sas-6 to centrioles (Dzhindzhev et al., 2014; Ohta et al., 2014). We reasoned therefore, that Ana2 was likely to also play an important part in determining the structure of the central cartwheel. We set out to investigate the potential structural features of Ana2 that might be important for centriole assembly.

It is now widely accepted that the structure of the centriole cartwheel is formed around a core of 9 Sas-6 dimers that homo-oligomerise to form a ring structure (Cottee et al., 2011). Sas-6 molecules can form such ninefold symmetric rings in the absence of any other proteins in vitro, and mutations that perturb the ability of Sas-6 to homo-oligomerise in vitro strongly perturb centriole assembly in vivo (Kitagawa et al., 2011; van Breugel et al., 2011, 2014). We previously showed, however, that overexpressed Sas-6 can only form cartwheel-like structures in fly spermatocytes when Ana2 is also overexpressed (Stevens et al., 2010b). Here we show that Ana2/STIL proteins also homo-oligomerise and that mutations that perturb the homo-oligomerisation of fly Ana2 in vitro also strongly perturb centriole assembly in vivo. Thus, Sas-6 homo-oligomerisation alone appears unable to drive efficient cartwheel assembly in vivo if Ana2 is unable to homo-oligomerise.

Our initial structure/function analysis of Ana2 revealed that the CCCD is important for the recruitment of Ana2 to centrioles. This is consistent with the recent discovery that the CCCD in human STIL interacts with Plk4 and is important for STIL recruitment to centrioles (Ohta et al., 2014; Kratz et al., 2015). In both flies and vertebrates, Sak/Plk4 can also phosphorylate the STAN domain of Ana2/STIL, promoting its interaction with Sas-6, and allowing it to recruit Sas-6 to the newly forming centriole (Dzhindzhev et al., 2014; Ohta et al., 2014; Kratz et al., 2015). Interestingly, we found that although the STAN domain could not localise Ana2 to centrioles in fly embryos in the absence of the CCCD, the centriolar localisation of Ana2 was much weaker in the absence of the STAN domain, suggesting that an interaction with Sas-6 is required for robust Ana2 localisation in flies. This result is in agreement with the finding that fragments of STIL containing both the CCCD and the STAN domain interact most strongly with Plk4 (Kratz et al., 2015), but contrasts with reports in human cells where deleting the STAN domain did not affect STIL localisation to centrioles (Ohta et al., 2014) and in fly cultured cells, where the depletion of Sas-6 by RNAi did not detectably perturb the centriole localisation of Ana2 (Dzhindzhev et al., 2014). These latter results suggest that the proper recruitment of Ana2/STIL to centrioles is independent of the STAN domain's interaction with Sas-6. Our findings suggest, however, that although Plk4 may initially recruit some Ana2 to centrioles without Sas-6, the subsequent incorporation of Ana2 into the new centriole is dependent upon the successful assembly of a cartwheel structure, and this cannot occur without Sas-6. In addition, although Plk4 can clearly recruit Ana2/STIL to centrioles in flies and humans, in worms ZYG-1 (the Plk4 functional homologue) can directly recruit SAS-6 (Lettman et al., 2013). Thus, the molecular detail of the interactions between Plk4/Sak/ZYG-1, Sas-6 and Ana2/STIL/SAS-5 involved in cartwheel assembly remain to be fully elucidated, and may vary between different cell types and species.

The Ana2 CCCD tetramerises as a symmetric, parallel four helical bundle. Such structures are relatively rare in cytoplasmic proteins: most examples in the PDB are either engineered peptides, extensions of larger domains such as tetrameric membrane-associated receptors, or occur within a single polypeptide (and so are not oligomerisation domains). Although Ana2/STIL/SAS-5 proteins are highly diverged, a CCCD is found in all family members described to date (Figure 2A). Our data suggests that the human STIL CCCD can also form tetramers, although it does so more weakly than the fly CCCD; we speculate that tetramerisation via the CCCD could be a common feature of Ana2/STIL proteins. A fragment of C. elegans SAS-5 also behaves as a tetramer, although the relevant oligomerisation domain has not been identified (Shimanovskaya et al., 2013). It will be particularly interesting to test whether the C. elegans SAS-5 CCCD forms a tetramer, as this organism appears to form a spiral cartwheel rather than a flat-ring cartwheel (Hilbert et al., 2013). It was recently proposed that fly Ana2 can form tetramers through a different mechanism that is dependent on an interaction with dynein light chain (LC8) (Slevin et al., 2014). This interaction may be important for spindle orientation, rather than centriole duplication (Wang et al., 2011), and our results suggest that the observed tetramerisation was likely driven by the CCCD rather than the interaction with LC8.

We also examined the structure of Drosophila Sas-6 and confirmed that, similar to other Sas-6 orthologues, it associates via two self-interaction interfaces, a C-terminal coiled-coil dimerization (C–C), and an N-terminal headgroup oligomerization (N–N). The interface between the headgroup and the C–C was similar to that seen in most other species (Kitagawa et al., 2011; van Breugel et al., 2011, 2014), and different to that observed in C. elegans (Hilbert et al., 2013) strongly suggesting that Drosophila Sas-6 assembles into a canonical, flat ring structure. In agreement with previous studies (Kitagawa et al., 2011; van Breugel et al., 2011), we found that mutations that perturb the ability of Drosophila Sas-6 to homo-oligomerise through the N–N interface in vitro (GFP-Sas-6-F143D) cannot support efficient centriole duplication in vivo, and this is also true for mutations that perturb the ability of Ana2 to tetramerise in vitro (Ana2-CCA-GFP). Most importantly, however, we note that both these mutant proteins can support the assembly of some centrosomes, or CLSs, that can recruit other centriole and centrosome proteins and that often concentrate at spindle poles. We suspect that this is because centriole assembly is normally driven by a complex set of interactions between proteins such as Plk4, Sas-6, Ana2/STIL, Sas-4 and Cep135/Bld10 so that some residual (although possibly abnormal) centriole assembly is still possible even if one of these interaction interfaces is perturbed.

We predicted that GFP-Sas-6-F143D might act as a dominant-negative in cells, forming hetero-dimers with the WT protein that can incorporate into the cartwheel through the WT subunit, but which cannot then support further cartwheel assembly (Figure 8A). Surprisingly, although GFP-Sas-6-F143D appears to be overexpressed in embryos, brains and testes, it only had a dominant-negative effect on centriole duplication in early embryos (although the centrioles in spermatocytes were slightly, but significantly, shorter in the presence of GFP-Sas-6-F143D). These data suggest that WT-mutant heterodimers can transiently incorporate into the cartwheel and perturb assembly but, if given enough time, the heterodimers will dissociate and eventually a cartwheel can assemble from the pool of WT–WT homodimers. This would explain why GFP-Sas-6-F143D has no dramatic dominant-negative effect in somatic cells (where centriole assembly can presumably occur relatively slowly during an S-phase period that can last several hours) but has a dramatic dominant-negative effect in syncytial embryos (where centriole assembly must be completed during an S-phase that lasts only a few minutes). If this interpretation is correct, it implies that partially assembled cartwheel rings must be relatively stable structures that can be maintained in the partially assembled state until enough WT–WT homodimers have been incorporated to complete ring assembly.

Interestingly, overexpressed Ana2-CCA-GFP had no detectable dominant-negative effect on centriole duplication, even in rapidly dividing syncytial embryos. We speculate that this is because Ana2 is recruited to centrioles as a tetramer; thus, monomeric Ana2-CCA-GFP molecules would not efficiently compete with WT tetramers for centriole binding sites, perhaps because they lack the avidity of the tetramer. The Ana2-CCA-GFP molecules would also not ‘poison’ the WT molecules, as they would be unable to tetramerise with them. It is tempting to further speculate that two dimers of Sak/Plk4 (Slevin et al., 2012; Park et al., 2014; Shimanovskaya et al., 2014) might function as the centriole binding sites for the Ana2 tetramer.

The Sas-6 crystal structure can be modelled with high precision into the Cryo-EM tomographic map of the Trichonympha cartwheel structure (Guichard et al., 2013). Ana2/STIL proteins contain essential CR2 and STAN domains that interact with Sas-4/CPAP and Sas-6, respectively: these domains are connected to the CCCD by extended, unconserved (and probably relatively unstructured) linkers. Thus, in flies, the Ana2 CCCD parallel tetramer tethers the CR2 and STAN domains in a particular geometry and stoichiometry. In Figure 9 we present several models of how the Ana2 tetramer might be incorporated into the Trichonympha cartwheel structure. An interesting feature of the cartwheel is that its basic building block is two Sas-6 rings assembled on top of each other that are held together by the convergence of their coiled-coil spokes at the outer region of the cartwheel (Figure 9). Every paired spoke comprises four molecules of Sas-6, so we favour the idea that the Ana2 tetramer might interact with these converged spokes, thus stabilising the basic, two ring, building block of the cartwheel (Figure 9A,B). If each Sas-6 ring has a strong, but not invariant, tendency to adopt a ninefold symmetric organization, having cartwheel assembly dependent on the simultaneous co-assembly of two rings, rather than just one, could dramatically increase the precision of ninefold symmetric ring assembly. Ana2 might also hold the Sas-6 molecules in an orientation that further favours the assembly of ninefold symmetric rings.

Figure 9

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Previous studies have indicated that STIL molecules can turnover at centrioles, suggesting that they are not ‘locked’ into the cartwheel structure (Vulprecht et al., 2012). In our favoured model (Figure 9A,B) Ana2 tetramers would help recruit Sas-6 dimers—and presumably also Sas-4 molecules (Cottee et al., 2013; Hilbert et al., 2013)—in a stoichiometry and conformation that would favour cartwheel assembly. Once incorporated, however, Sas-6 and Sas-4 molecules could quickly become ‘locked’ into the assembled cartwheel/centriole structure. Thus, Ana2 could be important for ensuring proper cartwheel assembly, but may not be required to maintain the assembled cartwheel structure. Although these models are attractive, several other arrangements of the Ana2 tetramer are also plausible (Figure 9), and more data will be required to identify the precise inter-molecular interactions that lead to the efficient assembly of the ninefold-symmetric cartwheel structure. Nevertheless, our findings indicate that the Ana2 tetramer plays an important part in cartwheel assembly in flies.

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Author details

1. Matthew A Cottee

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

   Contribution

   MAC, Conception and design, Purified protein constructs, Carried out biophysical analyses, Crystallised and solved the Ana2 CCCD and Sas-6 structures, Cloned and purified RNA constructs, Cloned Ana2 and Sas-6 constructs for generation of transgenic fly lines, Contributed to the writing of the manuscript

   Contributed equally with

   Nadine Muschalik

   Competing interests

   The authors declare that no competing interests exist.

2. Nadine Muschalik

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

   Present address

   Division of Cell Biology, MRC-Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   NM, Conception and design, Carried out the Drosophila in vivo work, Contributed essential unpublished data, Contributed to the writing of the manuscript

   Contributed equally with

   Matthew A Cottee

   Competing interests

   The authors declare that no competing interests exist.

3. Steven Johnson

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

   Contribution

   SJ, Crystallised and solved the Sas-6 structure, Experimental design, Carried out biophysical analyses, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Joanna Leveson

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

   Contribution

   JL, Cloned and purified RNA constructs, Contributed the RNA injection experiments, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Jordan W Raff

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

   Contribution

   JWR, Conception and design, Contributed to the writing of the manuscript, Analysis and interpretation of data

   For correspondence

   jordan.raff@path.ox.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

6. Susan M Lea

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

   Contribution

   SML, Conception and design, Crystallised and solved the Ana2 CCCD and Sas-6 structures, Contributed to the writing of the manuscript

   For correspondence

   susan.lea@path.ox.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

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