InteractiveFly: GeneBrief

spindle assembly abnormal 6: Biological Overview | References

Gene name - spindle assembly abnormal 6

Synonyms -

Cytological map position - 99D1-99D1

Function - cytoskeleton

Keywords - centriole assembly, cytoskeleton

Symbol - sas-6

FlyBase ID: FBgn0039731

Genetic map position - 3R:25,821,079..25,822,760 [+]

Classification - Smc domain

Cellular location - centriole

NCBI link: EntrezGene
sas-6 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.
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.
Jo, K. H., Jaiswal, A., Khanal, S., Fishman, E. L., Curry, A. N. and Avidor-Reiss, T. (2019). Poc1B and Sas-6 function together during the atypical centriole formation in Drosophila melanogaster. Cells 8(8). PubMed ID: 31387336
Insects and mammals have atypical centrioles in their sperm. However, it is unclear how these atypical centrioles form. Drosophila melanogaster sperm has one typical centriole called the giant centriole (GC) and one atypical centriole called the proximal centriole-like structure (PCL). During early sperm development, centriole duplication factors such as Ana2 and Sas-6 are recruited to the GC base to initiate PCL formation. The centriolar protein, Poc1B, is also recruited at this initiation stage, but its precise role during PCL formation is unclear. This study shows that Poc1B recruitment was dependent on Sas-6, that Poc1B had effects on cellular and PCL Sas-6, and that Poc1B and Sas-6 were colocalized in the PCL/centriole core. These findings suggest that Sas-6 and Poc1B interact during PCL formation. Co-overexpression of Ana2 and Sas-6 induced the formation of ectopic particles that contained endogenous Poc1 proteins and were composed of PCL-like structures. These structures were disrupted in Poc1 mutant flies, suggesting that Poc1 proteins stabilize the PCL-like structures. Lastly, Poc1B and Sas-6 co-overexpression also induced the formation of PCL-like structures, suggesting that they can function together during the formation of the PCL. Overall, these findings suggest that Poc1B and Sas-6 function together during PCL formation.
Persico, V., Migliorini, M., Callaini, G. and Riparbelli, M. G. (2020). The Singularity of the Drosophila Male Germ Cell Centriole: The Asymmetric Distribution of Sas4 and Sas6. Cells 9(1). PubMed ID: 31947732
Drosophila spermatocytes have giant centrioles that display unique properties. Both the parent centrioles maintain a distinct cartwheel and nucleate a cilium-like region that persists during the meiotic divisions and organizes a structured sperm axoneme. Moreover, the parent centrioles are morphologically undistinguishable, unlike vertebrate cells in which mother and daughter centrioles have distinct structural features. However, immunofluorescence analysis of the parent centrioles in mature primary spermatocytes revealed an asymmetric accumulation of the typical Sas4 and Sas6 proteins. Notably, the fluorescence intensity of Sas4 and Sas6 at the daughter centrioles is greater than the intensity found at the mother ones. In contrast, the centrioles of wing imaginal disc cells display an opposite condition in which the loading of Sas4 and Sas6 at the mother centrioles is greater. These data underlie a subtle asymmetry among the parent centrioles and point to a cell type diversity of the localization of the Sas4 and Sas6 proteins (Persico, 2020).
Gartenmann, L., Vicente, C. C., Wainman, A., Novak, Z. A., Sieber, B., Richens, J. H. and Raff, J. W. (2020). Sas-6, Ana2 and Sas-4 self-organise into macromolecular structures that can be used to probe centriole/centrosome assembly. J Cell Sci. PubMed ID: 32409564
Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins-Plk4, Ana2/STIL, Sas-6, Sas-4/CPAP or Asl/Cep152-leads to the absence of centrioles. This study used Sas-6/Ana2 Particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs/embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, while Asl helps to initiate SAP assembly, but is not required for SAP growth. Although not centrioles, SAPs recruit and organize many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl/Cep152 to efficiently recruit pericentriolar material (PCM), but Spd-2/Cep192 can promote some PCM assembly independently of Asl/Cep152. These observations provide new insights into the pathways of centriole and centrosome assembly.


Centrioles are microtubule-based cylindrical structures that exhibit 9-fold symmetry and facilitate the organization of centrosomes, flagella, and cilia. Abnormalities in centrosome structure and number occur in many cancers. Despite its importance, very little is known about centriole biogenesis. Recent studies in C. elegans have highlighted a group of molecules necessary for centriole assembly (Bettencourt-Dias, 2007; Leidel, 2005a). ZYG-1 kinase recruits a complex of two coiled-coil proteins, SAS-6 and SAS-5, which are necessary to form the C. elegans centriolar tube, a scaffold in centriole formation. This complex also recruits SAS-4, which is required for the assembly of the centriolar microtubules that decorate that tube. This study shows that Drosophila SAS-6 is involved in centriole assembly and cohesion. Overexpression of DSAS-6 in syncitial embryos leads to the de novo formation of multiple microtubule-organizing centers (MTOCs). Strikingly, the center of these MTOCs did not contain centrioles, as described previously for SAK/PLK4 overexpression. Instead, tube-like structures are present, supporting the idea that centriolar assembly starts with the formation of a tube-like scaffold, dependent on DSAS-6 (Pelletier, 2006). In DSAS-6 loss-of-function mutants, centrioles failed to close and to elongate the structure along all axes of the 9-fold symmetry, suggesting modularity in centriole assembly. It is proposed that the tube is built from nine subunits fitting together laterally and longitudinally in a modular and sequential fashion, like pieces of a layered 'hollow' cake (Rodrigues-Martins, 2007b).

Drosophila has one SAS-6 protein, named in this study DSAS-6. DSAS-6 localizes to centrioles and its absence leads to a reduction in centrosome number in S2 cells. A 16-fold reduction in the total number of centrosomes was observed in larval brains of a genetically null DSAS-6 mutant, together with the appearance of smaller centrosomes (Rodrigues-Martins, 2007b).

To further understand the role of DSAS-6 in centrosome biogenesis, Drosophila testes were examined. Whereas G2 wild-type spermatocytes contained four centrioles per cell, DSAS-6 mutant spermatocytes showed a reduced number of centrioles, consistent with a role for DSAS-6 in centriole duplication. The absence of centrioles in some of these cells was verified by transmission electron microscopy (TEM) (Rodrigues-Martins, 2007b).

Smaller centrioles were found in testes as judged by both the fluorescence of GFP-PACT and γ-tubulin, centriolar markers for spermatocytes in G2. These small centrioles seemed to result from a failure in centriole elongation rather than centriole fragmentation because more than the expected wild-type number of GFP-PACT bodies per cell was never observed, even at later stages of development, in contrast to what has been reported upon centriole fragmentation. To further test whether elongation was affected, the size of centrioles was measured in different developmental stages as they elongate. In contrast to the wild-type, DSAS-6 mutant centrioles showed no significant growth with development, indicative of defects in elongation. Additionally, it has been previously shown that despite the efficiency of DSAS-6 RNAi, centriole disappearance shows slow kinetics (Rodrigues-Martins, 2007b), similarly to previous reports for SAK/PLK4 (Bettencourt-Dias, 2005). This is consistent with a role for DSAS-6 in centriole assembly rather than in maintaining centrosome integrity. However, the possibility of slow loss of centriole integrity giving rise to nondetectable centriolar fragments cannot be ruled out (Rodrigues-Martins, 2007b).

TEM analysis of centrioles within mutant cysts revealed a variety of defects in centriole assembly. These included the absence of adjacent microtubule triplets and the inverted relative positioning of centriolar microtubules with respect to PCM. Examples were also observed of the 'transient' cilia that emanate from primary spermatocytes in which distal but not proximal parts of the basal body were missing, also pointing to a defect in centriole elongation (Rodrigues-Martins, 2007b).

Despite their small size, centrioles in DSAS-6 mutants acted as MTOCs, recruiting PCM proteins such as γ-tubulin and CNN and nucleating astral and spindle microtubules. Meiotic spindles lacking centrioles frequently showed abnormalities, leading to abnormal meiotic products. This reinforces earlier findings of the important role played by normal centrioles in male meiosis (Rodrigues-Martins, 2007b).

It was observed that 80% of the centriole pairs in DSAS-6 mutant cysts in G2 were disengaged and did not adopt the V-shape shown by 100% of their engaged wild-type counterparts. Such mutant centriole pairs might be expected to lead to the formation of multipolar spindles, as observed in DSAS-6 mutant brains. This suggests that DSAS-6 participates, directly or indirectly, in centriole engagement. DSAS-6 localized along centrioles, and its signal was stronger at the proximal and distal parts of the centrioles relative to the centriolar markers GFP-PACT and DSPD-2, consistent with it having roles in centriole engagement and elongation (Rodrigues-Martins, 2007b).

After meiosis, spermatocytes engage in their differentiation program whereby centrioles become basal bodies that are extended into the canonical structure of the flagellar axoneme. DSAS-6 mutants showed a variety of structural abnormalities throughout spermatid differentiation including abnormally shaped nuclei, abnormal flagella organization. 42% of the axonemes analyzed were incomplete, with 1 to 5 consecutive microtubule doublets missing. Interestingly, 9% of the axonemes analyzed showed loss of the 9-fold symmetry by the presence of additional doublets (Rodrigues-Martins, 2007b).

SAK/PLK4-induced de novo centriole biogenesis is dependent on DSAS-6 (Rodrigues-Martins, 2007a). These findings led to examination of whether overexpression of DSAS-6 in the female germline would be sufficient to induce the formation of multiple centrioles. Despite the presence of many GFP-DSAS-6 aggregates, these did not contain centriole or PCM markers and centrioles were lost normally during oogenesis. The acentriolar meiotic spindles had normal morphology with apparently no additional D-PLP bodies or microtubule asters, as would be expected if there were ectopic centrioles. When embryos and eggs overexpressing GFP-DSAS-6 were examined, microtubule asters were found unattached to spindles, containing centriole and centrosome markers, including GFP-DSAS-6 protein foci, γ-tubulin, D-PLP, DSAS-4, and CNN. Many GFP-DSAS-6 protein aggregates were found that did not colocalize with centrosome markers. These may reflect nonfunctional aggregates. GFP-DSAS-6 was able to form MTOCs de novo, because they appeared in unfertilized eggs. In embryos, these structures could b detected already by the end of the second mitosis, some of them in groups at the poles of the mitotic spindles, others forming far away (Rodrigues-Martins, 2007b).

By using TEM, converging cytoplasmic microtubules and dense PCM material were observed in embryos and unfertilized eggs overexpressing GFP-DSAS-6. Normal centrioles were not found at their foci, but instead tube-like structures, hollow at the TEM level, were detected in longitudinal sections, and were detected after serial transverse sectioning in both embryos and unfertilized eggs. Some of these had a small number of microtubule doublets associated. Some of the tubes showed a wrong curvature, as if parts of the tube were brought together in the wrong orientation. Together, this suggests that DSAS-6 overexpression leads to formation of abnormal structures possibly related to potential centriole assembly intermediates that can recruit PCM and thereby provide a focus for nucleation of cytoplasmatic microtubules (Rodrigues-Martins, 2007b).

A clear reduction was observed in the number of centrioles and centrosomes after RNAi in S2 cells and in a null Drosophila SAS-6 mutant, as expected from the described role of its orthologs in C. elegans and humans. Additionally, only a small proportion of the centrioles in DSAS-6 mutant testes was able to attain full size (~25% versus 87% in the wild-type), suggesting defects in the elongation of those structures. Whether the ability to form such partial structures reflects residual DSAS-6 protein in the mutant (maternal protein perdures to this stage in many Drosophila mitotic mutants) or partial redundancy of function with another molecule is not clear at present. The comparable structural defects in mutant axonemes are likely to reflect similar abnormalities in the basal bodies that act as their templates. However, the possibility of a role for DSAS-6 in axoneme formation cannot be fully discarded (Rodrigues-Martins, 2007b).

What might be the role of DSAS-6 in centriole formation? The fact that electron-dense, tube-like structures were observed upon overexpression of GFP-DSAS-6 and the fact that SAS-6 is required in C. elegans to form a tubular centriole scaffold (Pelletier, 2006) suggest a universal role for this molecule in the formation of a tube-like centriole intermediate. The appearance of nonclosed tubes and of several open tubes linked to each other at the edges suggests that tubes are assembled in a modular way. What might be the unit/module of assembly of such tubes? The analysis of centrioles and axonemes in DSAS-6 mutants allowed assessment of the degree of modular assembly, by using as a readout the structural microtubules associated with this putative inner tube. The most commonly observed incomplete centrioles and axonemes in the mutants were missing 1 to 5 adjacent triplets/doublets of microtubules. Also, axonemes were observed with 10 or 11 MT doublets. This suggests that the unit of assembly of the tube is the common denominator in those structures, one-ninth of the centriole (Rodrigues-Martins, 2007b).

The nature of the tube is at present not clear. The possibility that DSAS-6 is a structural component of the tube cannot be discarded because it is present there. However, an alternative possibility is that DSAS-6 is present in the structure not as the main structural component, but to regulate the binding of the modules in the correct orientation into the tube. DSAS-6 absence would lead to a failure of structural modules to bind, or to binding in the wrong orientation, as was observed. DSAS-6 overexpression would connect some of those modules in the wrong position, leading to the formation of abnormally shaped and larger tubes. The de novo formation of abnormal centriole-like structures with few centriole microtubules has been described before in a dominant-negative dynein Drosophila mutant, suggesting that dynein may be involved in the transport of centriole precursor structures and/or molecules involved in centriole duplication, such as DSAS-6 (Rodrigues-Martins, 2007b).

There is a lack of cohesion between both centrioles within each centrosome in DSAS-6 mutant testes and brains, suggestive of a weakness in the link between mother and daughter centrioles, which is only normally lost at the end of meiosis I/mitosis. DSAS-6 may regulate that link directly or indirectly. Drug treatments and molecular changes that affect tubulin polymerization give rise to phenotypes similar to the ones described in this study, suggesting that ultimately changes in tubulin stability may be at the origin of both assembly and cohesion phenotypes (Rodrigues-Martins, 2007b).

Previous work indicates that de novo MTOC formation is normally inhibited by the presence of centrosomes. Strikingly, DSAS-6 overexpression could induce the de novo formation of tube-like structures after fertilization, in the presence of a basal body/centriole, suggesting that excess of DSAS-6 can override normal blocks to de novo MTOC formation. The presence of supernumerary and abnormal MTOCs in many cancers serves as an alert to the importance of understanding possible negative outcomes of misregulation of DSAS-6 and other molecules involved in centriole biogenesis (Rodrigues-Martins, 2007b).

Revisiting the role of the mother centriole in centriole biogenesis

Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. Overexpression of SAK/PLK4 can induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two proteins required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly (Rodrigues-Martins, 2007a).

Centrioles are essential for the formation of cilia and flagella and for the organization of the centrosome. Normally, centrioles duplicate in coordination with the cell cycle. A new centriole, the daughter, arises orthogonally to each old one, the mother, in S phase. This led to the idea that the mother centriole templates the formation of the daughter. However, daughter centrioles do not incorporate a substantial proportion of the mother, and centrioles can also form de novo when existing centrioles are naturally lost during development or are physically removed, questioning the idea of the mother centriole as a template (Rodrigues-Martins, 2007a).

SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical centriole duplication and is necessary for centriole formation. The Caenorhabditis elegans ZYG-1 kinase, a homolog of SAK, is part of a conserved module of proteins, which also includes SAS-6 and SAS-4, necessary for the normal centriole duplication cycle. ZYG-1 is an upstream regulator in that process, a role consistent with formation of multiple centrioles in cultured cells following overexpression of active SAK kinase. The generation of multiple centrioles associated with high SAK expression also occurs physiologically in the olfactory mucosa. The Drosophila egg contains all the proteins necessary to make 213 centriole pairs (centrosomes). Centrioles are naturally eliminated from the oocyte cytoplasm in the course of development and provided to the egg in the form of the basal body of the sperm. Thus, the consequences of overexpressing SAK was studied in a cytoplasm that either contained centrioles (the embryo) or lacked them (the unfertilized egg) (Rodrigues-Martins, 2007a).

Embryos overexpressing SAK did not develop and were filled with free asters of microtubules not associated with spindles. Those asters were focused around Drosophila pericentrin-like protein (D-PLP)-containing structures, a centriolar and pericentriolar material (PCM) marker. These centrosomes first appeared in 15- to 30-min-old embryos and spread to fill the entire embryo after 2 to 3 hours. The observed supernumerary centrosomes led to abnormal mitotic progression and impaired embryonic development, as id observed upon microtubule depolymerization by colchicine treatment. To address the origins of those centrosomes, the very early stages of embryonic development were examined in embryos overexpressing SAK. Both the sperm aster around the incoming basal body and the first mitotic spindle were normal. However, at anaphase or telophase of the first mitosis, more than two centrosomes were observed at each pole, an indication of the onset of centrosome amplification. No other centrosomes were seen in the embryo at this stage. Moreover, it is estimated that a minimum of 3700 centrosomes (equivalent to 12 duplication cycles) were present after 60 min in embryos overexpressing SAK. After 60 min, a wild-type embryo only showed 128 centrosomes. Duplicating centrioles were observed in groups, suggesting they originated by duplication of a progenitor. Thus, upon fertilization of eggs overexpressing SAK, the basal body of the sperm enters an environment that promotes accelerated canonical duplication, overriding any existing controls that would normally couple the centrosome and chromosome cycles (Rodrigues-Martins, 2007a).

Uncoupling between centrosome and chromosome cycles occurs when embryos are arrested in S-phase-like conditions. However, this did not seem to be so in this case, because proliferating cell nuclear antigen (PCNA), which appears early in S phase, was not detected in DNA of SAK-overexpressing embryos (Rodrigues-Martins, 2007a).

It was next asked whether SAK could promote centriolar assembly in the absence of centrioles. Centrioles were lost normally in oocytes overexpressing SAK. Yet observations of unfertilized eggs at varying developmental intervals revealed free centrosomes in eggs overexpressing SAK that had exited meiosis II but never in wild-type eggs. Thus, in the absence of a basal body provided by the sperm, SAK can induce de novo formation of centrosomes. Whereas in embryos centrosomes appeared in a single cluster in the first mitotic spindle and spread throughout the cytoplasm, in unfertilized eggs they appeared scattered at random positions, including at the anterior and posterior poles. The formation of the first centrioles started later in eggs than in embryos [at 30 min, 0 amplification in eggs versus 51% amplification in embryos; after 1 hour, the amounts were 18% versus 89%, respectively], suggesting that centrioles take longer to be made in the absence of a template. However, once the first centrosomes had formed in eggs, their spreading in space and time was very similar to that seen in embryos, indicative of canonical biogenesis. Thus, once the first centrioles are formed de novo, they probably duplicate through the canonical pathway (Rodrigues-Martins, 2007a).

There is precedent for defects in de novo-formed centrioles. The presence of SAK and two other molecules required for centriole duplication was confirmed: DSAS-6 and DSAS-4. PCM components were also detected, including γ-tubulin, centrosomin (CNN), and centrosomal protein 190 (CP190). Moreover, electron microscopy showed that centrioles in both embryos and eggs overexpressing SAK were structurally normal. It also showed the presence of procentrioles next to the completed ones in both embryos and eggs, a result suggesting that SAK-induced centrioles can duplicate (Rodrigues-Martins, 2007a).

These results show that SAK is sufficient to induce both canonical and de novo centriole biogenesis. If both rely on self-assembly of the structure, the use of the same regulatory molecules would be predicted. The dependency of SAK-promoted centriole biogenesis on DSAS-4 and DSAS-6 was examined. Advantage was taken of the fact that centrioles can be eliminated from Drosophila tissue culture cells. After depletion of SAK in four rounds of RNA interference (RNAi) over a period of 16 days, more than 80% of the cells lacked centrioles, presumably because the remainder are diluted in each division cycle. Subsequent overexpression of SAK led to a clear increase in the number of cells with several centrosomes (from 4% to 48%). Depletion of DSAS-6 or DSAS-4 prevented SAK-induced centrosome biogenesis in cells with and without centrioles (Rodrigues-Martins, 2007a).

These results suggest that centriole biogenesis is a template-free self-assembly process that is locally triggered and regulated by molecules such as SAK, DSAS-6, and DSAS-4. What could be the role of the mother centriole? The presence of SAK at the centriole and the fact that assembly is faster in the presence of centrioles suggest that the mother centriole is not a bona fide template but a platform for regulatory molecules, hence catalyzing and regulating daughter centriole assembly. The establishment of that platform is probably less efficient in the absence of centrioles. The mother centriole could in principle establish a temporally and spatially regulated gradient of SAK activity, as demonstrated for RanGTP, a small guanosine triphosphatase involved in spindle assembly, perhaps counteracted in the cytoplasm by other molecules. These data also point to a role for centrioles in regulating total centriole number, because their presence precludes de novo formation. This is true even in a large embryo (~800 µm) containing very large amounts of SAK. Whether this indicates sequestering of active SAK or its substrates in existing centriolar structures or an active inhibitory effect of centrioles upon de novo assembly requires further study (Rodrigues-Martins, 2007a).

The regulation of SAK activity is essential in the control of centriole number and may be a parameter that is regulated according to cellular needs, because multiciliated cells of the respiratory tract have high SAK levels. The activity of SAK may be inhibited in the acentriolar female meiosis, as de novo centrosome formation only occurs after meiosis exit in eggs overexpressing SAK. Drosophila eggs and embryos should provide an ideal experimental system for further analyses of the control of centriole biogenesis and how it may go awry in cancer (Rodrigues-Martins, 2007a).

Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation

Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. This study investigated the effects of overexpressing the three proteins known to be required for centriole replication in Drosophila -- DSas-6, DSas-4, and Sak. By directly observing centriole replication in living Drosophila embryos, this study shows that the overexpression of GFP-DSas-6 can drive extra rounds of centriole replication within a single cell cycle. Extra centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of centriole-like structures in unfertilized eggs, which normally do not contain centrioles. These data suggest that the levels of centriolar DSas-6 determine the number of daughter centrioles formed during centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra centrioles in some tissues but not others, suggesting that centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of centriole-precursors that are normally present in the egg (Peel, 2007).

This study shows that DSas-6, like DSas-4 and Sak, is required for centriole duplication in Drosophila. Studying the effects of overexpressing each of the three proteins, the following is shown: first, the overexpression of DSas-6 in vivo can drive extra rounds of templated centriole replication within a single cell cycle. Second, the overexpression of these proteins induces the formation of extra centriole-like structures to varying extents in different tissues. Third, the overexpression of any of these proteins at high levels can drive the de novo formation of centriole-like structures in unfertilized eggs. The implications of each of these findings is discussed in turn (Peel, 2007).

It has previously been shown that the overexpression of Plk4/Sak in human cells leads to an accumulation of extra centrioles and HsSAS-6 appears to have a similar effect. Because these experiments were performed with fixed cultured cells, it was unclear how the extra centrioles formed and whether these proteins could drive centriole accumulation in vivo. In the current experiments, extra rounds of templated centriole replication driven by the overexpression of DSas-6 were directly visualized in vivo. Moreover, these extra centrioles appear to be fully functional because they organize PCM and MTs and, most importantly, they can undergo further rounds of replication in synchrony with the other centrioles in the embryo (Peel, 2007).

Recent studies in C. elegans have revealed that centriole replication requires the ordered activity of SPD-2, ZYG-1, SAS-5, and SAS-6, and finally SAS-4. The current findings demonstrate that DSas-6 levels are critical in determining the number of centrioles formed during centriole replication in Drosophila embryos. How might DSas-6 regulate centriole number during replication? One possibility is that, when overexpressed, DSas-6 is recruited normally to the mother centriole but is then inappropriately recruited to the newly formed daughter centriole, thereby inducing the formation of a 'granddaughter' centriole. Another possibility is that excessive recruitment of DSas-6 to the mother centriole expands the area where centrioles can form, thereby resulting in the generation of multiple daughter centrioles. Neither mechanism is mutually exclusive, and both of these configurations of centrioles have been observed in Drosophila somatic cells in which the inactivation of Cdk1 led to centriole overduplication (Vidwans, 2003; Peel, 2007 and references therein).

Extra rounds of templated centriole replication were not directly observed in Ubq-GFP-Sak embryos, but it is suspected that this is because the protein was expressed at very low levels in embryos. In larval brain cells and ovarian nurse cells, Sak was the most potent of the three replication proteins at inducing the formation of extra centriole-like structures. The formation of these extra structures required DSas-4, and the structures contained several centriole markers and could organize PCM markers and MTs. Nevertheless, EM studies will be required to confirm that these structures are true centrioles (Peel, 2007).

A priori, it is perhaps surprising that two different proteins can drive centriole overduplication, because only one protein would be expected to be rate limiting in any given system. The data suggest that it is the amount of DSas-6 at the centriole that determines the number of daughter centrioles formed during each round of replication (the 'litter' size), and it is suspected that overexpressed Sak can recruit extra DSas-6 to the centrioles even when DSas-6 is not overexpressed. The configuration of the extra centrioles in human cells overexpressing Plk4/Sak is consistent with this proposal, and the formation of these extra centrioles requires HsSAS-6. The observation that DSas-4 overexpression does not induce templated-centriole overduplication in any of the cell types examined is consistent with this hypothesis, because SAS-4 is recruited to centrioles only after ZYG-1 and SAS-6 in C. elegans. Overexpressed DSas-4 is presumably unable to recruit extra DSas-6 to the centrioles (Peel, 2007).

The results demonstrate that the overexpression of centriole duplication proteins can have different effects in different tissues. The overexpression of GFP-Sak or GFP-DSas-6 leads to an accumulation of extra centrioles in larval brain cells but not in larval spermatocytes. It seems unlikely that these differences result only from differing expression levels in the different tissues, because the Ubq promoter appears to drive higher levels of GFP-DSas-6 and DSas-4-GFP in the testes than in the brain. It is speculated, therefore, that additional mechanisms may regulate the activities of these proteins, and these mechanisms may differ between tissues (Peel, 2007).

Perhaps the most surprising of the observations is that the expression of any of the three fusion proteins at high levels can trigger the de novo formation of many hundreds of centriole-like structures in unfertilized eggs. EM studies will be required to see whether these structures are normal centrioles, but they all incorporate endogenous centriole markers and organize PCM and astral MTs. Nevertheless, there are clear morphological differences between the structures formed by the overexpression of GFP-DSas-6 and those formed by the overexpression of DSas-4-GFP and GFP-Sak. Interestingly, it has previously been shown that the expression of a dominant mutant form of dynein heavy chain, LaborcD, can lead to the rapid de novo formation of centriole-like structures in a manner very similar to that reported in this study. An EM analysis revealed that these structures were 'rudimentary centrioles' that consisted of hollow tubes that lacked any associated MTs. The de novo formation of centrioles in cultured cells also leads to the formation of centriole-like structures that, initially, do not have the normal appearance of centrioles at the EM level (Peel, 2007).

Whereas the de novo formation of centrioles in cultured cells is a slow process that occurs over several hours, the centriole-like structures that was observed in unfertilized eggs appear very rapidly upon egg deposition. Even in 30 min collections of both UAS-GFP-Sak and UAS-GFP-DSas-6 unfertilized eggs, it was found that >95% of the eggs had at least ~50 of these structures and most had several hundred structures that had already recruited PCM components and were nucleating MTs. Because the expression of these replication proteins does not lead to the abnormal persistence of centrioles during oogenesis, it is concluded that the centriolar components in these unfertilized eggs must be organized in such a way that they can be very rapidly assembled into centriole-like structures when the egg is deposited (Peel, 2007).

This is further supported by the observation that even DSas-4-GFP can induce the formation of centriole-like structures in unfertilized eggs. SAS-4 functions at a late step in centriole duplication, so it is unlikely that it could induce the de novo formation of centriole-like structures unless the centriolar components were already partially assembled. It is speculated that centriolar components normally have a tendency to transiently self-assemble into 'centriole precursors' in these eggs. The overexpression of any of the replication proteins can stabilize these precursors, allowing them to mature into centriole-like structures upon egg deposition (Peel, 2007).

These observations are consistent with the hypothesis that normal templated centriole replication may depend upon the presence of centriole-precursors in the cytoplasm. In this model, cells normally contain centriole precursors, but during replication only one of these becomes stabilized when it contacts the mother centriole, thereby allowing it to mature into a daughter centriole. In unfertilized Drosophila eggs, the overexpression of replication proteins may stabilize these centriole precursors throughout the egg, thereby circumventing the normal requirement that the centriole precursors contact the mother centriole to become stabilized (Peel, 2007).

SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole

Centrioles consist of nine-triplet microtubules arranged in rotational symmetry. This structure is highly conserved among various eukaryotic organisms and serves as the base for the ciliary axoneme. Recently, several proteins such as SAS-6 have been identified as essential to the early process of centriole assembly, but the mechanism that produces the 9-fold symmetry is poorly understood. In C. elegans and Drosophila, SAS-6 has been suggested to function in the formation of a centriolar precursor, a central tube that then assembles nine-singlet microtubules on its surface. However, the generality of the central tube is not clear because in many other organisms, the first structure appearing in the centriole assembly is not a tube but a flat amorphous ring or a cartwheel -- a structure with a hub and nine radiating spokes. This study shows that in Chlamydomonas the SAS-6 protein localizes to the central part of the cartwheel and that a null mutant of SAS-6, bld12, lacks that part. Intriguingly, this mutant frequently has centrioles composed of 7, 8, 10, or 11 triplets in addition to 9-triplet centrioles. It is presumed that, in many organisms, SAS-6 is an essential component of the cartwheel, a structure that stabilizes the 9-triplet structure (Nakazawa, 2007).

A new Chlamydomonas mutant, bld12, is deficient in establishing the 9-fold symmetry. Like other centriole-deficient mutants, this mutant displays abnormal nuclear segregation and lacks flagella when grown under normal conditions. However, ~10% of the cells produce one or two flagella when the cell walls are removed with autolysin. The motility of these flagella is variable, from complete paralysis to sporadic twitching to almost normal beating. When these flagella were observed by electron microcopy, a striking defect was found: The number of the outer-doublet microtubules frequently differed from nine. Of more than 10,000 axonemal cross sections examined, ~5% had 8 doublets, ~5% had 10 doublets, and ~0.1% had 11 doublets, whereas ~90% had 9 doublets. In contrast, a control measurement in pf14 (a mutant lacking radial spokes) detected only one abnormal axoneme in 1260 cross-section images. Striking defects were also found in the centriole, viewed either in whole cells or in the isolated nucleo-flagellar apparatus (NFAp) -- a complex of a nucleus, two mature centrioles (basal bodies), and their attached flagella. Most of the centrioles in the bld12 cells were split into fragments of one- to five-triplet microtubules; only < 20% of the total retained the circular arrangement of triplets. The number of triplets in the circular centrioles also varied: Of ~160 cross-section centriole images in an NFAp sample, ~3% had 7 triplets, ~13% had 8, ~70% had 9, ~13% had 10, and ~1% had 11. Serial-section analysis of the centriole-flagellum connection indicated that these circular centrioles, and not the split ones, apparently serve as the template for the assembly of axonemes with abnormal numbers of doublet microtubules. Nine-doublet axonemes were observed more frequently than nine-triplet centrioles, possibly because axonemes with aberrant numbers of doublets are short and therefore less frequently observed by thin-section electron microscopy than are nine-doublet axonemes. Another possibility is that axonemes with nine doublets are assembled on the basal bodies with seven or eight triplets by insertion of a doublet(s) that is not templated from a triplet. However, this latter mechanism cannot explain why the axonemes with 10 or 11 doublets occur less frequently than the centrioles with 10 or 11 triplets (Nakazawa, 2007).

The circular centrioles in bld12 have two classes of ultrastructural defects. First, triplets are often missing near the proximal end, indicating that some triplets are shorter than normal. This observation suggests that the triplets are partially depolymerized at the proximal ends. It might be that bld12 is deficient in some mechanism that stabilizes the proximal end of the centriolar microtubules. Second, these centrioles appear to lack the symmetrical organization of the cartwheel, a structure composed of three distinct parts: the hub, the inner spoke, and the spoke tip. In the bld12 centriole, the spoke tip, a distal part of the spoke that appears thicker than the rest, remains attached to the centriole as a protrusion from the A tubules of triplets. However, the hub at the center of the cartwheel is missing. It is also missing in immature centrioles (probasal bodies), suggesting that the central part of the cartwheel is not assembled at the onset of the centriole assembly process (Nakazawa, 2007).

Positional cloning via a PCR-based method determined that the bld12 phenotype is due to the loss of a homolog of SAS-6 (CrSAS-6). The mutation was mapped to a 600 kb region of Linkage Group XII/XIII, and a deletion of about 40 kb was detected in this region. Examination of the Chlamydomonas genome sequence database indicated that the deleted sequence included a gene coding for a protein that has been reported to be a Chlamydomonas homolog of the C.elegans protein SAS-6 (CrSAS-6) in addition to 12 other genes. When the genomic and cDNA clones of the CrSAS-6 gene were isolated and transformed into the bld12 cells, both rescued the flagella-less phenotype. In addition, another allele of bld12 (bld12-2) was isolated that also assembled flagella with various numbers of doublets. The genetic lesion in this mutant is a base-substitution mutation that is predicted to cause abnormal splicing and thereby produce a premature stop codon in frame. The PISA and the coiled-coil domains common to all known SAS-6 proteins (Leidel, 2005b) are conserved in CrSAS-6 (Nakazawa, 2007).

Immunoblots with an antibody raised against the whole protein detected a single band of CrSAS-6 in the wild-type cells and the bld12 cells rescued with genomic DNA or cDNA, but not in the bld12 cells. Immunofluorescence microscopy of wild-type cells localized CrSAS-6 exclusively to the centrioles, and immunoelectron microscopy of isolated NFAp clearly localized it to the central part of the cartwheel that surrounds the hub. This localization pattern is in good agreement with the part of the cartwheel missing in the bld12 centriole, suggesting that CrSAS-6 constitutes part of the inner spoke itself. This localization pattern is also in agreement with that of an SAS-6 homolog in the ciliary basal bodies of Tetrahymena (Kilburn, 2007). To examine the possible interaction of Bld12p with Bld10p, a component of the cartwheel spoke tip, the localization of CrSAS-6 was examined in bld10 mutants, as well as the localization of Bld10p in bld12 mutants. In contrast to bld12, which produces centrioles (albeit abnormal), the bld10 null mutant lacks a discrete centriolar structure. Western blotting showed that Bld10p is present in the bld12 cells, and immunofluorescence (IF) localized it to the centriole, although the IF signal was weaker than that in the wild-type cells. In the bld10 mutant, in contrast, CrSAS-6 signals were almost always absent, although very faint spots were observed in a small fraction of cells. These results suggest that Bld10p is recruited to the centriole independently of CrSAS-6 (Nakazawa, 2007).

On the basis of these results, a model is proposed for the mechanism that stabilizes the 9-fold symmetry of the centriole. During centriole assembly, the cartwheel appears at an early stage following the formation of the amorphous ring and serves as the scaffold for microtubule assembly. CrSAS-6 is necessary for cartwheel formation, especially for the radial arrangement of the cartwheel spokes. Bld10p, a homolog of mammalian centrosomal protein Cep135, functions in microtubule nucleation at the spoke tip (Hiraki, 2007). In the presence of SAS-6, the cartwheel is properly formed, Bld10p functions at each tip of the nine spokes, and the nine microtubules are formed. When SAS-6 is missing, the central part of the cartwheel is not formed, but Bld10p can somehow nucleate microtubules even if the cartwheel structure is completely disorganized. In this case, however, the number of microtubule nucleation sites is not fixed to nine, resulting in centrioles with variable numbers of triplet microtubules. Thus CrSAS-6 stabilizes the 9-fold symmetry of the centriole by determining the radial shape of the cartwheel (Nakazawa, 2007).

The importance of the cartwheel in establishing the 9-fold symmetry of the centriole has been suggested by experiments using truncated Bld10p (Hiraki, 2007). When a Bld10p molecule lacking the C-terminal 35% is expressed in the bld10 cells, the cartwheel spoke is shortened and cylindrical centrioles with eight triplets are frequently formed. This is probably because the cartwheel with short spokes, i.e., that with a small circumference, can accommodate only eight triplets. The number of the spokes radiating from the hub remains nine even in the eight-triplet centrioles, suggesting that the 9-fold symmetry originates from the hub. The centriole spokes must transmit the geometrical information to the sites where the triplets form. The loss of the radial arrangement of the spokes in bld12, therefore, naturally results in an instability of the 9-fold symmetry. Thus both of the two cartwheel proteins, CrSAS-6 and Bld10p, should be crucial for establishing the 9-fold symmetry of the centriolar structure (Nakazawa, 2007).

A recent study using electron tomography to investigate centriole assembly in C. elegans embryos showed that SAS-6 is required for the formation of the central tube, a hollow tube that appears in the first step of centriole assembly in this organism(Pelletier, 2006). Nine-singlet microtubules assemble around the tube, suggesting that the tube functions as the scaffold for microtubule assembly. A tube-like structure has also been observed in Drosophila embryos in which its SAS-6 (DSAS-6) is overexpressed (Rodrigues-Martins, 2007). Interestingly, a null mutant of DSAS-6 produces abnormal centrioles that lack a subset of triplets and assume unclosed cylindrical structures. Thus the central tube and SAS-6 must be crucial for the establishment of the centriolar microtubule arrangement in these organisms. However, a conserved role for the central tube in centriole assembly during evolution is not clear, because central tubes have not been observed in other organisms. In duplicating basal bodies of Paramecium and Chlamydomonas, for example, a flat amorphous ring rather than a tube appears first, and then the cartwheel follows. Although it is possible that previous electron microscopic studies have missed the presence of tubes in these organisms, the amorphous ring or the cartwheel may well perform the function that the central tube carries out in C. elegans and Drosophila (Dutcher, 2007). The finding that CrSAS-6 localizes to the cartwheel and stabilizes the 9-fold symmetry of the centriolar structure strongly suggests that CrSAS-6 is a component of the cartwheel and that the cartwheel, rather than the amorphous ring, performs a function in Chlamydomonas similar to the function of the central tube in C. elegans and Drosophila. However, the possibilities cannot be ruled out that Chlamydomonas centrioles also develop on an as-yet-unobserved central tube and that CrSAS-6 functions in both the tube and the cartwheel. The cartwheel has been observed in many organisms, including mammals, Paramecium, Tetrahymena, and Chlamydomonas, as a transient or stable component of the centriole or the ciliary basal body. It is presumed that the cartwheel-mediated mechanism may function in centriole assembly in a wide range of organisms (Nakazawa, 2007).

In these experiments the absence of the cartwheel resulted in the production of centrioles with aberrant numbers of triplet microtubules. However, it should be noted that ~70% of the circularly arranged centrioles in the bld12 cells were still composed of nine microtubules. Thus it is likely that a cartwheel-independent mechanism that exerts strong pressure toward 9-fold symmetry is also present. In other words, the 9-fold symmetry in the centriole structure is likely to be established by multiple factors. The presence of multiple factors might explain the invariance of the centriole structure through evolution (Nakazawa, 2007).

Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos

Despite being essential for proper cell division, the mechanisms governing centrosome duplication are incompletely understood and represent an important open question in cell biology. Formation of a new centriole next to each existing one is critical for centrosome duplication. In C. elegans embryos, the proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but the mechanisms underlying their requirement remain unclear. This study demonstrates that the kinase ZYG-1 phosphorylates the coiled-coil protein SAS-6 at serine 123 in vitro. This phosphorylation event is shown to be crucial for centriole formation in vivo. Furthermore, it was established that such phosphorylation ensures the maintenance of SAS-6 at the emerging centriole. Overall, these findings establish that phosphorylation of the evolutionarily conserved protein SAS-6 is critical for centriole formation and thus for faithful cell division (Kitagawa, 2009).

The centrosome is the major microtubule organizing center (MTOC) of animal cells and comprises two centrioles surrounded by pericentriolar material (PCM). Centrioles and the related basal bodies are microtubule-based structures that comprise nine microtubule blades arranged in a radial symmetric fashion. Centrioles recruit and organize the PCM from which most microtubules are nucleated. Duplication of the centrosome occurs once per cell cycle and the two resulting centrosomes assemble a bipolar spindle during mitosis. Formation of a new centriole next to each existing one is essential for centrosome duplication, but the mechanisms governing this process are incompletely understood (Kitagawa, 2009).

Time-resolved electron tomography in C. elegans embryos revealed that centriole formation begins with the assembly of a central tube, onto which microtubules are then added. The central tube is thought to be related to the cartwheel that is apparent at the onset of centriole or basal body formation in other species and that appears to impart the 9-fold radial symmetry. Forward genetic and functional genomic screens identified five proteins essential for centriole formation in C. elegans: the kinase ZYG-1 and the coiled-coil proteins SPD-2, SAS-6, SAS-5, and SAS-4. Molecular epistatic experiments indicate that SPD-2 is required for the centriolar localization of the four other proteins, whereas ZYG-1 is needed for centriolar SAS-6 and SAS-5. SAS-6 and SAS-5 physically interact and are themselves essential for SAS-4 loading onto centrioles and subsequent microtubule addition (Kitagawa, 2009 and references therein).

The ZYG-1 related Polo-like kinase 4 (Plk4, also known as SAK) is necessary for centriole formation in human cells and Drosophila. Moreover, Plk4 overexpression induces formation of multiple new centrioles in human cells, as well as amplification and de novo formation of centrioles in D. melanogaster. Although these studies established that ZYG-1/Plk4 is crucial for regulating centriole formation, the underlying mechanisms have not been identified to date (Kitagawa, 2009 and references therein).

Proteins of the SAS-6 family are invariably present in organisms with centrioles or basal bodies and are essential for their formation. Chlamydomonas reinhardtii and Tetrahymena thermophila SAS-6 homologs localize to the cartwheel, and the human protein likewise localizes to the proximal part of the new centriole. Suggestively, in addition, SAS-6 centriolar recruitment proceeds in parallel with elongation of the central tube in C. elegans. Together, these observations indicate that proteins of the SAS-6 family play a fundamental role in an early stage of centriole formation across evolution, but how they are regulated to perform this function is not known (Kitagawa, 2009 and references therein).

This work refines the working model of centriole formation in C. elegans, in particular regarding the contribution of ZYG-1 and SAS-6. An initial step leading to centriole formation entails centriolar recruitment of SAS-6, which occurs in an SAS-5-dependent manner. These findings indicate that this recruitment can occur in most embryos despite ZYG-1 depletion or the absence of SAS-6 phosphorylation at S123. Next, ZYG-1 phosphorylates SAS-6 at S123, which ensures that SAS-6 is maintained in the central tube. Thereafter, SAS-4 promotes the recruitment of microtubules to the fully formed central tube, thus completing the process of centriole formation (Kitagawa, 2009).

Since ZYG-1 localizes to centrioles just before the initial recruitment of SAS-6, a model is favored in which ZYG-1 phosphorylates SAS-6 in the vicinity of the existing centriole. However, the possibility cannot be excluded that this phosphorylation event takes place in the cytoplasm. Although ZYG-1 has been postulated to be essential for the presence of SAS-6 at centrioles, this conclusion was based primarily on examining embryos during mitosis. The present analysis reveals in addition that the recruitment of SAS-6 can occur upon ZYG-1 inactivation. Even though it cannot be formally rule out that such initial recruitment reflects residual ZYG-1 function, this is viewed as unlikely, notably because recruitment is observed not only for SAS-6 and GFP-SAS-6 upon ZYG-1 depletion, but also for GFP-SAS-6RR[S123A] upon depletion of endogenous SAS-6. Interestingly, the relationship between ZYG-1 and SAS-6 uncovered in this study in C. elegans embryos mirrors that observed between the related proteins in human cells, where HsSAS-6 is recruited, but not maintained, at centrioles in cells depleted of Plk4. This analogous relationship raises the possibility that phosphorylation of SAS-6 proteins by ZYG-1/Plk4-related kinases is an evolutionarily conserved mechanism that promotes centriole formation (Kitagawa, 2009).

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, anastral spindle 2 (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 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. 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, 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, 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. 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. Surprisingly, however, overexpression of Sak, DSas-6, or DSas-4 cannot drive centriole overduplication in primary spermatocytes, 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. 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. 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 and Drosophila pericentrin-like protein (D-PLP), continue to localize along the basal body. In contrast, Ana2, like the conserved duplication proteins, 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 to a suggeston 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. 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. 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. Second, STIL mutant mice show defects characteristic of aberrant cilia function, such as randomized left-right asymmetry and neural tube abnormalities. Most importantly, it has recently been shown that mutations in human STIL cause primary microcephaly (MCPH), a congenital disorder characterized by reduced brain size. 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, 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).

Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation

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).

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).

Superresolution characterization of core centriole architecture

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

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

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

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

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

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

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

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

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication

The duplication and 9-fold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. This study used hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its sub-cellular localization. A core stabilization sequence within Gorab's C-terminal coiled-coil domain was identified that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an anti-parallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable hetero-trimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a 16-fold reduced binding affinity for Gorab that can not support centriole duplication. Thus Gorab dimers at the Golgi exist in equilibrium with Sas-6 associated monomers at the centriole to balance Gorab's dual role (Fatalska, 2021).

Centrioles are the ninefold symmetrical microtubule arrays found at the core of centrosomes, the bodies that organize cytoplasmic microtubules in interphase and mitosis. Centrioles also serve as the basal bodies of both non-motile and motile cilia, and flagellae. The core components of centrioles and the molecules that regulate their assembly are highly conserved. The initiation of centriole duplication first requires that the mother and daughter pair of centrioles at each spindle pole disengage at the end of mitosis. Plk4 then phosphorylates Ana2 (Drosophila)/STIL(human) at its N-terminal part, which promotes Ana2 recruitment to the site of procentriole formation, and at its C-terminal part, which enables Ana2 to bind and thereby recruit Sas6. The ensuing assembly of a ninefold symmetrical arrangement of Sas6 dimers provides the structural basis for the ninefold symmetrical cartwheel structure at the procentriole's core. Sas6 interacts with Cep135 and in turn with Sas4 (Drosophila)/CPAP (human), which provides the linkage to centriole microtubules (Fatalska, 2021).

An unexpected requirement has been identified for the protein, Gorab, to establish the ninefold symmetry of centrioles (Kovacs, 2018). Flies lacking Gorab are uncoordinated due to basal body defects in sensory cilia, which lose their ninefold symmetry, and also exhibit maternal effect lethality due to failure of centriole duplication in the syncytial embryo. Gorab is a trans-Golgi-associated protein. Its human counterpart is mutated in the wrinkly skin disease, gerodermia osteodysplastica. By copying a missense mutation in gerodermia patients that disrupts the association of Gorab with the Golgi, this study was able to create mutant Drosophila Gorab, which was also unable to localize to trans-Golgi. However, this mutant form of Gorab was still able to rescue the centriole and cilia defects of gorab null flies. It was also found that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype that can be overcome by a second mutation preventing Golgi targeting. Thus, centriole and Golgi functions of Drosophila Gorab are separable (Fatalska, 2021).

The Golgi apparatus both delivers and receives vesicles to and from multiple cellular destinations and is also responsible for modifying proteins and lipids. Gorab resembles a group of homodimeric rod-like proteins, the golgins, which function in vesicle tethering. The golgins associate through their C-termini with different Golgi domains, and their N-termini both capture vesicles and provide specificity to their tethering. There is known redundancy of golgin function, reflected by the overlapping specificity of the types of vesicles they capture. Gorab is rapidly displaced from the trans-side of the Golgi apparatus by Brefeldin A, suggesting that its peripheral membrane association requires ARF-GTPase activity (Fatalska, 2021).

Previous studies of human Gorab indicated its ability to form a homodimer in complex with Rab6 and identified its putative coiled-coil region as a requirement to localize at the trans-Golgi (Egerer, 2015; Witkos, 2019). Studies on its Drosophila counterpart supported Gorab's ability to interact with itself, potentially through the predicted coiled-coil motif. However, this region was also found to overlap with the region required for Gorab's interaction with Sas6 (Kovacs, 2018). These findings raised the questions of how Gorab's putative coiled-coil region could facilitate interactions with the Golgi, on the one hand, and its Sas6 partner, on the other. To address this, s hydrogen-deuterium exchange (HDX) was employed in conjunction with mass spectrometry (MS). HDX enables the identification of dynamic features of protein by monitoring the exchange of main chain amide protons to deuteria in solution. This study used HDX-MS to monitor the retarded exchange of amide protons localized between interacting regions of Gorab and Sas6 to identify the interacting surfaces within the Gorab-Sas6 complex. Together with other biophysical characterizations, this has revealed that Gorab is able to form a homo-dimer through its coiled-coil region but that it interacts as a monomer with the C-terminal coiled-coil of Sas6. Mutation of a critical amino acid in Sas6's Gorab-binding domain generates a variant of Sas6 with a sixteenfold reduced binding affinity for Gorab that is no longer able to support centriole duplication (Fatalska, 2021).

Together, these findings indicate that Gorab exists at the trans-Golgi network as a homodimer. Dimerization requires its coiled-coil motif (residues 200-315) within which is a core sequence (residues 270-287) that represents the most stable part of this dimerization region. Dimerization enables Gorab to interact with Rab6, and this in turn enables its association with the trans-Golgi. In contrast, Gorab interacts with Sas6 as a monomer. Gorab's binding to Sas6 occurs with a higher affinity than its homodimerization, enabling a Gorab monomer to associate with the Sas6 dimer. Thus, the relatively small number of Sas6 molecules at the centriole would more avidly bind the Gorab monomer, allowing greater excess of Gorab to accumulate as dimers at the trans-Golgi. Sas6 and Gorab interact through short interfaces within their coiled-coil regions. Disruption of this region of Sas6 through mutation of a single conserved leucine residue, L447, results in a failure of Gorab to bind to Sas6 and localize to the centriole. While the possibility cannot be formally excluded that the L447A mutation affects some other aspect of Sas6 function, the finding that expression of this mutant phenocopies a strong gorab hypomorph in its effects upon both co-ordination and centriole duplication suggests that failure to recruit Gorab is responsible for the Sas6-L447A defect. The finding of some residual apparent Gorab-like function in Sas6-L447A-expressing flies may reflect the overexpression of the protein due to the technical requirements of the experiment and the fact that Sas6-L447A still binds Gorab but with a sixteenfold reduced affinity compared to wild-type Sas6. Given that Sas6-L447A greatly diminishes the interaction with Gorab, whereas the mutation, M440A, in the adjoining 'a' position of the 'a-g' coiled-coil heptad repeat does not, leads to the conclusion that Gorab binds to a narrow region near the C-terminus of the coiled coil of Sas6 (Fatalska, 2021).

Gorab shows many of the properties typical of golgins, a family of tentacle-like proteins that protrude from the Golgi membranes to capture a variety of target vesicles. Redundancy between golgins in their ability to bind target vesicles could act as a functional safeguard and might explain why loss-of-function gorab mutants display no obvious Golgi phenotype, contrasting to the Golgi defects shown by the C-terminally tagged Gorab molecule (Kovacs, 2018). Gorab is similar to other golgins, which also associate with the Golgi membranes through their C-terminal parts in interactions that require Rab family member proteins to interact with the C-terminal part of the golgin dimer. The N-terminal parts of the golgins interact with their vesicle targets. Human GORAB's N-terminal part interacts with Scyl1 to promote the formation of COPI vesicles at the trans-Golgi (Witkos, 2019). However, its precise role in the transport of COPI vesicles is not clear, particularly why loss of human GORAB affects Golgi functions in just bone and skin when COPI function is required in multiple tissues. Drosophila Gorab also co-purifies and physically interacts with both Yata, counterpart of Scyl1, and COPI vesicle components, and its importance for transport of COPI vesicles in Drosophila is similarly unclear (Fatalska, 2021).

This study offers a perspective on how Gorab interacts with Sas6 at the centriole and suggests the possibilities for why this interaction is essential to establish the centriole's ninefold symmetry. The heterotrimeric structure formed by a Sas6 dimer and the Gorab monomer will together constitute a single spoke plus central hub unit of the centriole's cartwheel. The C-terminal part of Gorab would be expected to lie in a tight antiparallel association with the C-terminal part of Sas6's coiled-coil region. Gorab's N-terminus might thus be expected to extend towards the centriolar microtubules and their associated proteins. As the microtubules of Drosophila's somatic centrioles exist as doublets of A- and B-tubules, it is tempting to speculate that Gorab interacts with the centriole wall in a region occupied in other cell types by the C-tubule. This could account for the lack of any requirement for Sas6-Gorab interaction in the male germ-line, where centrioles have triplet microtubules and a C-tubule occupies this space. Gorab's partner proteins interacting with its N-terminal region are therefore of great interest at both the Golgi and in the centriole, and it will be key to understand the nature of these interactions in future studies (Fatalska, 2021).


Search PubMed for articles about Drosophila Sas-6

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

Bettencourt-Dias, M. et al. (2005). SAK/PLK4 is required for centriole duplication and flagella development. Curr. Biol. 15: 2199-2207. PubMed ID: 16326102

Bettencourt-Dias, M., and Glover, D. M. (2007). Centrosome biogenesis and function: centrosomics brings new understanding. Nat. Rev. Mol. Cell Biol. 8: 451-463. PubMed ID: 17505520

Dutcher, S.K. (2007). Finding treasures in frozen cells: New centriole intermediates. Bioessays 29: 630-634. PubMed ID: 17563074

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

Egerer, J., Emmerich, D., Fischer-Zirnsak, B., Chan, W. L., Meierhofer, D., Tuysuz, B., Marschner, K., Sauer, S., Barr, F. A., Mundlos, S. and Kornak, U. (2015). GORAB missense mutations disrupt RAB6 and ARF5 binding and Golgi targeting. J Invest Dermatol 135(10): 2368-2376. PubMed ID: 26000619

Fatalska, A., Stepinac, E., Richter, M., Kovacs, L., Pietras, Z., Puchinger, M., Dong, G., Dadlez, M. and Glover, D. M. (2021). The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication. Elife 10. PubMed ID: 33704067

Hiraki, M., Nakazawa, Y., Kamiya, R., and Hirono, M. (2007). Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole. Curr. Biol. 17: 1778-1783. PubMed ID: 17900905

Kilburn, C. L., et al. (2007). New Tetrahymena basal body protein components identify basal body domain structure. J. Cell Biol. 178: 905-912. PubMed ID: 17785518

Kitagawa, D., Busso, C., Flückiger, I., Gönczy, P. (2009). Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos. Dev. Cell 17(6): 900-7. PubMed ID: 20059959

Kovacs, L., Chao-Chu, J., Schneider, S., Gottardo, M., Tzolovsky, G., Dzhindzhev, N. S., Riparbelli, M. G., Callaini, G. and Glover, D. M. (2018). Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles. Nat Genet. PubMed ID: 29892014

Leidel, S. and Gonczy, P. (2005a). Centrosome duplication and nematodes: recent insights from an old relationship. Dev. Cell 9: 317-325. PubMed ID: 16139223

Leidel, S., Delattre, M., Cerutti, L., Baumer, K. and Gonczy, P. (2005b). SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells. Nat. Cell Biol. 7: 115-125. PubMed ID: 15665853

Nakazawa, Y., Hiraki, M., Kamiya, R. and Hirono, M. (2007). SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole. Curr. Biol. 17(24): 2169-74. PubMed ID: 18082404

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

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(10): 834-43. PubMed ID: 17475495

Pelletier, L., et al. (2006). Centriole assembly in Caenorhabditis elegans. Nature 444(7119): 619-23. PubMed ID: 17136092

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

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

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

Vidwans, S. J., Wong, M. L. and O'Farrell, P. H. (2003). Anomalous centriole configurations are detected in Drosophila wing disc cells upon Cdk1 inactivation. J. Cell Sci. 116: 137-143. PubMed ID: 12456723

Witkos, T. M., Chan, W. L., Joensuu, M., Rhiel, M., Pallister, E., Thomas-Oates, J., Mould, A. P., Mironov, A. A., Biot, C., Guerardel, Y., Morelle, W., Ungar, D., Wieland, F. T., Jokitalo, E., Tassabehji, M., Kornak, U. and Lowe, M. (2019). GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation. Nat Commun 10(1): 127. PubMed ID: 30631079

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date revised: 20 August 2021

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