SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells

The mechanisms that ensure centrosome duplication are poorly understood. In Caenorhabditis elegans, ZYG-1, SAS-4, SAS-5 and SPD-2 are required for centriole formation. However, it is unclear whether these proteins have functional homologues in other organisms. Here, we identify SAS-6 as a component that is required for daughter centriole formation in C. elegans. SAS-6 is a coiled-coil protein that is recruited to centrioles at the onset of the centrosome duplication cycle. Our analysis indicates that SAS-6 and SAS-5 associate and that this interaction, as well as ZYG-1 function, is required for SAS-6 centriolar recruitment. SAS-6 is the founding member of an evolutionarily conserved protein family that contains the novel PISA motif. We investigated the function of the human homologue of SAS-6. GFP-HsSAS-6 localizes to centrosomes and its overexpression results in excess foci-bearing centriolar markers. Furthermore, siRNA-mediated inactivation of HsSAS-6 in U2OS cells abrogates centrosome overduplication following aphidicolin treatment and interferes with the normal centrosome duplication cycle. Therefore, HsSAS-6 is also required for centrosome duplication, indicating that the function of SAS-6-related proteins has been widely conserved during evolution.     

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 melanogaster 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. Here, we find that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. We show 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. We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery.     

Sequential protein recruitment in C. elegans centriole formation

Formation of the microtubule-based centriole is a poorly understood process that is crucial for duplication of the centrosome, the principal microtubule-organizing center of animal cells . Five proteins have been identified as being essential for centriole formation in Caenorhabditis elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2 . The relationship between these proteins is incompletely understood, limiting understanding of how they contribute to centriole formation. In this study, we established the order in which these five proteins are recruited to centrioles, and we conducted molecular epistasis experiments expanding on earlier work. We find that SPD-2 is loaded first and is needed for the centriolar localization of the four other proteins. ZYG-1 recruitment is required thereafter for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for the presence of SAS-4, which is incorporated last. Our results indicate in addition that the presence of SAS-5 and SAS-6 allows diminution of centriolar ZYG-1. Moreover, astral microtubules appear dispensable for the centriolar recruitment of all five proteins. Several of these proteins have homologs in other metazoans, and we expect the assembly pathway that stems from our work to be conserved.     

SAS-4 is essential for centrosome duplication in C elegans and is recruited to daughter centrioles once per cell cycle

The mechanisms governing centrosome duplication remain poorly understood. We identified a gene called sas-4 that is essential for this process in C. elegans. SAS-4 encodes a predicted coiled-coil protein that localizes to a tiny dot in the center of centrosomes throughout the cell cycle. FRAP experiments with GFP-SAS-4 transgenic embryos reveal that SAS-4 is recruited to the centrosome once per cell cycle, at the time of organelle duplication. Additional evidence indicates that SAS-4 is recruited to the daughter centriole or a closely associated structure. These findings identify SAS-4 recruitment as a key step in the centrosome duplication cycle.     

The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

The centriole in eukaryotes functions as the cell''s microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei, an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei.     

Identification of compounds that bind the centriolar protein SAS-6 and inhibit its oligomerization

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal “head” domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.     

SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans

Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome.     

Self-assembling SAS-6 Multimer Is a Core Centriole Building Block

Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a “cartwheel” with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with ∼6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles.     

PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans embryos

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Highlights? PP2A phosphatase is essential for centriole formation in C. elegans ? PP2A subunits genetically and physically interact with the SAS-5/SAS-6 complex ? PP2A-mediated dephosphorylation of SAS-5 is required for SAS-5/SAS-6 centriolar targeting ? Human PP2A is required for HsSAS-6 centriolar targeting and centriole formation     

DSAS-6 organizes a tube-like centriole precursor, and its absence suggests modularity in centriole assembly

Centrioles are microtubule-based cylindrical structures that exhibit 9-fold symmetry and facilitate the organization of centrosomes, flagella, and cilia [1]. Abnormalities in centrosome structure and number occur in many cancers [1, 2]. 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 [1, 3]. 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 [4, 5]. This complex also recruits SAS-4, which is required for the assembly of the centriolar microtubules that decorate that tube [4, 5]. Here we show that Drosophila SAS-6 is involved in centriole assembly and cohesion. Overexpression of DSAS-6 in syncitial embryos led 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 [6]. Instead, tube-like structures were present, supporting the idea that centriolar assembly starts with the formation of a tube-like scaffold, dependent on DSAS-6 [5]. 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. We propose 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.     

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. Here we show 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. We presume that, in many organisms, SAS-6 is an essential component of the cartwheel, a structure that stabilizes the 9-triplet structure.     

ATG8 localization in apicomplexan parasites: Apicoplast and more?

The ATG genes are highly conserved in eukaryotes including yeasts, plants, and mammals. However, these genes appear to be only partially present in most protists. Recent studies demonstrated that, in the apicomplexan parasites Plasmodium (malaria parasites) and Toxoplasma, ATG8 localizes to the apicoplast, a unique nonphotosynthetic plastid with 4 limiting membranes. In contrast to this established localization, it remains unclear whether these parasites can induce canonical macroautophagy and if ATG8 localizes to autophagosomes. Furthermore, the molecular function of ATG8 in its novel workplace, the apicoplast, is totally unknown. Here, we review recent studies on ATG8 in Plasmodium and Toxoplasma, summarize both consensus and controversial findings, and discuss its potential role in these parasites.     

Protein phosphatase 2A-SUR-6/B55 regulates centriole duplication in C. elegans by controlling the levels of centriole assembly factors

Centrioles play a crucial role in mitotic spindle assembly and duplicate precisely once per cell cycle. In worms, flies, and humans, centriole assembly is dependent upon a key regulatory kinase (ZYG-1/Sak/Plk4) and its downstream effectors SAS-5 and SAS-6. Here we report a role for protein phosphatase 2A (PP2A) in centriole duplication. We find that the PP2A catalytic subunit LET-92, the scaffolding subunit PAA-1, and the B55 regulatory subunit SUR-6 function together to positively regulate centriole assembly. In PP2A-SUR-6-depleted embryos, the levels of ZYG-1 and SAS-5 are reduced and the ZYG-1- and SAS-5-dependent recruitment of SAS-6 to the nascent centriole fails. We show that PP2A physically associates with SAS-5 in vivo and that inhibiting proteolysis can rescue SAS-5 levels and the centriole duplication defect of PP2A-depleted embryos. Together, our findings indicate that PP2A-SUR-6 promotes centriole assembly by protecting ZYG-1 and SAS-5 from degradation.     

DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement

Centrioles form cilia and centrosomes, organelles whose dysfunction is increasingly linked to human disease. Centriole duplication relies on a few conserved proteins (ZYG-1/Sak/Plk4, SAS-6, SAS-5/Ana2, and SAS-4), and is often initiated by the formation of an inner "cartwheel" structure. Here, we show that overexpressed Drosophila Sas-6 and Ana2 coassemble into extended tubules (SAStubules) that bear a striking structural resemblance to the inner cartwheel of the centriole. SAStubules specifically interact with centriole proximal ends, but extra DSas-6/Ana2 is only recruited onto centrioles when Sak/Plk4 kinase is also overexpressed. This extra centriolar DSas-6/Ana2 induces centriole overduplication and, surprisingly, increased centriole cohesion. Intriguingly, we observe tubules that are structurally similar to SAStubules linking the engaged centrioles in normal wild-type cells. We conclude that DSas-6 and Ana2 normally cooperate to drive the formation of the centriole inner cartwheel and that they promote both centriole duplication and centriole cohesion in a Sak/Plk4-dependent manner.     

SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.     

Naegleria gruberi De Novo Basal Body Assembly Occurs via Stepwise Incorporation of Conserved Proteins

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, γ-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally γ-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.The beautiful and enigmatic pinwheel structures of centrioles and basal bodies have captured the imaginations of cell biologists for over a century. These small (∼1-μm) organelles are composed largely of a cylinder of nine microtubule triplets (11). The surrounding amorphous material harbors the microtubule-organizing activities of the centrosome, placing centrioles at the hub of the microtubule cytoskeleton. Metazoan centrosomes define mitotic spindle poles, and their centrioles are called basal bodies when used to form cilia (29). Moreover, in 1900 Meeves showed in a series of classical experiments that centrioles and basal bodies are interconvertible structures (34). Centrioles must replicate exactly once per cell cycle, as duplication errors can lead to problems with chromosome segregation and cell morphology (17).Virtually all animal cells have a pair of centrosomal centrioles that duplicate via “templated” assembly, with the new centriole developing perpendicular and attached to a preexisting centriole (4). Centrioles can also be formed “de novo” in cytosol devoid of preexisting centrioles and basal bodies (20). In addition to many in vivo examples (20), terminally differentiated fibroblasts held in S phase can assemble centrioles de novo after removal of preexisting centrioles by laser microsurgery (15).The amoeboflagellate Naegleria gruberi grows as an amoeba that completely lacks a cytoplasmic microtubule cytoskeleton. However, when exposed to stressors such as temperature, osmotic, or pH changes, Naegleria rapidly differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton from scratch, including two basal bodies and flagella (8). This differentiation occurs synchronously, with approximately 90% of cells growing visible flagella in a 15-min window (T₅₀ = 65 min after initiation of differentiation). As part of this differentiation, Naegleria has been shown to assemble the pinwheel structure of the basal bodies de novo, about 10 min before flagella are seen (11).Two centrosomal proteins that have been studied during Naegleria differentiation are centrin and γ-tubulin. Centrin is a calcium-binding phosphoprotein that is an integral component of the wall and lumen of basal bodies and of the pericentriolar lattice in many organisms (4, 19). During differentiation, Naegleria induces synthesis of centrin protein, which then localizes specifically to basal body structures throughout differentiation (18). γ-Tubulin is a general microtubule nucleation factor that localizes to microtubule-organizing centers (MTOCs) of many types. Surprisingly, Naegleria''s γ-tubulin homolog has been reported to localize to basal body precursor complexes and then move to the other end of the cell before disappearing completely (32).A third protein that has come under recent scrutiny for its role in centriole duplication is SAS-6, a functionally conserved coiled-coil protein required for the formation of diverse basal body precursor structures (7, 21,–23, 31). In Caenorhabditis elegans and Drosophila melanogaster, SAS-6 is recruited at S phase to form the “central tube,” a cylindrical basal body precursor that lacks microtubules (22, 23). SAS-6 is also required for the formation of the flat ring or cartwheel with nine radiating spokes, which is the first structure to be formed in the Chlamydomonas basal body (21).To determine if Naegleria is likely to have typical basal body components, we identified conserved basal body genes in the Naegleria genome. We also made antibodies to and localized Naegleria''s homologs of SAS-6 and γ-tubulin. Finally, we have determined the order of expression and incorporation of these proteins, as well as centrin, during Naegleria de novo basal body assembly.     

SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament

The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.     

SAS‐6 coiled‐coil structure and interaction with SAS‐5 suggest a regulatory mechanism in C. elegans centriole assembly

The centriole is a conserved microtubule‐based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS‐5 and SAS‐6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS‐5 C‐terminal domain (residues 390–404) specifically binds to a narrow central region (residues 275–288) of the SAS‐6 coiled coil. This was supported by the crystal structure of the SAS‐6 coiled‐coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS‐6 CCD, which gives rise to an anti‐parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS‐5 and SAS‐6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero‐complex to facilitate the correct assembly of the nine‐fold symmetric centriole.