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Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9 fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.

Centrioles are evolutionarily conserved multi-protein organelles essential for forming cilia and centrosomes. Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. The importance of this architecture has been difficult to decipher notably because of the lack of precise tools to modulate the underlying assembly reaction. Here, we developed monobodies against Chlamydomonas reinhardtii SAS-6, characterizing three in detail with X-ray crystallography, atomic force microscopy and cryo-electron microscopy. This revealed distinct monobody-target interaction modes, as well as specific consequences on ring assembly and stacking. Of particular interest, monobody MBCRS6-15 induces a conformational change in CrSAS-6, resulting in the formation of a helix instead of a ring. Furthermore, we show that this alteration impairs centriole biogenesis in human cells. Overall, our findings identify monobodies as powerful molecular levers to alter the architecture of multi-protein complexes and tune centriole assembly., Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. Here, the authors develop monobodies against Chlamydomonas reinhardtii SAS-6 and use X-ray crystallography, atomic force microscopy and cryo-electron microscopy to reveal insights into ring assembly and stacking.

Centrioles are polarized microtubule‐based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS‐6 proteins. A cryo‐tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub‐tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A‐C linker. Moreover, we report ~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS‐6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS‐6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms., Ultrastructural studies of centrioles from termite flagellates delineate the architecture of the centriole proximal region, including new insights into stacking of cartwheel‐forming SAS‐6 rings.

The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle – from assembly to disappearance – focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.

Gene targeting was used to ablate Cep135, a component of the centriolar cartwheel, in chicken DT40 cells. Cep135-deficient cells showed no major defects in centrosome composition or function, although centrosome amplification after HU treatment increased significantly. EM revealed an unusual structure in the lumen of Cep135-null centrioles., Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase–arrested cells.

The centriole is an evolutionarily conserved macromolecular structure that is crucial for the formation of flagella, cilia and centrosomes. The ultrastructure of the centriole was first characterized decades ago with the advent of electron microscopy, revealing a striking ninefold radial arrangement of microtubules. However, it is only recently that the molecular mechanisms governing centriole assembly have begun to emerge, including the elucidation of the crucial role of spindle assembly abnormal 6 (SAS-6) proteins in imparting the ninefold symmetry. These advances have brought the field to an exciting era in which architecture meets function.

International audience; Centrosome duplication occurs once per cell cycle and ensures that the two resulting centrosomes assemble a bipolar mitotic spindle. Centriole formation is fundamental for centrosome duplication. In Caenorhabditis elegans, the evolutionarily conserved proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but how they function is not fully understood. Here, we demonstrate that Protein Phosphatase 2A (PP2A) is also critical for centriole formation in C. elegans embryos. We find that PP2A subunits genetically and physically interact with the SAS-5/SAS-6 complex. Furthermore, we show that PP2A-mediated dephosphorylation promotes centriolar targeting of SAS-5 and ensures SAS-6 delivery to the site of centriole assembly. We find that PP2A is similarly needed for the presence of HsSAS-6 at centrioles and for centriole formation in human cells. These findings lead us to propose that PP2A-mediated loading of SAS-6 proteins is critical at the onset of centriole formation.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo‐electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel‐bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2‐rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A‐C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis., In situ and ex vivo cryo‐ET of centrioles from Chlamydomonas, Paramecium, Naegleria and humans describe the centriole's proximal region and reveal evolutionarily‐conserved structures as well as differences between species.