Isabelle Flückiger

Structural Basis of the 9-Fold Symmetry of Centrioles

Summary The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

Spindle positioning in human cells relies on proper centriole formation and on the microcephaly proteins CPAP and STIL

Patients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes centrosome protein 4.1 associated protein (CPAP; also known as centromere protein J, CENPJ). CPAP is a large coiled-coil protein enriched at the centrosome, a structure that comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains crucial for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects correlate with a randomization of spindle position. Moreover, we demonstrate that the MCPH protein SCL/TAL1 interrupting locus (STIL) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH because of aberrant spindle positioning in progenitor cells during brain development.

Native Architecture of the Centriole Proximal Region Reveals Features Underlying Its 9-Fold Radial Symmetry

BACKGROUND Centrioles are cylindrical microtubule-based structures whose assembly is critical for the formation of cilia, flagella, and centrosomes. The centriole proximal region harbors a cartwheel that dictates the 9-fold symmetry of centrioles. Although the cartwheel architecture has been recently analyzed, how it connects to the peripheral microtubules is not understood. More generally, a high-resolution view of the proximal region of the centriole is lacking, thus limiting understanding of the underlying assembly mechanisms. RESULTS We report the complete architecture of the Trichonympha centriole proximal region using cryotomography. The resulting 3D map reveals several features, including additional densities in the cartwheel that exhibit a 9-fold symmetrical arrangement, as well as the structure of the Pinhead and the A-C linker that connect to microtubules. Moreover, we uncover striking chiral features that might impart directionality to the entire centriole. Furthermore, we identify Trichonympha SAS-6 and demonstrate that it localizes to the cartwheel in vivo. CONCLUSIONS Our work provides unprecedented insight into the architecture of the centriole proximal region, which is key for a thorough understanding of the mechanisms governing centriole assembly.

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 Caenorhabditis 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. Here, we demonstrate that the kinase ZYG-1 phosphorylates the coiled-coil protein SAS-6 at serine 123 in vitro. Importantly, we show that this phosphorylation event is crucial for centriole formation in vivo. Furthermore, we establish that such phosphorylation ensures the maintenance of SAS-6 at the emerging centriole. Overall, our findings establish that phosphorylation of the evolutionarily conserved protein SAS-6 is critical for centriole formation and thus for faithful cell division.

Discovering Regulators of Centriole Biogenesis through siRNA-Based Functional Genomics in Human Cells

Centrioles are essential for forming cilia, flagella, and centrosomes and are thus critical for a range of fundamental cellular processes. Despite their importance, the mechanisms governing centriole biogenesis remain incompletely understood. We performed a high-content genome-wide small-interfering-RNA-based screen to identify genes regulating centriole formation in human cells. We designed an algorithm to automatically detect GFP-Centrin foci that, combined with subsequent manual analysis, allowed us to identify 44 genes required for centriole formation and 32 genes needed for restricting centriole number. Detailed follow-up characterization uncovered that the C2 domain protein C2CD3 is required for distal centriole formation and suggests that it functions in the basal body to template primary cilia. Moreover, we found that the E3 ubiquitin ligase TRIM37 prevents centriole reduplication events. We developed a dynamic web interface containing all images and numerical features as a powerful resource to investigate facets of centrosome biology.

Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry

Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel–microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

Cell-free reconstitution reveals centriole cartwheel assembly mechanisms

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

Correlative multicolor 3D SIM and STORM microscopy

Within the last decade, super-resolution methods that surpass the diffraction limit of light microscopy have provided invaluable insight into a variety of biological questions. Each of these approaches has inherent advantages and limitations, such that their combination is a powerful means to transform them into versatile tools for the life sciences. Here, we report the development of a combined SIM and STORM setup that maintains the optimal resolution of both methods and which is coupled to image registration to localize biological structures in 3D using multicolor labeling. We utilized this workflow to determine the localization of Bld12p/CrSAS-6 in purified basal bodies of Chlamydomonas reinhardtii with utmost precision, demonstrating its usefulness for accurate molecular mapping in 3D.

Identification of Chlamydomonas Central Core Centriolar Proteins Reveals a Role for Human WDR90 in Ciliogenesis

Summary Centrioles are evolutionarily conserved macromolecular structures that are fundamental to form cilia, flagella, and centrosomes. Centrioles are 9-fold symmetrical microtubule-based cylindrical barrels comprising three regions that can be clearly distinguished in the Chlamydomonas reinhardtii organelle: an ∼100-nm-long proximal region harboring a cartwheel; an ∼250-nm-long central core region containing a Y-shaped linker; and an ∼150-nm-long distal region ending at the transitional plate. Despite the discovery of many centriolar components, no protein has been localized specifically to the central core region in Chlamydomonas thus far. Here, combining relative quantitative mass spectrometry and super-resolution microscopy on purified Chlamydomonas centrioles, we identified POB15 and POC16 as two proteins of the central core region, the distribution of which correlates with that of tubulin glutamylation. We demonstrated that POB15 is an inner barrel protein within this region. Moreover, we developed an assay to uncover temporal relationships between centriolar proteins during organelle assembly and thus established that POB15 is recruited after the cartwheel protein CrSAS-6 and before tubulin glutamylation takes place. Furthermore, we discovered that two poc16 mutants exhibit flagellar defects, indicating that POC16 is important for flagellum biogenesis. In addition, we discovered that WDR90, the human homolog of POC16, localizes to a region of human centrioles that we propose is analogous to the central core of Chlamydomonas centrioles. Moreover, we demonstrate that WDR90 is required for ciliogenesis, echoing the findings in Chlamydomonas. Overall, our work provides novel insights into the identity and function of centriolar central core components.