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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SAS6-Cep135-CPAP (Towards a molecular understanding of the centriole assembly process)

Teaser

Summary

Centrioles are crucial organelles in animal cells where they direct formation of the microtubule network, the mitotic spindle, cilia and flagella. They comprise large, protein-based, cylindrical assemblies that form centrosomes and basal bodies in cells. Centrioles are essential for diverse cellular processes including division, sensing and locomotion. The wide-reaching contributions of these organelles are best appreciated when errors in centriole assembly occur; these lead, for example, to male sterility and ectopic pregnancies due to immotile sperm, primary microcephaly, cancer and ciliopathies that can affect the liver, kidneys, gut and the respiratory track. Thus, understanding centriole assembly is a crucial question both for cell biology and clinical applications with direct implications to life quality and health.

The centriole architecture is conserved and comprises cylinders typically ~500 nm long and ~250 nm in diameter with characteristic 9-fold radial symmetry. Structurally, the best-studied region of centrioles is the cartwheel, which is the first region forming during centriole biogenesis. The cartwheel consists of a circular hub from which nine spokes emanate. Recently, the protein SAS-6, which is essential for normal centriole assembly was shown to form cartwheels in vitro in the absence of other components. Cartwheel layers stack with ~8.5 nm periodicity at their central hubs, while spokes from successive layers merge in pairs to give ~17 nm periodicity at the cartwheel periphery (Fig 1). Importantly, stacking of cartwheel layers is not maintained through direct interactions of the central hubs, which do not connect to one another, but by peripheral associations that ensure correct spacing (Fig 2). Despite the importance of centriolar structure which is directly linked to organelle function and organism health our understanding of this system remains limited. In this proposal we combined multidisciplinary approaches in an effort to address important biological questions:
â€¢ How SAS-6 stack along the length of centrioles, thereby providing an initial scaffold for subsequent recruitment of further centriole components
â€¢ Furthermore, individual cartwheel hubs, resolved by Xray crystallography, are symmetric along the ring plane (have identical â€˜topâ€™ and â€˜bottomâ€™ surfaces). How then do non-symmetric, polar cartwheels and centrioles emerge?

To ensure good organisation and implementation of the proposal, we subdivided the proposed research in distinct experimental objectives.

To ensure good organisation and implementation of the proposal, we subdivided the proposed research in distinct experimental objectives.
Obj1: Resolving the SAS-6 coiled coil molecular architecture
Obj2 Evaluation of the SAS-6 coiled-coil interactions
Obj3 Cartwheel reconstitution with CrSAS-6 variants in vitro

Work performed

We discovered a new self-association interaction in SAS-6 mediated by the coiled-coil domain of this protein. The new interaction is relatively weak but drives two coiled-coil dimers to associate in a parallel fashion, corresponding to pairwise-merging spokes at the cartwheel periphery (Fig. 1B). Merging of spokes allows two SAS-6 rings to form a ring dimer (Fig. 2), which is known to be the incremental block of cartwheel assembly. Crystallographic structures of the interacting SAS-6 coiled coils showed two possible arrangements: a symmetric positioning with the two coiled coils exactly aligned (Fig. 3A), or an asymmetric arrangement, where
the two SAS-6 coiled coils have a ~1 nm linear offset (Fig. 3B). Crucially, the difference between these two arrangements holds a fundamental implication for cartwheel structure, as an asymmetric SAS-6 interaction could give rise to asymmetric (polar) SAS-6 ring dimers, which in turn would build polar cartwheels To assess which of the two interaction arrangements of SAS-6 coiled coils is found in cartwheels, we tested the ability of SAS-6 mutants that selectively disrupt the symmetric or asymmetric arrangement to form cartwheels in vitro. As seen in Fig. 4, SAS-6 mutants that disrupt the symmetric coiled coil arrangement form cartwheels similar to the wild-type (WT) protein, whereas mutations disrupting the asymmetric arrangement abrogate cartwheel formation. Thus, our results demonstrate that an asymmetric SAS-6 interaction is essential for cartwheel formation.

Research results has been presented during the weekly meetings of the research group, in annual meetings with collaborators, to colleagues in departmental seminars, in the Departmentâ€™s Annual Recess. Moreover results has been disseminated in International Conferences and Workshops and exploited in different outreach activities with the aim to communicate research advancements to non specific audience and raise awareness about the importance of research to society. Any future results will be disseminated through publication in peer-reviewed, international scientific journals,following an â€œOpen Accessâ€ policy. Protein structures of single components or complexes, and the data underpinning them (crystallographic structure factors, EM datasets), will be deposited at the RCSB, EMDB databases and they will be released upon publication.
All disseminated results and outputs of the Fellowship acknowledged the funding received from the Marie Sklodowska- Curie programme (Article 29.4 of the grant agreement) under Horizon 2020. We commit that all future publications and other outputs (in print/ or in preparation) will also comply with this obligation.

Final results

Studies to date have noted the very high conservation of centriole components at the structural and mechanistic level. Thus, it is highly likely that an asymmetric SAS-6 oligomer, similar to that seen in Chlamydomonas is at the root of cartwheel polarity in vertebrates. However, SAS-6 sequence conservation is weak, especially in the coiled-coil
segment (25% identity between human and Chlamydomonas), which makes transferring amino acid level information for functional assays across systems difficult. Thus, to understand the molecular basis of cartwheel polarity in vertebrate centrioles we first need to resolve the structure of the underlying asymmetric SAS-6 interaction. We have obtained 2.6 Ã… resolution diffracting crystals from the human SAS-6 (hSAS-6) coiled coil domain and 2.3 Ã… resolution diffracting crystals from the zebrafish SAS-6 coiled coil (67% identical to the human). Analysis of crystallographic parameters suggests the asymmetric units of both crystals contain two coiled-coil dimers, and an interaction between SAS-6 coiled-coils has been corroborated by SEC-MALS. We will resolve the structures of these SAS-6 coiled-coil arrangements to understand the basis of SAS-6 oligomer asymmetry, and engineer mutations that specifically disrupt this interaction as performed previously.

Elucidating the structural and molecular mechanisms behind the origin of organelle polarity will advance our understanding of centriole assembly, a fundamental cellular process with clear implications in biology and for life quality, and of great interest to both cell and structural biologists. Such an understanding would place centrioles in the forefront of an emerging field in structural cell biology: to resolve the architecture of large cell assemblies and organelles.