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:
• How SAS-6 stack along the length of centrioles, thereby providing an initial scaffold for subsequent recruitment of further centriole components
• Furthermore, individual cartwheel hubs, resolved by Xray crystallography, are symmetric along the ring plane (have identical ‘top’ and ‘bottom’ 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
