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Core - Structure and Assembly Mechanisms of Centrioles

Mark Van Breugel

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Medical Research Council (MRC)
Centrioles are barrel shaped, 9-fold symmetric cell organelles that constitute the base of cilia and flagella and are essential for their formation. Centrioles also form the core of centrosomes that they help to structure. Due to their important cellular roles, centrioles and their dysfunctions are associated with numerous human diseases ranging from microcephaly and ciliopathies to cancer and infertility. In the past, successful efforts have been made to identify the key proteins involved in the assembly and function of centrioles. However, what is so far largely lacking is a detailed understanding of how these components are structurally organized within the centriole, what their exact assembly mechanisms are and how their assembly leads to a faithful formation of functional centrioles. My lab aims to shed light on these questions by combining biochemical and biophysical approaches with the structural characterisation of centriolar components using X-ray crystallography and electron microscopy. Specifically, we try to make key centriolar components or fragments of these recombinantly using different expression systems. The purified components are characterised biochemically and biophysically to obtain information on their ability to self-associate and associate with each other. The components or their complexes are also subjected to protein crystallographic methods to derive high-resolution structures or, if diffraction grade crystals cannot be obtained, are studied by electron microscopy. The combined information from these approaches is then used to derive detailed models of the self-association and mutual interactions of the centriolar components. Subsequently, the significance of these interactions for the formation of centrioles is tested using targeted mutations of these components in vivo and the effect of these mutations on the architecture and function of centrioles is assessed using light microscopy and electron microscopy. The nature of the resulting assembly defects and specifically the resulting presence or absence of other centriolar components will then reveal the hierarchy of interactions that make up centrioles. The power of this approach is highlighted by our previous characterization of the conserved centriolar protein SAS-6. We could show that SAS-6 self-associates to organise a key assembly intermediate in centriole formation, elucidate its assembly mechanism and show how it contributes to the establishment of centrioles of the right symmetry in vivo. A detailed study of other key centriolar components can therefore be expected to yield information about how this assembly intermediate is extended and how the downstream assembly steps of centrioles are achieved on a molecular level.

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