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Title: Re-engineering Caenorhabditis elegans SAS-6 : from spirals to rings
Author: Ford, Jodie
ISNI:       0000 0004 8503 2406
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Centrioles have conserved 9-fold symmetry. This symmetry is templated by a scaffold of SAS-6. SAS-6 oligomerises to form a stack of rings, each with 9-fold radial symmetry, that forms the cartwheel, which imparts symmetry to the organelle. However, crystallographic and electron microscopy studies suggest that C. elegans is unique in that it forms a spiral SAS-6 scaffold instead of a cartwheel. Thus, there are two competing architectures of SAS-6 scaffold. Inconclusive electron microscopy of C. elegans centrioles mean it is unclear which architecture is present in vivo. C. elegans is used as a model for centriole biology, it is therefore important to determine which SAS-6 oligomer structure is present at the centre of the centriole because a spiral scaffold would have implications for centriole duplication and elongation. To probe the nature of the C. elegans SAS-6 scaffold, I use a protein engineering approach to convert spiral-forming SAS-6 into a ring oligomer. Molecular dynamics simulations of SAS-6 are used to screen the architecture of the SAS-6 mutants. The conformation of the engineered SAS-6 oligomers in vitro is determined using atomic force microscopy. I show that there is flexibility in the architectures sampled by wild-type C. elegans SAS-6 and that two point mutations are sufficient to increase the number of rings formed by C. elegans SAS-6 by >20%.
Supervisor: Vakonakis, Ioannis Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available