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Title: Regulation of γ-tubulin ring complex recruitment in Drosophila
Author: Tovey, Corinne
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Microtubule nucleation is catalysed by γ-tubulin ring complexes (γ-TuRCs) which are recruited to specific microtubule organising centres (MTOCs) within cells. The recruitment and activation of γ-TuRCs allows regulation of microtubule nucleation in both time and space. Mozart1 (Mzt1) is a recently-discovered component of the γ-TuRC which is important for the recruitment of γ-TuRCs to MTOCs, and therefore for cell division, in all systems in which it has so far been studied. Work in yeasts and cultured human cells suggests that Mzt1 is important for the binding of tethering proteins to the γ-TuRC, driving oligomerisation of the ring or activation of the γ-TuRC, and Mzt1 has been proposed to stabilise a hinge region in GCP3 which moves upon tethering-protein binding. In this thesis, I show that Mzt1 is a member of the γ-TuRC in Drosophila but that, surprisingly, its expression is restricted to the testes. This relatively simple finding has a major impact on the field, as it answers the longstanding question of whether γ-TuRC heterogeneity exists. Consistent with testes-specific expression, mzt1 null mutants are viable but develop male sterility as they age. Thus, unlike in other organisms, Drosophila Mzt1 is dispensable for cell division but is required for the production of sperm. Intriguingly, I found that Mzt1 is important for the recruitment of γ-TuRCs to the basal bodies but not to the mitochondrial derivatives of spermatids, demonstrating that γ-TuRC heterogeneity can exist even within the same cell at the same developmental stage. I have also found that differences between the centrosomal and testes-specific mitochondrial isoforms of the γ-TuRC tethering protein Centrosomin (Cnn) represent a means of regulating γ-TuRC recruitment and activity in a tissue-specific manner, and that this mechanism may be conserved in humans. I demonstrate that the extended N-terminus of centrosomal isoforms of Cnn is inhibitory to the CM1-domain-γ-TuRC interaction and that mutations that mimic phosphorylation within the N-terminus confer binding ability. Given that the centrosomal Cnn isoform is known to be phosphorylated specifically at centrosomes during mitosis, I propose that centrosome-specific phosphorylation events within the N-terminus relieve the CM1-domain inhibition. In contrast, the absence of the inhibitory N-terminal extension within the testes-specific Cnn isoforms allows these isoforms to bind γ-TuRCs at mitochondria, which lack mitotic kinases. This would provide cycling cells with control over when and where their γ-TuRCs are activated, contributing to the precise spatiotemporal regulation of spindle formation.
Supervisor: Conduit, Paul Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Microtubules ; ?-TuRC ; Mozart1 ; Centrosomin