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Role of the Rcd4:Ana3 sub-complex in Drosophila centriole duplication



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Complementary studies in several model systems have played a significant role in establishing a framework of conserved proteins that govern the centriole duplication cycle. In an attempt to search for additional molecular components, two genome-wide RNA interference screens were conducted in cultured Drosophila cells (Dobbelaere et al., 2008; Goshima et al., 2007). While these studies significantly advanced the cataloguing of centriolar and centrosomal proteins in flies, the precise molecular contributions of several of these proteins has remained unknown. Reduction in Centrosomin dots 4 (Rcd4) and Anastral spindle 3 (Ana3) are two such poorly characterised centriolar proteins. Thus, the aim of my thesis was to investigate the molecular functions of these two proteins in Drosophila centriole biogenesis. My biochemical studies demonstrated a direct interaction between Rcd4 and the C-terminal part of Ana3, identifying a new centriolar sub-complex. This laid the foundation for me to further examine the function of the Rcd4:Ana3 sub-complex in the centriole duplication cycle. To gain insights into the centriolar roles of the sub-complex, I turned to generating CRISPR/Cas9 mutants for their two genes. Analysis of mutant phenotypes revealed similar functions for both proteins in centriole duplication in somatic tissues and for the formation of basal bodies and cilia in the neurosensory organs. Moreover, I found that both proteins are recruited to the procentriole in interphase where Rcd4 loading is dependent upon the prior loading of Ana3. The assembly of the Rcd4:Ana3 sub-complex is a pre-requirement for the recruitment of Ana1 and consequently for its function in initiating the final stage of centriole to centrosome conversion. These studies provide insight into the molecular mechanism of Rcd4:Ana3’s role in somatic cells of the fly. However, I found differing requirements for the two components for centriole development in the male germ line. Ana3 is essential for centriole formation during spermatogenesis but Rcd4 is not. My preliminary results indicate that Rcd4 may potentially contribute to the stabilisation of the cartwheel of spermatocyte centrioles. Nevertheless, further experiments are required to understand the precise mechanism of Rcd4’s role in the male germ line. Taken together, my thesis work contributes towards the identification of a new centriolar sub-complex between Rcd4 and Ana3 which functions as a pre-requirement for the initiation of centriole to centrosome conversion in the final stage of the duplication cycle in somatic cells.





Glover, David M


Rcd4, Ana3, Centriole, Centrosome, Basal Body, Drosophila


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge