Structural and Functional Analysis of the Protein Network Controlling Centriole Biogenesis

Schneider, Manuela Sandra (2017). Structural and Functional Analysis of the Protein Network Controlling Centriole Biogenesis. PhD thesis The Open University.



Centrosomes function as microtubules organising centres in mitosis or as basal bodies in cilia and flagella formation. The duplication of centrioles, which are at the core of each centrosome, is tightly regulated and occurs only once per cell cycle, which allows for correct segregation of chromosomes. In C. elegans, only five proteins are essential for centriole duplication. How their Drosophila counterparts interact with each other on a molecular level still remains largely unknown.

To further our understanding of the protein interactions governing centriole duplication, I applied in vivo and in vitro methods to design a protein interaction network at the centriole. I analysed mass spectrometry data from purifications of ProteinA-tagged centriole duplication proteins to identify in vivo protein complexes; and performed direct in vitro and in vivo protein-protein interaction assays.

Additionally, I studied the Drosophila proteins Ana2 and Sas6; showing that Plk4 phosphorylates S318, S365, S370, and S373 of the Ana2-STAN motif; which identifies a direct substrate of Plk4. By in vitro interaction studies I show that Sas6 aa276-432 and Ana2-STAN interact directly, if the latter is phosphorylated by Plk4. The depletion of Ana2 or expression of Ana2-4A causes loss of centrosomes in Drosophila cell culture, which was not observed when expressing Ana2-4D. This supports the importance of Plk4-mediated phosphorylation of Ana2-STAN in centriole duplication.

Mass spectrometry analysis of Sas6 purifications from cultured Drosophila cells identified the uncharacterised protein CG33052 (Dragon) and reciprocal purifications of tagged Dragon from Drosophila cell culture and syncytial embryos identified Sas6. By in vitro interaction studies I confirm that Dragon aa191-318/GoRab and Sas6 aa351-462/HsSas6 interact directly. The deletion mutant DragonΔaa260-286 does not interact with Sas6 in vitro or in vivo, and fails to rescue centriole duplication after Dragon depletion. Dragon localises to the trans-Golgi and co-localises with Sas6 at the centriole throughout the cell cycle.

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