INVESTIGATION OF THE DISTAL APPENDAGE PROTEIN CEP164 IN Trypanosoma brucei
Cilia and flagella are highly conserved organelles required for eukaryotic cells to perform sensory and motility functions. They are found in many cells of the human body and are essential for the survival of single celled protozoans. These organelles are nucleated from a basal body after docking to the plasma membrane and are structurally similar to centrioles. Docking of the basal body requires the presence of transitional fibres, which mature from distal appendages on the centriole. These structures rely on the presence of the Cep164 protein to carry out docking and ciliogenesis. Understanding of basal body docking and flagellum length regulation in the Trypanosoma brucei cell is limited and is thought to involve the transitional fibres and surrounding structures. Three diverse Cep164 orthologues in the T. brucei parasite have been identified, but knowledge on their function is limited as functional analysis of these proteins has not been carried out. This project, in collaboration with the TrypTag project used a bioinformatic approach to identify basal body proteins in the T. brucei cell. Candidate proteins were confirmed as basal body components through endogenous tagging and co-localisation studies. The Cep164 orthologues (Cep164A, Cep164B and Cep164C) localised to the distal section of the mature basal body only, with Cep164C showing a cell cycle dependent localisation. Functional analysis of the Cep164 proteins was performed through the generation of inducible RNAi cell lines. Ablation of these proteins showed a functional role of flagellum length regulation, or formation of the transitional fibres and correct basal body docking. This work provides further understanding on the three Cep164 proteins in the T. brucei cell and proposes how the cell regulates its flagellum length.
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