Functional Investigation of Trypanosoma brucei Microtubule Associated Proteins and Their Role in Cellular Morphogenesis.

Towers, Katie (2010) Functional Investigation of Trypanosoma brucei Microtubule Associated Proteins and Their Role in Cellular Morphogenesis. PhD thesis, Lancaster University.

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Abstract

The Trypanosoma brucei cytoskeleton is generated by an elaborate array of subpellicular microtubules. This corset of microtubules requires extensive remodelling during cell growth and division. Microtubule nucleation/outgrowth and coordinated severing/re-establishment of inter-microtubule cross-links is orchestrated by microtubule associated proteins (MAPs). The T. brucei genome encodes a discrete set of trypanosomatid specific MAPs but functional data for most of these proteins is sparse. Through bioinformatic analysis we have identified a novel trypanosomatid-specific protein (GB4L). GB4L has a functional role in trypanosome morphogenesis and microtubule organisation in the procyclic and bloodstream form of the parasite. RNAi ablation of GB4L causes a cytokinetic defect, as does depletion of TCP86 (another novel and trypanosomatid-specific MAP recently identified in the McKean laboratory). Electron microscopy was used to examine both the GB4L and TCP86 RNAi cell lines, demonstrating that the phenotypes observed after GB4L and TCP86 protein depletion are very distinct. However, in both cases protein depletion causes morphological abnormalities at the posterior end of cells. ganisation of subpellicular microtubules was interrogated through localisation of canonical plus tip binding proteins (+TIPs) EB1 and XMAP215. Microtubule plus ends are organised in a highly reproducible pattern throughout the cell cycle. This organisation becomes disrupted when GB4L or TCP86 are depleted, showing that GB4L and TCP86 play critical yet distinct roles in orchestrating cytoskeletal remodelling. RNAi ablation of GB4L and TCP86 also has effects on other MAPs due the concerted roles these proteins play in cytoskeletal remodelling. Investigation into MAP interdependency relationships suggests that MAPs assemble as distinct complexes in a defined temporal order on subpellicular microtubules. This work provides further insight into the complexities of trypanosome morphogenesis and indicates that disruption of critical MAP interactions could conceivably provide valid targets for the development of novel chemotherapeutic strategies against human and animal trypanosomiasis.