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Identification and characterization of flagellar tip proteins in Trypanosoma brucei
Pavlisková, Hana ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee) ; Hashimi, Hassan (referee)
The tip of the eukaryotic flagellum is one of the most important regions of the flagellum in many eukaryotic cells. Several important functions have been associated with the flagellar tip. However, its protein composition remains largely unknown. The main aim of this thesis was to identify flagellar tip proteins and characterize them. Using the parasitic model organism Trypanosoma brucei and the TrypTag.org project, a unique resource localizing every protein encoded in the trypanosome's genome, we were able to identify, for the first time, the complete catalog of flagellar tip proteins in a eukaryotic organism. In T. brucei the full complement comprises 78 proteins localizing exclusively to the flagellar tip or being highly enriched there. To characterize these proteins, we established new reagents and approaches. First, we developed antibody markers labeling the tip of an assembling trypanosome flagellum via recognizing the flagella connector. This enabled us to study the tip-specific processes. Second, we developed a rapid and cloning-free approach for tagging and inducible overexpression of trypanosome proteins. We demonstrated that the approach is well suited for overexpression of large proteins, such as some of the flagellum tip proteins. This enables the study of overexpression phenotypes and...
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