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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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Searching for the common function of the BBSome across the evolution and development
Mašková, Kristýna ; Štěpánek, Ondřej (advisor) ; Čajánek, Lukáš (referee)
The BBSome is a protein complex whose function is associated with ciliary trafficking. It has been found that the BBSome is evolutionarily conserved among ciliated organisms. The disruption of the BBSome leads to cilia dysfunctions and affects many signalling pathways. In humans, genetic defects in the BBSome are the cause of Bardet-Biedl Syndrome, which is a pleiotropic disease. The BBSome is studied separately in various model organisms and cell lines to understand the molecular functions of the BBSome. These studies have not been compared to see if there is a common BBSome function, which could be why the BBSome is evolutionarily conserved among ciliated organisms. In this bachelor's thesis, the knowledge about the BBSome was summarized and compared to identify a putative common function of the BBSome among various model organisms or cell types. It seems that there has not been found any BBSome function that could be identified as the common function because the BBSome has many specialized functions in different organisms and tissues. The only distant similarity is BBSome-dependent ciliary retrograde transport, which has been described in most of the studied model organisms and cell types.
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Stability of protein complexes in the cytoskeleton of the eukaryotic flagellum
Pružincová, Martina ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee)
The cilium/flagellum is a complex organelle protruding from the cell body and functioning in motility, sensing, and signalling. It is composed of hundreds of protein constituents, the majority of which comprise the flagellar cytoskeleton - the microtubule-based axoneme. Because the flagellum lacks ribosomes, its protein constituents have to be imported from the cell body and delivered to proper locations. Moreover, these proteins have to retain their function over a considerable length of time, despite the mechanical stress caused by flagellar beating and due to environmental exposure. This raises the question whether and where protein turnover occurs. Previously, it was established that Chlamydomonas reinhardtii flagella are dynamic structures (Marshall & Rosenbaum, 2001). In contrast, in the Trypanosoma brucei flagellum axonemal proteins are remarkably stable (Vincensini et al., 2018). However, the questions of axonemal assembly and stability were so far investigated only for a small number of proteins and during relatively short periods. Moreover, in these experiments expression of studied proteins was controlled by non-native regulatory elements. To elucidate the site of incorporation of proteins from all major axonemal complexes and to find out if and where the protein turnover occurs, T....
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Stability of protein complexes in the cytoskeleton of the eukaryotic flagellum
Pružincová, Martina ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee)
The cilium/flagellum is a complex organelle protruding from the cell body and functioning in motility, sensing, and signalling. It is composed of hundreds of protein constituents, the majority of which comprise the flagellar cytoskeleton - the microtubule-based axoneme. Because the flagellum lacks ribosomes, its protein constituents have to be imported from the cell body and delivered to proper locations. Moreover, these proteins have to retain their function over a considerable length of time, despite the mechanical stress caused by flagellar beating and due to environmental exposure. This raises the question whether and where protein turnover occurs. Previously, it was established that Chlamydomonas reinhardtii flagella are dynamic structures (Marshall & Rosenbaum, 2001). In contrast, in the Trypanosoma brucei flagellum axonemal proteins are remarkably stable (Vincensini et al., 2018). However, the questions of axonemal assembly and stability were so far investigated only for a small number of proteins and during relatively short periods. Moreover, in these experiments expression of studied proteins was controlled by non-native regulatory elements. To elucidate the site of incorporation of proteins from all major axonemal complexes and to find out if and where the protein turnover occurs, T....
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