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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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The influence of the RACK1 scaffold protein and the ERK signalling pathway on cell polarity
Klímová, Zuzana ; Vomastek, Tomáš (advisor) ; Brábek, Jan (referee) ; Varga, Vladimír (referee)
The establishment of cell polarity is an essential step in cell migration, as it provides cells with information about the direction of migration. It is a highly dynamic process that leads to an asymmetric distribution of cytoskeletal networks, cell organelles, protein complexes and signalling pathways, which is reflected in the typical polarised cell shape. The cell shape is determined by the interplay between the dynamics of the actin cytoskeleton, cell adhesions and the cell membrane towards the extracellular surface. Cell adhesion and spreading on the extracellular matrix is a morphogenetic process in which cells initially spread isotropically from the point of first contact and then spontaneously break their radial symmetry and develop a migratory polarity with spatially separated protruding cell front and non-protruding cell rear. It is unclear how these symmetry break events, both complex and stochastic, are organised and regulated. In this study, I show that symmetry breaking in isotropically spreading fibroblasts begins with the establishment of a non-protruding cell rear delineated by large but sparse focal adhesions. Development of the non-protruding regions requires the scaffold protein RACK1, which promotes adhesion-mediated activation of ERK2. ERK2 and RACK1 inhibit p190RhoGAP...
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Molecular mechanisms of the assembly and function of BBSome
Prasai, Avishek ; Huranová, Martina (advisor) ; Varga, Vladimír (referee) ; Bosáková, Michaela (referee)
Bardet Biedl syndrome is a genetic disorder caused by the dysfunction of the BBSome, an octameric cargo adaptor protein complex. The BBSome facilitates the transport of signaling receptors into and out of the primary cilium, a microtubule based sensory organelle of the cell. The first part of this thesis focuses on the elucidation of the assembly of the BBSome in living cells. We generated a library of human and mouse cells lines deficient in the individual BBSome subunits and transduced them with the other YFP tagged subunits. We employed biochemical assays, immunofluorescence and quantitative fluorescence microscopy techniques to analyze the individual steps in the BBSome assembly pathway. We revealed that the BBSome assembly occurs sequentially in spatially regulated steps. We showed that BBS4 nucleates the assembly of a pre-BBSome at the pericentriolar satellites. The translocation of the pre-BBSome to the ciliary base is facilitated by BBS1. We also revealed that in a BBS chaperonin deficient cell line, BBS12 KO cells, a small fraction of the BBSome and/or BBSome sub-complexes are still able to form and localize to the cilium. This could suggest that the BBS chaperonins might act later in the BBSome assembly pathway providing a means for quality control for the BBSome. Ciliary ectocytosis...
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Intermediate filament proteins of Preaxostyla flagellates
Švagr, Ezra ; Hampl, Vladimír (advisor) ; Varga, Vladimír (referee)
5 Abstract Monocercomonoides exilis and Paratrimastix pyriformis are protists from within the group Preaxostyla (Metamonada), they possess an excavate morphology that is presumed to be an ancestral cytoskeletal organisation of eukaryotic cells. A significant part of eukaryotic but specifically excavate morphology is fibers composed of unidentified proteins. The hypothesis on which this thesis builds upon is that these fibers are composed of Intermediate Filament proteins (IF proteins). IF proteins are a polyphyletic group of proteins involved in the assembly of mechano-elastically important fibers in eukaryotes. The most widespread group of these proteins is a family called SF-assemblins, homologues of which were identified first in Chlamydomonas reinhardii, and giardins a protein family first discovered in Giardia intestinalis, which were also found to be related to this group. Nested into the bigger hypothesis is an idea, that SF-assemblins are present in M. exilis and P. pyriformis, further strengthening their position as a universally present eukaryotic feature. The goal of this work was to find support for the morphological hypothesis that LECA possessed an excavate morphology in protein composition of the cytoskeleton. Two approaches were employed. First, identifying proteins in cytoskeletal fraction...
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Identification and characterization of ciliary tip proteins
Gorilák, Peter ; Varga, Vladimír (advisor) ; Lánský, Zdeněk (referee) ; Dean, Samuel (referee)
The distal tip of the cilium/flagellum, also known as the ciliary tip domain (CTD), is critical for the structure and function of the eukaryotic cilium. The limited knowledge of its protein constituents hinders a better understanding of the domain. In this thesis, we set out to verify the localization of a subset of known mammalian CTD constituents and to assess the localization of candidate CTD proteins, orthologs of which localize to the tip of the flagellum of evolutionary distant protozoan Trypanosoma brucei. Using our localization pipeline, we identified two proteins that robustly localize to the CTD of the primary cilium. One of these proteins (ZC2HC1C), in addition, also localizes to stationary foci along the axoneme, positions of which coincide with sites of intraflagellar train pausing and turning. We hypothesize that these may be ends of sub-distally terminating axonemal microtubules. We further show that the protein ULK4 localizes to the CTD of motile ependymal cilia but not to the CTD of primary cilia, consistent with previously published phenotypes in ULK4 depleted mice and exemplifying differences in the composition of CTDs of the two types of cilia. Finally, we demonstrate that Expansion microscopy, a rapid and robust super-resolution technique, is well suited for ultrastructural and...
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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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Monoxenous trypanosomatids of invertebrates
Havlová, Jolana ; Votýpka, Jan (advisor) ; Varga, Vladimír (referee)
The class Kinetoplastea contains free-living and parasitic species. One of the most dominant group within the class is the order Trypanosomatida which includes obligate parasites (Trypanosoma, Leishmania) infecting a wide range of hosts. Some species are serious pathogens of humans and domestic animals and cause considerable losses. However, the majority of trypanosomatids belongs to monoxenous parasites of insect which are usually harmless to their hosts. Monoxenous trypanosomatids predominantly infect Hemiptera and Diptera. This diploma thesis is focused on the detection of monoxenous trypanosomatids in cockroaches captured in the Czech Republic and cockroaches from different breedings. Cockroaches are very suitable mechanical vectors of many different pathogens (including parasites) and are significant health threat for humans and animals. First trypanosomatids in cockroaches were documented at the beginning of the 20th century, but there is no study focused on this topic specifically. Another aim of this thesis is morphological and ultramicroscopic analysis and the study of the host specificity of the recently described species Herpetomonas tarakana, isolated from a cockroach. My findings were partly used in the already published study "Diversity of trypanosomatids in cockroaches and the...
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Searching for microtubule inner proteins
Bočan, Václav ; Libusová, Lenka (advisor) ; Varga, Vladimír (referee)
Diploma thesis Searching for microtubule inner proteins Bc. Václav Bočan Abstract Microtubules (MTs) - cylindrical polymers of α- and β-tubulin - maintain numerous irreplaceable func- tions in all eukaryotic cells. For this complex involvement of MTs in many cellular processes, precise tuning of their post-translational modifications, polymerization state, and interactome is crucial. Recently, a new mode of interactions with MTs was discovered - several microtubule inner proteins (MIPs) can enter the lumen of MTs. Little is known about MIPs in dynamic MTs in the cytoplasmic network. Only two proteins have been shown to bind to the inside of dynamic MTs so far: αTAT1 and MAP6; other proteins have been suggested to. Stabilised MTs, like the axoneme of the flagellum, contain dozens of orderly bound MIPs in the lumen and new ones are being added. MIPs are believed to play a role during axonemal assembly and to increase the stiffness required for flagellar beating. This diploma thesis investigated MIPs in both dynamic and axonemal MTs. In the first part of the thesis, the goal was to identify candidates for new MIPs in the dynamic MTs by two independent approaches - proximity-labelling by promiscuous biotin ligase using αTAT1 and MAP6 as baits, and direct isolation of MTs from cells and washing away outer...
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Regulation of microtubule dynamics revealed by single-molecule TIRF and IRM microscopy
Zhernov, Ilia ; Lánský, Zdeněk (advisor) ; Cifra, Michal (referee) ; Varga, Vladimír (referee)
The microtubular cytoskeleton is a ubiquitous and highly diverse biopolymer network present in all eukaryotic cells. Microtubules stochastically alternate between phases of growth and shrinkage. Cells take advantage of this dynamicity to generate forces for essential processes, such as cell division, motility or morphogenesis. Regulating the microtubule dynamics enables cells to adaptively respond to a wide range of tasks and conditions. Molecular mechanisms underpinning the regulation are not fully understood. Using a bottom-up approach and the combination of single molecule total internal reflection fluorescence (TIRF) microscopy and interference reflection microscopy (IRM), we here reconstituted and explored two dynamic cytoskeletal systems. (i) Microtubule doublets, comprising incomplete B-microtubule on the surface of a complete A- microtubule, provide an essential structural scaffold for flagella. Despite the fundamental role of microtubule doublets, the molecular mechanism governing their formation is unknown. We here demonstrate an inhibitory role of tubulin C-terminus in microtubule doublet assembly. By partial enzymatic digestion of polymerized microtubules followed by the addition of free tubulin in the presence of a stabilizing agent, we assembled microtubule doublets and revealed the B-...
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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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