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Membraneless organelles in eukaryotic cells
Beránková, Pavla ; Libusová, Lenka (advisor) ; Bařinka, Cyril (referee)
Membraneless organelles (MLOs) are a newly described type of cellular compartments. They consist of protein and nucleic acid molecules that undergo liquid-liquid phase separation (LLPS). MLOs are able to fulfill unique biological roles, because they are highly dynamic and their composition can be effectively regulated. Composition and function of these formations are swiftly being elucidated. The work summarizes the basic principles of LLPS in living organisms and further focuses on several types of MLOs functionally connected to microtubules (MTs). Their recurrent feature is the ability to nucleate MTs. This eventual role corresponds well with their high temporal and spatial dynamics.
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Plant tubulin code
Ničová, Klára ; Schwarzerová, Kateřina (advisor) ; Cvrčková, Fatima (referee)
Microtubules, which are made of polymers of α- and β-tubulin, are an integral part of the cytoskeleton. Both types of tubulin share a considerable sequential homology across eukaryotic organisms. Tubulins are encoded by relatively large gene families. The expression of these genes produces different tubulin isotypes, some of which may exhibit different properties. Tubulin isotypes can be further posttranslationally modified. The best known posttranslational modifications of tubulin include acetylation, phosphorylation, tyrosination, polyglutamylation and polyglycylation. The tubulin code arises from the combination of expression of different tubulin isotypes and their posttranslational modifications. As a consequence, microtubules in cells can be composed of a "mixture" of different tubulins with distinct functions and properties. Even though the existence of the tubulin code has been proven in every model organism, plants included, the precise understanding of the meaning of microtubules being composed of different tubulins is still subjected to research. Much of the research on the tubulin code has been carried out on animal models. In contrast, relatively little is known about the existence of the tubulin code in plants. This theses summarizes current knowledge on the localization and regulation...
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Protein composition of the cytoskeleton of protists
Švagr, Eva ; Hampl, Vladimír (advisor) ; Pánek, Tomáš (referee)
While we have a good understanding of the roles of actin and tubulin filaments in the cell cytoskeleton, intermediate filaments (IFs) are often overlooked. However, the importance of IFs becomes quite apparent, as proteins similar to IF proteins have been identified in many protist cells. This holds particularly for the cells of protists, where even the functions of some members of the of actin and tubulin superfamilies remain unclear. Intermediate filaments are still not well established as components of protist cytoskeletons, in contrast to their more thoroughly studied counterparts in Metazoa. Protist and metazoan IF proteins are dissimilar in their sequence; however, they share similarities in structure, and they assemble autonomously into analogous filaments. IF-like proteins have been localized to striated fibers or unique cytoskeletal components in several organisms, even though not much is known about the protein composition of these striated fibers to date. This suggests that IF-like proteins might be a universally present component of these striated fibers often seen in protist cells. Keywords: Protists, cytoskeleton, microtubules, microfilaments, intermediate filaments, fibrils, protein composition
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Sperm centrioles and their role in reproduction
Vlčková, Monika ; Frolíková, Michaela (advisor) ; Liška, František (referee)
Centrioles are evolutionarily conserved protein structures composed of microtubules. In somatic cells, centrioles serve as the basal body of cilia and flagella and allow the assembly of pericentriolar material, thereby creating the centrosome. Without centrosome, animal cells are not capable of nuclear division. Centrioles do not arise de novo and their formation always requires the presence of a preexisting centriole. Since there are no centrioles in the egg at the time of fertilization, unlike spermatozoa, sperm is the carrier of centrioles and therefore all of the centrioles of the emerging organism are of paternal origin. There are two centrioles in the sperm - a cylindrical shape proximal and a distal one, which is perpendicular to the proximal. The sperm centrioles are the basis for the formation of the sperm flagellum and after fertilization form the mitotic spindle of the zygote, necessary for equal DNA and cell distribution. It follows from the above that the presence of centrioles in sperm is essential in mammals and defects in their structure may lead to male sterility or embryo development disorders. However, sperm centrioles differ from somatic centrioles in their structure and behavior and understanding these differences is one of the important tasks of reproductive biology.
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Microtubule inner proteins
Bočan, Václav ; Libusová, Lenka (advisor) ; Sulimenko, Vadym (referee)
Microtubules are a prominent part of the cytoskeletal network in eukaryotic cells. They are involved in nearly all cellular processes, e.g. in vesicular trafficking, signal transduction, locomotion, or cell morphogenesis. To discharge that many functions, precise regulation of microtubule dynamics and architecture is essential. Such regulation is maintained by various microtubule-associated proteins, which usually bind from the outside. However, several proteins were found to bind in the lumen of microtubules. These microtubule inner proteins were shown to function either as post-translational modifiers of tubulin or stabilizers in time- persistent microtubular structures. A few inner proteins were identified, but our understanding of their attributes is still incomplete. This thesis summarizes current knowledge of microtubule inner proteins. The scope is focused on their enzymatic and structural features. Tubulin acetyltransferase represents the enzymatic MIPs. Possible ways of lumen entry and impact on the tubulin lattice are described. Next, the structural roles of proteins inside microtubules, most prominent in the axoneme, are outlined. The relevance of microtubule inner proteins for cytoskeletal functions, flagellar motility, and future perspectives are discussed at the end.
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Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica
Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...
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Interakce viru klíšťové encefalitidy s cytoskeletem hostitelských buněk
PRANČLOVÁ, Veronika
This thesis is focused on the role of host cytoskeleton, primarily microtubules and microfilaments, during tick-borne encephalitis virus infection in human neuroblastoma cell line SK-N-SH and tick cell line IRE/CTVM19. The importance of cytoskeletal integrity and dynamics to the viral replication cycle were examined using specific chemical inhibitors showing the virus utilizes studied structures in both cell lines. Immunofluorescence microscopy revealed structural changes in the actin cytoskeleton during late infection in SK-N-SH cells. Moreover, differences in expression of cytoskeleton-associated genes in both cell lines were compared. Several genes with up-regulated expression in SK-N-SH cells were identified during late infection.
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Major structural protein of Polyomaviruses: Interactions with host cell structures
Mrkáček, Michal ; Horníková, Lenka (advisor) ; Němečková, Šárka (referee)
The main structural protein VP1 is the product of late polyomaviral genes and it is the largest and the most abundant protein of the whole polyomaviral capsid. Because of the low coding capacity of the polyomaviral genomes, it is considered that in addition to its structural role the VP1 protein might have some additional functions in the late phase of the infectious cycle. This diploma thesis is exactly on these additional functions. In the case of the VP1 protein of mouse polyomavirus, it was observed that the protein is capable of binding to the structure of cellular microtubules. The first objective of this work was to test whether pentamers of the VP1 protein are able of this binding without the participation of other cellular (or viral) proteins. Based on an in vitro experiment, we showed that protein VP1 binds to the structure of microtubules very inefficiently. The second objective of this work was to prepare a detection system that would allow an identification of potential interaction partners of BK polyomavirus VP1 protein. Therefore, expression plasmids producing the N and C-terminally tagged VP1 protein were prepared. These tagged proteins had the property of being biotinylated whilst being produced in the transfected cells. By using affinity chromatography, the entire protein complexes...
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Comparative phenotypic study of selected Arabidopsis formin mutants
D'Agostino, Viktoria ; Cvrčková, Fatima (advisor) ; Tylová, Edita (referee)
Actin filaments and microtubules are involved in cell development and morphogenesis. Plant Class II formins regulate both cytoskeletal polymers. However their function has not yet been fully described. This study examines effects of LOF mutations in Arabidopsis thaliana FH13 (AT5G58160) and FH14 (AT1G31810) genes on early root system development using a pharmacological approach. Since measuring root length of numerous mutant lines in multiple conditions is laborious and time consuming, this thesis also involves optimization of this process with the aim to establish a reliable method of fast visualisation and measurement of Arabidopsis seedlings in a time series in the laboratory. Furthermore, statistical analysis for a large amount of data gathered in multiple conditions had to be optimized. While no significant phenotype in terms of root length was found in fh13, fh14 and double fh13 fh14 LOF mutants under standard conditions, treatment with cytoskeletal drugs revealed possible changes in lateral root branching in an fh14 mutant. Nevertheless, specific function of FH13 and FH14 remains a question.
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Molecular mechanism of microtubule severing by katanin
Podhájecký, Roman ; Lánský, Zdeněk (advisor) ; Libusová, Lenka (referee)
Microtubule cytoskeleton is a dynamic filamentous network, whose reorga- nization underpins important cellular processes, such as cell motility or cell di- vision. This remodeling highly depends on microtubule-associated proteins that can remodel individual microtubules, such as katanin. Katanin is a microtubule- associated protein that employs ATP hydrolysis to sever microtubules. This function can rapidly reorganize microtubule networks by providing fast amplification of the microtubule polymer number as well as microtubule fast degradation. The exact mechanism of microtubule severing and its regulation by katanin is largely dis- cussed nowadays. In this project I would like to describe katanin's mechanism of action and its regulation on a molecular level. Key words: cytoskeleton, remodelling of microtubule networks, microtubules, microtubule se- vering enzymes, katanin 1
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