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MAP code and regulation of microtubule-based processes
Karhanová, Adéla ; Lánský, Zdeněk (advisor) ; Tomášová, Štěpánka (referee)
Microtubule associated proteins (MAPs) are considered as key regulators of molecular trafficking in cells. Even though their malfunctioning results in severe pathologies, such as neurodegenerative disorders, the regulatory roles of these proteins remain under debate. Since MAPs bind to the cytoskeleton, this structure has to be vital for the function of MAPs. Microtubules, a highly dynamic type of cytoskeletal structure, have been given extra attention due to their association with cell division and vital functions in neurons. Microtubules can undergo post-translational modifications that affect molecular motors as well as binding of other proteins, such as MAPs. Whether post-translational modifications of microtubules regulate the distribution of MAPs is so far not sufficiently documented. However, MAPs have been shown to cooperatively form cohesive envelopes on the microtubules and thereby regulate the access of motors and severing enzymes. As there are many types of MAPs and they are mutually exclusive, a hypothesis of a regulatory 'MAP code' emerged recently in the literature. Using available literature, this review will try to introduce the new model of MAP code and provide some background information on previous research on this topic.
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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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The role of anillin in the growth cone of neurons
Tomášová, Štěpánka ; Libusová, Lenka (advisor) ; Vinopal, Stanislav (referee)
During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...
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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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