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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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Role of NAV3 in glioblastoma cells invasiveness
Legátová, Anna ; Brábek, Jan (advisor) ; Libusová, Lenka (referee)
The invasion of tumor cells from the primary lesion and the formation of metastases are the main reasons for the severe impact of cancer diseases. An option for dealing with this poor impact is the development of drugs (so-called migrastatics) that would target these processes and thus limit the spread of tumor cells from the site of the primary tumor. However, to develop such drugs, it is essential to clarify the molecular mechanisms that control or promote cell migration. One of the possible strategies for migrastatics development is the targeting of cytoskeletal structures, which play an indispensable role in cell migration. This work is focused on Neuron navigator 3 (NAV3), a protein that binds to + ends of microtubules (MTs), participates in their stabilization, and is able to mediate crosstalk between MTs and the actin network. The function of NAV3 is important for directing MTs into growing axons and proper neurite outgrowth, which is necessary for brain development. The results of this thesis suggest that NAV3 could act as a pro-tumor factor, which localizes not only to the + ends of MTs, but also to the cell protrusions, and whose presence supports the cell expansion and increases the invasive potential of glioblastoma cell lines. Key words: neuron navigator 3, microtubules, cancer,...
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Tubulin post-translational modifications and microtubule associated proteins in neural development and disease.
Belyaeva, Polina ; Balaštík, Martin (advisor) ; Siahaan, Valerie (referee)
Microtubules (MTs) are essential components of the cytoskeleton in all eukaryotic cells. Their function is particularly important in neurons where MTs stabilize their long processes and are responsible for the precisely regulated anterograde and retrograde, intra-axonal and intra- dendritic transport over long distances. MTs are essential also during development of the vertebrate brain and all its major steps: neurogenesis, neuronal migration and neuronal differentiation. MTs are regulated at multiple levels, but two seem to be particularly important: 1. posttranslational modifications of tubulin (PTMs) have been shown to control several MT properties as stability or MT-based transport. 2. microtubule-associated proteins (MAPs) that bind soluble MT subunits, MT lattice as well as MT ends and control MT-based transport and MT dynamics by either stabilizing, destabilizing or severing MTs. Consequently, deregulation of either tubulin PTMs or MAPs may induce severe changes in neuronal cytoskeleton. Bachelor's thesis summarizes current knowledge on how PTMs (especially polyglutamylation) and MAPs (especially microtubule cleaving proteins such as spastin) regulate MT and neuronal development and degeneration. Keywords: Microtubules, tubulin post-translational modifications, polyglutamylation,...
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Cytoskeletal orchestration of early mammalian development
Novotný, Jan ; Libusová, Lenka (advisor) ; Mašek, Jan (referee)
Structures of the cytoskeleton play a critical role in early mammalian development. Microtubules, intermediate filaments and actin microfilaments provide structural and mechanical basis for major developmental transitions of preimplantation development. Microvilli supported by bundles of actin filaments become restricted to one pole of the blastomere during the process of polarisation. All components of the cytoskeleton network undergo apparent reorganisation in embryo compaction. Cells flatten and adhere to each other thanks to the formation of cytoskeleton-linked intercellular junctions and oscillatory contractions generated by actomyosin complexes. Filopodia, actin based protrusions, are debated for their potential role in blastomere apposition and cell signalling. The first process of cell line specification in mammalian development is that which defines outer cells of morula as presumptive trophectoderm and inner cells as presumptive inner cell mass. Proteins and structures of cytoskeleton are asymmetrically distributed between these two populations helping to determine the fate of each blastomere. The aim of the thesis is to summarise the roles of cytoskeleton in blastomere polarisation, embryo compaction and specification of inner and outer cell populations in mammalian preimplantation...
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Role of tau phosphorylation in formation of tau envelopes
Karhanová, Adéla ; Lánský, Zdeněk (advisor) ; Štěpánek, Luděk (referee)
Tau is an intrinsically-disordered microtubule-associated protein important for axonal development and a critical regulator of microtubule functions in axons. Tau activity is controlled by phosphorylation and its deregulation resulting in tau hyperphosphorylation and aggregation has been linked to multiple neurodegenerative disorders, collectively termed tauopathies. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive "envelopes" around microtubules. Tau envelopes regulate the action of other microtubule-associated proteins, such as the motility of molecular motors, and protect microtubules against degradation by microtubule-severing enzymes. How the formation, dynamics, and function of tau envelopes are regulated, however, is unknown. Here we show that tau phosphorylation impedes the formation and functioning of protective tau envelopes. Using a combination of reconstitution experiments and live cell imaging, we show that phosphorylated tau incorporates into tau envelopes and that it slows down the envelope growth. Importantly, we demonstrate that phosphorylated tau also destabilizes already existing envelopes leading to their disassembly. Together, our results demonstrate...
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The role of cytoskeleton in movement and navigation of neuronal growth cones
Olekšák, Adam ; Libusová, Lenka (advisor) ; Weissová, Romana (referee)
Growth cone is a dynamic structure localized at the tips of growing neurites. It detects guidance cues in the extracellular environment and enables growing neurite to properly respond to them. Cytoskeleton plays a key role in navigation of growing neurite. Microtubules and microfilaments enable the motion of the growth cone by generating force acting on the cytoplasmic membrane as well as on the substrate on which the neuron grows. Microfilaments are located mainly in the periphery of the growth cone. They are more dynamic than microtubules, and their polymerization and depolymerization enable formation of dynamic processes on the growth cone. These are used by neurons to gain information about their surroundings and for formation of adhesions on permissive substrates. Adhesions facilitate the growth of neurites. Direction, timing and speed of formation of the dynamic processes are regulated by a number of actin-associated proteins. These proteins are common targets of signalling pathways that are induced by activation of growth cones' cytoplasmic membrane-bound receptors by guidance cues. Along the microfilament bundles, microtubules enter the periphery of the growth cone aided by crosslinking and motor proteins. Microtubules are necessary for reducing the growth cone dynamics, the first step in...
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Roles of tubulin post-translational modifications in regulation of microtubule-based processes
Šliková, Pavlína ; Novák, Petr (advisor) ; Libusová, Lenka (referee)
Microtubular cytoskeleton plays crucial roles during diverse cellular processes, such as intracellular transport, cell motility and chromosome segregation during cytokinesis. Tubulin, the building block of microtubules, undergoes numerous post-translational modifications which affect microtubular dynamics and organization as well as their interaction with associated proteins. Understanding the role post-translational modifications play in the diversification of functions and properties of microtubules is key for our comprehension of the dynamics of the complex microtubule cytoskeleton. However, mechanisms behind the effect of post-translational modifications on microtubule cytoskeleton are not fully understood. In this work, we focus on the influence of post-translational modifications on microtubule polymerization and interaction with molecular motor kinesin-1. Using total internal fluorescence and interference reflection microscopy techniques, we here show that high levels of post-translational modifications on microtubules decrease the time of microtubule-kinesin interaction whereas binding affinity and median velocity are not significantly different on modified and unmodified microtubules. Further, we show that the absence of polyglutamylation on tubulin isotypes leads to a faster microtubule...
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Induction and course of programmed cell death in cancer cells after taxane application."
Kábelová, Adéla ; Jelínek, Michael (advisor) ; Gemperle, Jakub (referee)
The taxanes are a class of commonly used anticancer agents, which are very effective in treatment of breast, ovarian, prostate or lung cancer. Taxanes bind to the β-tubulin subunit of microtubules and lead to their stabilization and inhibition of depolymerization. Such microtubules lose their function to form mitotic spindle, thus arresting cells in G2/M phase and resulting in apoptosis. Unfortunately some cells are able to escape from taxanes-induced apoptosis by developing various mechanisms of resistance including alteration in taxanes target microtubules or upregulation of specific transporters that pump the drug out of cells. Other types of resistance are connected with process of programmed cell death (PCD), especially with proteins that after taxane application participate in its successful progress. Taxanes can directly or indirectly modify the activity of Bcl-2-family proteins that control mitochondrial and endoplasmic reticulum integrity, thus regulating the initiation of PCD. Caspases are executioners of PCD and caspase-2 activated by cytoskeletal disruption seems to be especially important in taxanes- induced apoptosis. In some cases can taxane treatment also result in caspase-independent cell death. Special role has protein p53 that seems to be involved only in apoptosis caused by low taxanes...
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Molecular base of plant HSP90-MT interaction
Benáková, Martina ; Krtková, Jana (advisor) ; Malcová, Ivana (referee)
Microtubules (MTs) are one of the essential cell structure that participate in a number of key events in the plant cells and their properties and functions are influenced and modified by many other proteins. These proteins belong to a group of microtubule- associated proteins (MAPs, microtubule-associated proteins). One of the MAPs, the molecular chaperone Hsp90, examines and fulfills a large number of different functions in the cell. Its colocalization with MTs has been demonstrated previously by Freudenreich and Nick (1998) and Petrášek et al. (1998). However, direct interaction with MTs was described only recently using cosedimentation assay. The specific cytosolic isoform of tobacco Hsp90 bound to MTs was called Hsp90_MT due to its ability to bind MTs. It has been also found that the binding to MTs is independent on the activity of ATP (Krtková et al., 2012). The authors also described a positive effect of Hsp90_MT on MT recovery after their exposure to cold stress. Although MT cytoskeleton dynamics is influenced by a large number of MAPs, it is surprising that the molecular mechanism of MAPs interaction with MTs and their MT-binding domains have not been described yet. Therefore, we decided to determine the tobacco Hsp90_MT MT-binding domain by production of a set of recombinant proteins...
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Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Novák, Petr (referee)
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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