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Characterization of perinuclear actin fibers and their role in cell migration
Hlaváčková, Tereza ; Vomastek, Tomáš (advisor) ; Binarová, Pavla (referee)
Cell migration is crucial for such physiological and pathological processes as wound healing, emryonal development, immune response, and methastasizing of the cancer cells. It is tightly coupled with cell polarization, nuclear traslocation, and turnover of actin cytoskeleton. Substantial, but so far poorely explored, part of actin cytoskeleton is perinuclear actin cap - dome-like structure above the nucleus costructed from perinuclear actin fibers. At the apical side of the nucleus perinuclear actin fibers are associated with LINC complex through nesprin proteins; at the edges of the cell they are anchored to focal adhesions. In the literature there were assumptions that this type of actin fibers can generate traction forces for nuclear reorientation during cell migration. The aim of this thesis is to elucidate the mechanism involved in the attachment of perinuclear actin to the LINC complex and the nucleus, thereby regulating the formation of the perinuclear actin cap. In addition, we aimed to establish a semi- automatic tool for perinuclear actin fibers quantification. Rat2 fibroblasts were used as the model cell line because they contain well-developed perinuclear actin cap. We focused on the inactivation of LINC complex components, namely Giant nesprin proteins (nesprin 1 and nesprin 2) and...
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The characteristics of stress granules in yeast Saccharomyces cerevisiae
Slabá, Renata ; Hašek, Jiří (advisor) ; Binarová, Pavla (referee)
9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...
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Functional characterization of selected microtubule regulatory
Vinopal, Stanislav ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
Microtubules (MTs) play crucial roles in intracellular organization and transport, cell polarity, motility, signalling, division and differentiation. MTs form complex arrays, which are, due to their highly dynamic nature, capable of rapid reorganization in response to cellular requirements. Dynamics, stability and spatial organization of MTs are regulated by many factors including MT regulatory proteins. In the presented study we functionally characterized three selected MT regulatory proteins: Ca2+ -sensor STIM1, MT severing protein spastin and γ-tubulin that is essential for MT nucleation. We found out that activation of bone marrow mast cells (BMMCs) leads to the formation of plasma membrane protrusions containing MTs. Formation of these MT protrusions is dependent on an influx of extracellular Ca2+ regulated by protein STIM1, located in endoplasmic reticulum. STIM1 associates with MTs and its depletion prevents formation of MT protrusions. This indicates that Ca2+ ions might be involved in MT regulation. Since STIM1 depletion also causes defects in chemotaxis, we propose that MT protrusions might be involved in sensing of external signals recognized by BMMCs. Glioblastoma multiforme is the most common and most aggressive malignant primary brain tumor in humans. We demonstrated that MT severing...
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Following the Arp2/3-based processes in plant cells
Fišerová, Jindřiška ; Opatrný, Zdeněk (advisor) ; Binarová, Pavla (referee) ; Dráber, Pavel (referee)
1. ABSTRACT An actin cytoskeleton comprises an essential cytoskeletal structure that organizes a cytoplasm during number of processes occurring in animal and plant cells, such as cell division and growth, formation of membrane protrusions and cellular movement or cytoplasmic streaming. A dynamic actin net and an exact spatial and temporal actin filament organization is, therefore, necessary for correct growth processes and development of multicellular organism. Depolymerization and re-establishment of actin arrays is mediated by actin associated proteins. Among them, an Arp2/3 complex (actin related protein 2 and 3) plays an important role as a catalyst of the actin filament nucleation and, thus, enables the formation of dynamic actin network. In non-plant cells, Arp2/3 complex-dependent polymerization of actin filaments is required for movement of whole cells (fibroblasts or keratocytes), it drives organels and pathogens throughout the cell or mediates endocytic processes. The role of the Arp2/3 complex in non-motile plant cells is despite the considerable effort over last three years far from being understood. Unlike animals, where loss-of-function mutations in the Arp2/3 complex are lethal, only limited number of cell types show significant phenotype in Arabidopsis mutants. The evidence exists that the...
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Analysis of Rpg1/eIF3a mutation in Saccharomyces cerevisiae.
Luxová, Pavla ; Malcová, Ivana (advisor) ; Binarová, Pavla (referee)
Rpg1/Tif32/eIF3a is an essential and the largest subunit of translation initiation factor eIF3 in yeast Saccharomyces cerevisiae. Besides interactions within the eIF3 complex it has been shown to interact with microtubules. Preliminary data of the laboratory obtained using strains of the W303 genetic background indicated that there is a synthetic phenotype between rpg1-2 mutant and microtubule inhibitor nocodazole. Aim of this work to elucidate this "microtubule phenotype" of the rpg1-2 mutant and its dependency on used genetic background. I confirmed that independently on genetic background (W303, BY, SEY) all mutants rpg1-1, rpg1-2 and rpg1-3 were temperature- sensitive. I found that in contrast to published data on rpg1 mutants of the W303 background these mutants of the BY and the SEY backgrounds do not arrest the cell cycle in G1 phase during cultivation at the restrictive temperature (37řC, 4 hours). In addition, all three mutants did not show an increased sensitivity to benomyl and none of them affects microtubule rearrangement after a release of cells from the nocodazole treatment. I constructed new strains with a combination of the BUB1 gene deletion with the particular rpg1 mutation. Phenotypic analyses of new double mutants revealed that simultaneous dis-function of Bub1 and Rpg1 results...
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Funkční charakterizace podjednotky Sec5 komplexu exocyst u rostlin.
Sekereš, Juraj ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
Exocyst is a protein complex involved in tethering of secretory vesicles to cytoplasmic membrane before SNARE-mediated fusion event. Its presence and function in secretory pathway has been confirmed in yeasts, animals and plants. This thesis describes some properties of Sec5, one of the exocyst subunits, in plant model Arabidopsis thaliana. Microscopic methods, including VAEM/TIRF microscopy, were used to study subcellular localization and dynamics of Sec5-GFP fusion protein. Sec5 is cytoplasmic protein that also localizes to cytoplasmic membrane, particulary in cells with high secretory activity. It strongly localizes to maturating cell plates during late cytokinesis and its localization to cytoplasmic membrane partially depends on actin cytoskeleton. Generally, obtained results are in agreement with corresponding observations of behavior of other exocyst subunits in plant cells, suggesting, that Sec5 executes its function as part of the exocyst complex.
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Specificity of selected exocyst subunits in trichome development
Glanc, Matouš ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
Trichomes are fine epidermal outgrowths covering aerial organs of most land plants. Although unicellular trichomes of Arabidopsis thaliana have long been used as a model system in plant cell and developmental biology, surprisingly little is known about the processes involved in cell wall biogenesis during the last stage of trichome maturation. A role of EXO70H4, a putative subunit of the vesicle tethering complex exocyst, in trichome maturation has recently been identified in our laboratory. Image analysis, histochemical detection and FT-IR spectroscopy methods were used in this study to analyze cell wall defects of the exo70H4 LOF mutant, revealing the mutation causes altered deposition of pectins and possibly also lignins and hemicelluloses. Transgenic lines with EXO70 paralogues driven by the EXO70H4 promoter were prepared and their analysis revealed that the closest paralogue EXO70H3, unlike EXO70A1 and EXO70B1, can complement the exo70H4 mutation. Based on the results, questions concerning trichome cell wall composition, the role of EXO70H4 in trichome maturation and functions of the plant exocyst complex are discussed. Keywords: Arabidopsis, trichome, cell wall, secretory pathway, exocyst complex, EXO70H4, FT-IR spectroscopy
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Role and regulation of nuclear membrane during meiotic maturation of mammalian oocyte
Končická, Markéta ; Kubelka, Michal (advisor) ; Petr, Jaroslav (referee) ; Binarová, Pavla (referee)
Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...
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Functional studies of selected members of the Arabidopsis formin family
Oulehlová, Denisa ; Cvrčková, Fatima (advisor) ; Binarová, Pavla (referee) ; Černý, Jan (referee)
Formins are multidomain proteins containing a conserved formin-homology 2 (FH2) domain, which catalyzes de novo nucleation of actin filaments. In yeast and animal cells, both mechanisms and regulation of formin function have been extensively studied, yet much less is known about action of plant formins, which considerably differ from yeast and animal ones in the domain composition. In higher plants, formins are classified into two groups, Class I and Class II, and so far, experimental data are available only for the first group members. Here I present results of experimental study of several members of the large formin family in Arabidopsis, including the characterization of a Class II formin AtFH16. Arabidopsis genome contains 21 formin-encoding genes, and though they greatly differ in their expression levels and pattern, all of them are transcriptionally active. We selected 17 homozygous T-DNA insertional mutants in 14 formin genes. Under standard cultivation conditions, no obvious phenotypic discrepancies between wild type and mutant plants were found. To impair two dominant pollen formins, an atfh3atfh5 double-mutant was prepared and even in this case, both microspore development and pollen tube growth remained unaffected. Consistently, polarized growth of tobacco pollen tubes was not altered...
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Role and regulation of nuclear membrane during meiotic maturation of mammalian oocyte
Končická, Markéta ; Kubelka, Michal (advisor) ; Petr, Jaroslav (referee) ; Binarová, Pavla (referee)
Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...
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