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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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Role of GIT-PIX signaling cassette in cytoskeleton regulation
Beránek, Karel ; Dráber, Pavel (advisor) ; Libusová, Lenka (referee)
4 Abstract The cytoskeleton is a cellular structure comprised of three types of protein filaments called microfilaments, intermediate filaments and microtubules respectively. These filaments are highly dynamic and can change their organisation and properties according to the current needs of a cell. This system of cytoskeletal polymers is regulated by a plethora of interacting proteins, among which belong the GIT proteins. GIT proteins contain a great amount of interaction domains, and they form oligomers which function as a scaffold for the binding of associated proteins. In this way they facilitate specific subcellular localisation of many proteins and their complexes. GIT proteins contain a catalytic ARF-GAP domain that regulates the function of ARF GTPases. GIT proteins interact with PIX proteins. These proteins function as GEFs for Rho GTPases and thus regulate signal transduction influencing the cytoskeleton. GIT and PIX interact with each other by the means of their coiled-coil domains and form complexes called "GIT-PIX signalling cassettes". The cassettes regulates mostly microfilaments during the establishment of cell polarity, formation of membrane protrusions, and in cell motility and adhesion. Results of experiments published in the last few years suggest that the GIT-PIX signalling cassette...
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The effect of the mast cell activation on the microtubule organisation
Hájková, Zuzana ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee)
The activation of bone marrow-derived mast cells (BMMCs) induces a number of cell processes such as degranulation, proliferation and cytoskeleton rearrangements. Although microtubules are important in these processes, molecular mechanisms that control changes in microtubule organisation during cell activation are unknown. Activation of BMMCs can be achieved in several ways. Under physiological conditions, the aggregation of IgE receptors (FcRI) on the surface of BMMCs leads to the initiation of specific signaling pathways. Cells can be also activated nonspecifically by a tyrosine phosphatase inhibitor pervanadate, or by thapsigargin that inhibits Ca2+ ATPase pumps located on the endoplasmic reticulum. In this diploma thesis it was found out that rapid morphological changes can be monitored when BMMC are immobilised on the fibronectin before their activation. It was proved that specific and nonspecific activation events lead to microtubule reorganization, as well as to generation of a large number of microtubule-dependent protrusions. In the course of FcRI aggregation, generation of microtubule protrusions depends on the activity of Src family protein tyrosine kinases and on the intracellular Ca2+ concentration. STIM1, an endoplasmic reticulum Ca2+ sensor, which participates in the activation of...
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The role of evolutionarily conserved proteins BIR-1/Survivin and SKP-1 in the regulation of gene expression
Kostrouch, David ; Kostrouch, Zdeněk (advisor) ; Dráber, Pavel (referee) ; Pacák, Karel (referee)
SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...
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Dynamic and role of cytosekeletal proteins in mammalian sperm cells
Paleček, J. ; Dvořáková, K. ; Pěknicová, Jana ; Kubátová, Alena ; Dráber, Pavel
The cytoskeleton of sperm head consists of resistant structural proteins of the nucleus and of perinuclear theca, which are largely rosponsible for the shaping of the nucleus. three mahjor cyteskeletal proteins, actin, tzubnulin and spectrin, are present in the head of mammalian spermatohzoa. Maoreober, the mictotubular cytoskeleton also contains other dynamic proteins, which have a morphological function within the cell and which are responsible for cell signalling processes.
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