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Studium úlohy proteinkinázy C alfa v améboidní invazivitě nádorových buněk
Vaškovičová, Katarína ; Brábek, Jan (advisor) ; Pajer, Petr (referee)
1. Abstract Protein kinase C α (PKCα) is a serine/threonine protein kinase regulating many different signaling pathways. The aim of this study was to investigate the potential role of PKCα in amoeboid morphology and invasion of cancer cells. It was observed, that expression of PKCα as well as its phosphorylation on Thr497 remained unchanged upon amoeboid-mesenchymal transition of A375m2 cells (induced by inhibition of ROCK kinase) both in 3D and in 2D environment. However, activation of PKCα by PKC activator treatment resulted in mesenchymal- amoeboid transition of K2 and MDA-MB-231 mesenchymal cell lines, although it did not change overall invasivity ability of cells to invade 3D collagen. Notably, PKCα activation significantly reduced matrix degrading abilities of A375m2 cells. Conversely, inhibition of PKCα by PKCα inhibitor treatment caused amoeboid-mesenchymal transition of amoeboid A375m2 cells and it was associated with decreased invasiveness of all three cell lines used. PKCα inhibitor did not have any effect on gelatin degradation area of A375m2 cells. Consistently, specific siRNA mediated downregulation of PKCα lead to transition from amoeboid to mesenchymal morphology of A375m2 cells and reduced invasiveness of cells into 3D collagen. Moreover, gelatin degrading abilities of A375m2 cells were...
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The role of the exocyst in development and maintaining of cell migration structures
Vaškovičová, Katarína ; Brábek, Jan (advisor) ; Žárský, Viktor (referee)
The exocyst is a hetero-octameric protein complex which mediates tethering secretory vesicles to specific sites of plasma membrane for polarized exocytosis. The exocyst was long known to contribute to processes such as yeast budding, cytokinesis, epithelia polarization and neurite outgrowth. Recently, the role of the exocyst in regulation of actin cytoskeleton and cell migration was discovered. It was shown, that the exocyst is important for formation of cell migration structures such as lamellipodia and filopodia in motile cells and invadopodia in invasive cancer cells. These structures are all actin-based membrane protrusions and the exocyst can through its Exo70 subunit interact with the Arp2/3 complex, the activator of actin nucleation. By binding and activating the Arp2/3 complex, the exocyst mediates actin polymerization resulting in formation of these membrane protrusions. Furthermore, the exocyst probably targets the Arp2/3 complex to specific sites of plasma membrane that are intended to become membrane protrusions. In addition, the exocyst mediates secretion of matrix metalloproteinases (MMPs) in invadopodia. MMPs are important for degradation of the extracellular matrix, an essential process in cancer cell invasion. The exocyst seems to be part of the cascade downstream of cytokines...
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Changes in domain organization of the plasma membrane in the stress response
Vaškovičová, Katarína ; Malínský, Jan (advisor) ; Zimmermannová, Olga (referee) ; Cvačková, Zuzana (referee)
MCC/eisosomes are yeast plasma membrane microdomains that respond to changes in both extracellular and intracellular conditions and activate important stress-related signaling pathways. In this study, we investigated the function of MCC/eisosomes under the conditions of chronic glucose depletion. We found that MCC/eisosomes regulate mRNA decay under these conditions. Specifically, we demonstrated that the sequestration of the evolutionarily conserved Xrn1 exoribonuclease at MCC/eisosomes leads to the attenuation of its enzymatic activity. Modulation of activity by the enzyme localization may represent a novel and effective mechanism in regulation of biochemical pathways. Moreover, our results suggested that an MCC protein Nce102 might play a role in vacuolar fusion and lipid droplets degradation. We demonstrated that prolonged chronic glucose depletion induces the translocation of Nce102 from MCC to sterol-enriched microdomains in the vacuolar membrane. Deletion mutants lacking Nce102 and its functional homologue Fhn1 exhibited significant delay in vacuole maturation and in turnover of a lipid droplet marker Erg6. The function of MCC/eisosomes in the stress response have been demonstrated in many fungal species. Similar to the microdomain function, also individual protein components of...
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Studium úlohy proteinkinázy C alfa v améboidní invazivitě nádorových buněk
Vaškovičová, Katarína ; Brábek, Jan (advisor) ; Pajer, Petr (referee)
1. Abstract Protein kinase C α (PKCα) is a serine/threonine protein kinase regulating many different signaling pathways. The aim of this study was to investigate the potential role of PKCα in amoeboid morphology and invasion of cancer cells. It was observed, that expression of PKCα as well as its phosphorylation on Thr497 remained unchanged upon amoeboid-mesenchymal transition of A375m2 cells (induced by inhibition of ROCK kinase) both in 3D and in 2D environment. However, activation of PKCα by PKC activator treatment resulted in mesenchymal- amoeboid transition of K2 and MDA-MB-231 mesenchymal cell lines, although it did not change overall invasivity ability of cells to invade 3D collagen. Notably, PKCα activation significantly reduced matrix degrading abilities of A375m2 cells. Conversely, inhibition of PKCα by PKCα inhibitor treatment caused amoeboid-mesenchymal transition of amoeboid A375m2 cells and it was associated with decreased invasiveness of all three cell lines used. PKCα inhibitor did not have any effect on gelatin degradation area of A375m2 cells. Consistently, specific siRNA mediated downregulation of PKCα lead to transition from amoeboid to mesenchymal morphology of A375m2 cells and reduced invasiveness of cells into 3D collagen. Moreover, gelatin degrading abilities of A375m2 cells were...
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The role of the exocyst in development and maintaining of cell migration structures
Vaškovičová, Katarína ; Žárský, Viktor (referee) ; Brábek, Jan (advisor)
The exocyst is a hetero-octameric protein complex which mediates tethering secretory vesicles to specific sites of plasma membrane for polarized exocytosis. The exocyst was long known to contribute to processes such as yeast budding, cytokinesis, epithelia polarization and neurite outgrowth. Recently, the role of the exocyst in regulation of actin cytoskeleton and cell migration was discovered. It was shown, that the exocyst is important for formation of cell migration structures such as lamellipodia and filopodia in motile cells and invadopodia in invasive cancer cells. These structures are all actin-based membrane protrusions and the exocyst can through its Exo70 subunit interact with the Arp2/3 complex, the activator of actin nucleation. By binding and activating the Arp2/3 complex, the exocyst mediates actin polymerization resulting in formation of these membrane protrusions. Furthermore, the exocyst probably targets the Arp2/3 complex to specific sites of plasma membrane that are intended to become membrane protrusions. In addition, the exocyst mediates secretion of matrix metalloproteinases (MMPs) in invadopodia. MMPs are important for degradation of the extracellular matrix, an essential process in cancer cell invasion. The exocyst seems to be part of the cascade downstream of cytokines...
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