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Analysis of functional interaction between PKN3 kinase and CARMIL1 protein
Novotná, Petra ; Rösel, Daniel (advisor) ; Groušl, Tomáš (referee)
Cancer cell motility and cytoskeletal rearrangements are crucial for metastasis formation. These complex changes involve multiple cellular processes affected by many different proteins. One such protein is the Ser/Thr kinase PKN3. This kinase has been shown to be essential for metastasis formation in some aggressive types of breast and prostate cancer. Interestingly, the PKN3 kinase is not only important in malignant cancers but also in normal tissues. In endothelial cells, the PKN3 kinase can alter their adhesion, or in osteoclasts it helps to promote bone resorption. The effects of the PKN3 kinase on cancer malignancy and cell motility are well documented, but the mechanism behind these effects is still unclear. Therefore, our laboratory seeks to identify novel substrates and interaction partners of the PKN3 kinase. This work focuses on a novel potential substrate of the PKN3 kinase, CARMIL1. This protein is involved in actin cytoskeleton rearrangements by regulating actin polymerisation and thus cell motility. Here we provide evidence that the PKN3 kinase interacts with CARMIL1. Key words: PKN3, CARMIL1, actin cytoskeleton, cancer, invasion
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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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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 role of NG2 glycoprotein in regulation of Rho/ROCK signaling
Kratochvílová, Magdalena ; Rösel, Daniel (advisor) ; Kuželová, Kateřina (referee)
NG2 is a transmembrane glycoprotein, which takes part in cellular processes such as adhesion, migration or invasivity, i.e., in processes important in tissue development but also in tumor and metastasis formation. Among other things, NG2 leads to an inhibition of neurite growth, and probably plays an important role in amoeboid type of cell invasion. These processes are in many respects similar. Both in inhibition of neurite growth and in mesenchymal-amoeboid transition occur morphological changes which lead to a loss of cell protrusions and a transition to a rounded shape. In both of these processes Rho/ROCK signaling also plays a crucial role. Connection between NG2 and the Rho/ROCK signaling pathway has been indicated in the process of inhibition of neurite growth. The mechanism of Rho/ROCK signaling regulation by NG2 glycoprotein is, however, still unknown. In this thesis is proposed a molecular mechanism of Rho/ROCK pathway activation by glycoprotein NG2 which relies on the NG2/MUPP1/Syx signaling complex where the scaffold protein MUPP1, bound to activated NG2, enables binding and activation of the Syx protein. Syx then as RhoGEF activates Rho/ROCK signaling, and the activated Rho/ROCK pathway leads to inhibition of neurite growth, increased cell contractility and traction forces. These processes are...
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Effect of polyploidization on species invasive success
Líblová, Zuzana ; Münzbergová, Zuzana (advisor) ; Rooks, Frederick (referee)
Polyploid variants of many species of plants are strikingly frequently found among alien plants on all continents. They also very often have a much larger distribution range of its occurrence, compared to diploid plants in the place of their origin. In many cases, the polyploid cytotype also has increased tolerance to various stress factors or a physiological and morphological characteristics that allow them to survive the conditions in which the diploid plants would have little chance to survive. All this suggests that polyploidy is likely to bring plants an evolutionary advantage over their diploid ancestors, and polyploids therefore can successfully colonize new territories. This thesis summarizes the findings about the possible consequences of polyploidy at different levels in relation to their effects on the properties supporting plant invasive ability. It presents also known hypotheses dealing with possibilities of why plants become invasive after introduction. This is followed by sections devoted to flow cytometry, an important modern method for determining genome size and ploidy level. In conclusion it briefly describes the model species bird vetch (Vicia cracca) and the results of measurements of the degree of ploidy of seeds of this plant from Alaska and Japan.
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The role of cell polarity signaling in the plasticity of cancer cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Cvrčková, Fatima (referee)
Throughout the last few years cancer research has focused on studying the origin of secondary tumors, i.e. metastases, which are a direct outcome of the ability of cancer cells to disseminate from the primary tumor and invade the adjacent tissue. Generally, cancer cells migrate by two distinct mechanisms- amoeboid or mesenchymal. Whereas the mesenchymal migration mode can be described as "path generating", the amoeboid mode resembles a "path finding" way of migration. Both types of invasion are regulated by divergent signaling pathways that are closely related to cell polarity and cytoskeleton reorganization. Responsible for cell polarization are not only the polarity complexes Par, Scribble and Crumbs, but also phosphoinositides and Rho GTPases Rac, Rho and Cdc42, which, additionally, regulate the dynamics of the cytoskeleton. By a mutual interplay they regulate cell motility. It cannot come as a surprise that their deregulation commonly results in tumorigenesis. A more thorough comprehension of the signaling pathways leading to cancer cell invasiveness is a necessary step towards understanding the complex problem of metastasis. Key words: invasiveness, amoeboid, mesenchymal, cell polarity, motility, Rho GTPases, polarity complexes
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The biological importance of CAS SH3 domain tyrosine phosphorylation
Janoštiak, Radoslav ; Brábek, Jan (advisor) ; Dvořák, Michal (referee)
Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...
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The use of CAM assay for characterization and study of cancer cell invasive properties
Vágnerová, Lenka ; Dvořák, Michal (advisor) ; Geryk, Josef (referee)
The chorioallantoic membrane (CAM) of chicken embryos belongs to the in vivo model systems frequently used for the study of angiogenesis and cell invasiveness. Using CAM assay we have tested selected chicken sarcoma cell lines characterized by different angiogenic properties and different ability to form metastasis. In addition to CAM assay, several other methods have been used to characterize the phenotype of these cell lines. We have selected a few proteins which could significantly influence the angiogenic and metastatic properties of investigated cell lines. We have established cell lines stably overexpressing these genes and compared their phenotypes with parental cell lines. We have shown that genes encoding ISL1, ARNT2, PROM1, HOXA11 proteins participate, in our experimental model, in activation of programes controlling angiogenesis and cell invasion.
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Structural and regulatory aspects of Src kinase activation
Koudelková, Lenka ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee) ; Hejnar, Jiří (referee)
Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...
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The role of mitochondrial respiratory chain in invasiveness and metastasis of cancer cells and possible therapeutic interventions
Legátová, Anna ; Brábek, Jan (advisor) ; Truksa, Jaroslav (referee)
The mitochondrial respiratory chain, also called the electron transport chain (ETC), has a pivotal role in key features of cancer cells e.g., proliferation, the metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, or the ability to form metastases. This review summarizes current knowledge about ETC and its relationship to cancer, especially to invasiveness and metastases formation. Firstly, it deals with a process called the Warburg effect and with metabolic complexity in the tumor microenvironment. Then it shows how OXPHOS activity affects invasiveness of cancer cells and metastases formation, and it points out the connection between invasiveness and increased levels of ETC-generated reactive oxygen species. At the end, the review deals with possible use of ETC inhibitors in anticancer therapy.
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