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The ERK pathway is an evolutionarily conserved three-tier signaling cascade comprised of protein kinases Raf, MEK, and ERK. These core kinases are arranged in a hierarchical order and the signal is transduced from Raf to MEK to ERK. The ERK pathway is activated by diverse extracellular signals and in response regulates many cellular processes including cell proliferation, differentiation, apoptosis, migration or epithelial plasticity. Given the role of the ERK pathway in regulating such fundamental cellular processes, the ERK pathway signaling is tightly controlled and its dysregulation has pathological consequences such as cancer development and progression. Although much is known about mechanisms underlying the signal transduction by the ERK signaling pathway, much less is known about how two highly homologous ERK1 and ERK2 isoforms contribute to the signaling by this pathway. In this thesis, I studied isoform-specific functions of ERK1 and ERK2 using epithelial Madin- Darby Canine Kidney (MDCK) cells overexpressing either ERK1 or ERK2. Obtained data show that overexpression of ERK2, but not ERK1, had significant effects on the morphology and functional phenotype of MDCK cells. Both ERK1 and ERK2 expressing cells were able to form cohesive clusters, but the only ERK2 overexpression affected...

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...

Colorectal carcinoma is one of the most commonly diagnosed tumor diseases worldwide and is the cause of more than nine percent of deaths due to neoplasia. Colorectal cancer develops through different ways and one of them is the so-called serrated pathway, which is characterized by the presence of the BRAF V600E oncogenic mutation. Tumors arising through serrated pathway do not respond to classical therapy, and therefore are currently being studied at the molecular level. The oncogenic variant of the BRAF kinase activates MAPK signaling and is considered to be the main cause of serrated intestinal tumor formation. However, the mere presence of this oncogene is not sufficient for tumor development that requires further changes within the genome of the cell. In this thesis, we try to clarify what effect the BRAF V600E mutation has on the cells of the intestinal epithelium. In addition, we try to identify a possible cooperation between BRAF gene mutation and disruption of p53 and Wnt signaling, whose components are also frequently mutated in colorectal cancer. As a model for studying the processes associated with BRAF V600E activation, we use a mouse strain with conditional expression of a mutant variant of the Braf gene. We isolate intestinal organoids from these mice and subsequently perform in vitro...