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Intermediate filaments in mammalian cell motility
Čermáková, Kateřina ; Libusová, Lenka (advisor) ; Pelantová, Markéta (referee)
Cell migration is crucial for the formation and maintenance of a multicellular mammalian organism, contributing to important processes such as embryonic development, tissue renewal, and immune surveillance. It is a complex phenomenon involving a plethora of processes, including relevant signalization. An impairment of those processes could be projected into innumerable pathological states, from wound healing disruption to tumour metastasis and invasiveness, thus the interest of many researchers has turned toward migration. The migration of mammalian cells is dependent on a cytoskeleton, which is being considerably rearranged in motile cells. This thesis aims to summarise the role of intermediated filaments in cell motility - the less understood cytoskeletal network in this context. Cytoplasmic, as well as nuclear intermediate filaments, due to their unique mechanical properties, affect cell mechanics. They protect against physical stresses (as cells squeeze through confined pores in a complex intercellular microenvironment) and modulate and direct actomyosin-generated forces, which are the main driving force of migration. In addition, they contribute to important migration- involved steps, such as cell polarisation, cell adhesion to surrounding surfaces, cohesion in collective migration, and...
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Analysis of Src dynamics in cellular structures
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Rozbeský, Daniel (referee)
Src kinase is a key element in many signaling pathways affecting cellular processes such as differentiation, proliferation, motility, or migration. Deregulation of its activity is associated with the promotion of cancer. Therefore, understanding its cellular function is vital. Src activity directly correlates with its structure; when Src is active, it adopts opened conformation, when inactive, it is in closed conformation stabilized by intramolecular interactions. Detection of the conformation can be used to analyze Src activity. In this thesis, conformation-sensitive FRET-based Src biosensor was improved using mNeonGreen as a new acceptor fluorophore in the existing design and the properties of the new biosensor were compared with the original Src biosensor. The new biosensor is able to detect changes in Src conformation and can be stably expressed in cells. Src activity in focal adhesion was analyzed and higher Src activity in these structures was confirmed. Although the new biosensor did not exhibit significantly better sensitivity to Src conformational changes, it still proved to be a useful tool to study Src activity, and mNeonGreens higher brightness makes it more suitable for microscopic experiments. Key words: Src, FRET, biosensor, live-cell imaging, mNeonGreen
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Genetically encoded biosensors of cellular tension and their use in cellular biology
Pelantová, Markéta ; Rösel, Daniel (advisor) ; Lánský, Zdeněk (referee)
1 Abstract and key words Mechanical forces have great impact on the life of cells. They influence cell proliferation, migration or differentiation and defects in cellular mechanosensing were reported to be the cause of various diseases, such as deafness, atherosclerosis or cancer. However, mechanisms of mechanical sensing are not thoroughly examined and not many tools for doing such research are available. Genetically encoded FRET-based biosensors are one of the existing methods for studying transfer of mechanical signal in cells. It is a non-invasive method allowing to observe changes in mechanical tension across proteins in living cells. In this thesis, different types of existing genetically encoded FRET-based tension biosensors are introduced together with the process of their development and knowledge gained by their use in research. Key words: mechanical force, mechanosensing, FRET, tension sensor, biosensor development
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