The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing

Pacemaker implantation can be associated with several complications, including myocardial perforation with or without pericardial effusion, venous thrombosis, vegetations of the tricuspid valve (TV) or pacing lead, and tricuspid regurgitation (TR). The TR is thought to be derived from deformity or perforation of the TV by the pacing lead or secondary to atrioventricular discordance with asynchronous ventricular pacing. Severe TR can be deleterious to the patient because it raises the central venous pressure by increasing the right sided preload. Chronically, the increase in right sided blood volume can result in an increase in the right atrial pressure leading to a decrease in venous return and low cardiac output. Severe TR from leaflet adhesion to the pacemaker lead has not been reported before. With the aging of the population and the expanding use of pacemakers and implantable cardioverter defibrillators (ICD) in clinical practice, this complication may be seen more frequently. We present a patient diagnosed with severe TR, years after his pacemaker implantation. His TR was thought to be caused by adhesion of the tricuspid valve to his pacemaker lead.

The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing

No description available.

Förster Resonance Energy Transfer (FRET)-based techniques are gaining an increasing importance in cell biology and cell-matrix adhesion studies because they allow both the detection of conformational changes of target proteins and their localisation in cells. Frequency Domain-Fluorescence Lifetime Microscopy (FD-FLIM) is currently considered one of the most reliable methods to measure FRET in live cells. However, due to its dependence on many technical prerequisites, its use is not yet widespread. The purpose of this work was to first establish FD-FLIM measurements of FRET on a new FD-FLIM microscope module. Then we aimed to apply FD-FLIM-FRET measurements to the study of conformational changes of the cell matrix-adhesion proteins vinculin and integrin and of the growth factor receptor Tie-2. In the first part of the work, published FRET probes including distance-sensors and two sets of vinculin-based probes were extensively tested with FD-FLIM, sensitised emission and ratiometric FRET. FD-FLIM was shown to be the most accurate method in approximating molecular distances between fluorophores. Moreover this study unveiled specific caveats associated with both existing vinculin FRET probes. FD-FLIM was then used to study conformational changes of the extracellular matrix receptor alphavβ3 integrin and of the angiopoietin receptor Tie-2 using specific FRET probes designed by us. While data showed that the alphav-integrin-FRET probe localised to adhesion sites, more experiments will be required to evaluate its full functionality. The Tie-2-FRET probe was fully functional and, upon ligand binding, allowed the detection of a bending movement of the extracellular domain towards the cell membrane. Finally, a combination of FRET, immunofluorescence and tension release experiments were used to show that intracellular tension is not required to maintain integrins in their activated conformation. However, intracellular tension is required to recruit other key proteins such as vinculin, talin and tensin to adhesions sites. Overall this work demonstrates the importance of FD-FLIM-FRET as a tool to investigate conformational changes of adhesion proteins and transmembrane receptors within the cell environment.

No description available.

Focal adhesions are important subcellular structures that are composed of many signaling and scaffolding proteins. They serve not only for anchoring the cell to the substratum but they are also important signaling centers that regulate various cellular behavior such as migration, invasiveness, proliferation and survival. Focal adhesion signaling needs to be strictly regulated because alteration in activity or expression of many focal adhesion proteins leads to tumorogenesis and metastasis formation. One of the most important scaffolding protein associated with focal adhesion is p130Cas. The importance of p130Cas in regulation of cell migration and invasiveness has been well established. P130Cas also plays important role in regulation of cell survival and proliferation. Moreover, high protein levels of human ortholog of p130Cas - BCAR1, has been linked to more aggressive breast tumors and poor prognosis. During my doctoral studies, I focused on the role of p130Cas in integrin signaling. At the beginning we characterized the role of tyrosine 12 phosphorylation within its SH3 domain. We confirmed that this phosphorylation is increased in Src527F transformed mouse embryonic fibroblasts compared to non-transformed counterparts and also in some human cancer cell lines. We showed that this phosphorylation...

Adherens junctions are essential for embryogenesis and tissue homeostasis. The major transmembrane adhesion receptors in adherens junctions are the cadherins, which mediate cell-cell adhesion by binding to cadherins on adjacent cells. Cadherin function is regulated by the protein complexes that assemble at its cytoplasmic tail. Vinculin is one cytoplasmic component of the cadherin adhesion complex, but unlike other junction components, it also is enriched in cell-matrix adhesions. The presence of vinculin in cellmatrix adhesions has commanded the most attention, while little is known about its role in cell-cell adhesions. To define the role of vinculin in adherens junctions, I established a short hairpin RNA-based knockdown/substitution system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cell-cell adhesion was reduced owing to a lack of cadherin on the cell surface. I investigated the mechanism for this effect and found that vinculin must bind to beta-catenin to regulate E-cadherin surface expression. Having established a role for vinculin in cell-cell adhesions, the critical question became how vinculin recruitment to and activation at cell-cell junctions are regulated. I found that á-catenin triggers activating vinculin conformational changes. Unlike all of the known vinculin activators in cell-matrix adhesions, alpha-catenin binds and activates vinculin independently of an A50I substitution. Thus, adherens junction activators and cell-matrix activators bind to distinct regions of vinculin to activate this molecule. Using mutant vinculins that cannot be tyrosine phosphorylated, I found that vinculin recruitment to cell-cell adhesions, but not cell-matrix adhesions, requires phosphorylation at Y822. Furthermore, this residue is phosphorylated by Abl tyrosine kinases during the assembly of cell-cell adhesions. Taken together, these studies explain how vinculin is differentially recruited to adherens junctions and cell-matrix adhesions and describes the first known role for vinculin at cell-cell adhesions.

A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.

A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.