|Full text PDF:||https://edoc.ub.uni-muenchen.de/19694/|
Cell adhesion is an essential mechanism involved in many cellular processes, such as migration, proliferation and differentiation. The mechanical linkage between extracellular matrix and the f-actin cytoskeleton is mediated in specialized protein complexes, called focal adhesions. Key components of these cellular compartments are members of the integrin protein family; transmembrane proteins that connect to ligands in the extracellular matrix and recruit intracellular focal adhesion proteins. However, integrins have no catalytic function and cannot bind cytoskeletal components. The association with f-actin is mediated by intracellular adaptor molecules that link integrin tails and the cytoskeleton. These adhesion complexes not only mediate association with the extracellular matrix, but also serve as the mechanosensitive units of the cell. It has been known for some time that mechanical stimuli – as for example tissue rigidity – are epigenetic factors, regulating processes like organ development and stem cell differentiation. However, even though single components of the adhesion complex have been demonstrated to be involved in mechanosensitive processes, central mechanisms in mechanosensing through focal adhesions remained unknown. One of the major components responsible for the integrin-f-actin connection is the focal adhesion protein talin-1. Talin-1 directly binds intracellular integrin tails but also carries three f-actin binding sites and thus directly mediates the connection between extracellular Matrix and the cytoskeleton. Besides ist important role as integrin activator – and thus important promotor of integrin mediated adhesion – talin-1 has long been suspected as a mechanosensitive component in focal adhesions. Still, evidence of a regulatory role of talin-1 in mechanosensing in adhesive cells is still missing due to the lack of appropriate techniques. Using two single-molecule-calibrated FRET (Förster resonance energy transfer) based tension sensors, it could be demonstrated in this work that talin-1 is indeed subject to low-piconewton (pN) forces in integrin mediated adhesion processes. When localized in focal adhesion talin-1 bears forces of 7-10 pN. Regulation of talin-1 forces occurs through association with f-actin, either direct or indirect via binding of vinculin, a talin-1 interactor that strengthens the connection of talin-1 with the actin cytoskeleton. Disturbing the mechanical linkage of integrins to f-actin via talin-1 does not prevent integrin activation, but leads to defects in cell spreading and focal adhesion reinforcement. Furthermore, it could be shown that mechanical resilient linkages through talin-1 in focal adhesions are important for extracellular rigidity sensing.Taken together this work provides strong evidence that talin-1 mediated mechanical linkage between the extracellular matrix and the actin cytoskeleton is essential in mechanosignaling processes. For the first time it could be shown that talin-1 is subject to pN forces in living cells and that the force… Advisors/Committee Members: Fässler, Reinhard (advisor).