Molecular Counting in Traction Force Microscopy

Abstract

The mechanical respons of the micro environment of a cell is of great influence for its differentiation and mobility. However, the mechanism that a cell uses to sense these mechanical properties remains largely unkown. Mechanosensing could happen by direct coupling to the nucleus via the actin cytoskeleton or by activating molecular signaling cascades through protein complexes.A recent combination of traction force microscopy with super resolution imaging allowed for novel research into the nanoscale architecture of force-bearing focal adhesions. Here we apply this technique to investigate the relation between local force exertion by the cell and the number of signal transduction proteins inside the integrin adhesome.Integrin adhesomes are mechanosensory protein complexes that couple the intracelular force bearing actomyosin structures to the extracelular environment. These complexes contain signal transduction proteins like paxillin, vinculin, talin and FAK, that change conformation when force is exerted on the complex.By immunostaining these proteins with Alexa647 their position can be localized to 20nm in fixed cells. However, due to the photophysical properties of Alexa647, and the binding stoichiometry in antibody staining the number of localizations does not scale linearly with the number of signal transduction proteins. To overcome this problem we present a novel analysis method to relate the number of localizations to the minimal number of signal transduction proteins.