Mechanosensitive protein-protein interactions in nascent focal adhesions determined by three-colour FRET sensing using multiphoton fluorescence lifetime imaging microscopy (Conference Presentation)

Abstract

A tension-sensitive biosensor was used to detect changes in applied force across the mechanosensitive focal adhesion protein vinculin. When the biosensor is under tension, two fluorescent proteins separate, decreasing the amount of Förster Resonance Energy Transfer (FRET) observed. By measuring FRET using time-correlated single-photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) we observe the loss of FRET, as a direct consequence of an applied intracellular force across the biosensor. Mouse embryonic fibroblasts (MEFs) transfected with a vinculin construct encoding the Tension Sensing Module (TSM), demonstrate how force-transduction changes within maturing adhesions in both fixed and live cells.