A biomimetic system to reconstitute and test the actomyosin-dependent mechanosensitive protein complexes

Abstract

Many biological processes are governed by mechanical cues. For example, during migration, cells sense and respond to the mechanical stress of their environment by remodeling their shape, dynamics and adhesion to the extracellular matrix (ECM). Focal adhesions (FAs) sense the force generated by the actomyosin cytoskeleton and respond by recruiting new actin binding proteins ( ABPs). Although up to date several mechanosensitive path­ways have been identified, the biochemical characterization of the individual mechanosensitive proteins is currently limited by the available techniques. We developed a microscopy assay, with the ABP talin immobilized in 5-μm-diameter discs, regularly spaced by 35 μm and micropatterned on a glass coverslip. The self-organized actomyosin network exerts force on ABP and in response talin reveals cryptic vinculin-binding sites (VBSs). Finally, we observed that the activation of vinculin by talin induces a positive feedback that strengthens the actin-talin-vinculin association. This biomimetic system and the activity of the fluorescent proteins are visualized by total internal refection fluorescence (TIRF) microscopy. This technique can be used to study the ABPs that sense and respond to the mechanical force generated by the actomyosin cytoskeleton in a variety of physiological and pathological processes or for screening pharmacological inhibitors of mechanosensitive protein interactions.