Detection of fluorescent protein mechanical switching in cellulo

The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, determining the mechanisms by which forces affect protein function inside cells remains challenging. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated whether force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo within a synthetic actin crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cell force generation, external stiffness, and force-sensitive bond dynamics of the biosensor. This work describes a framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells. [Display omitted] •An approach to probe the effect of cellular forces on fluorescent protein (FP) function•FP function is sensitive to load magnitude and dynamics (e.g., duration)•FP function in biosensors is affected by bond dynamics and substrate stiffness•Force-induced changes in FP function can be used to indirectly probe mechanosensing Cells sense mechanical cues via force-induced alterations in protein structure and function, but elucidation of the molecular mechanisms is hindered by the lack of approaches to probe the effect of forces on protein structure and function inside cells. Motivated by in vitro observations of reversible fluorescent protein mechanical switching, we developed an approach for detecting fluorescent protein mechanical switching in cellulo. This enables the visualization of force-sensitive protein function inside living cells. Shoyer et al. develop an approach to probe how mechanical forces affect the function of fluorescent proteins inside living cells and leverage it to study mechanosensitive molecular processes. Fluorescent protein mechanical switching is manipulated by perturbations to force-activate bonds and substrate stiffness, consistent with its sensitivity to protein loading dynamics.