{Reference Type}: Journal Article
{Title}: Detection of fluorescent protein mechanical switching in cellulo.
{Author}: Shoyer TC;Collins KL;Ham TR;Blanchard AT;Malavade JN;Johns BA;West JL;Hoffman BD;
{Journal}: Cell Rep Methods
{Volume}: 4
{Issue}: 7
{Year}: 2024 Jul 15
暂无{DOI}: 10.1016/j.crmeth.2024.100815
{Abstract}: 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.