VALIDATION OF A GENETICALLY ENCODED SINGLE-FLUOROPHORE RATIOMETRIC SENSOR TO MONITOR RHO-ASSOCIATED KINASE (ROCK) ACTIVITY IN CELLS

Mechanical cues are important regulators of cell and tissue behavior. Among these, extracellular matrix (ECM) stiffness is a universal signal that controls both physiological and pathological functions. Cells measure ECM stiffness by actively developing contractile forces through the actin cytoskeleton and integrin-mediated adhesions. Rho-associated kinases (ROCKs) play a major role in mediating the remodeling of the contractile actomyosin cytoskeleton downstream of forces and of the Rho small GTPase. Forces are then translated into biochemical signals to enable a coherent biological response, including the rapid reinforcement of the actomyosin cytoskeleton and focal adhesions, and the regulation of gene expression. Measuring force mechano-sensing and the status of actomyosin contractility in cells is complicated because it is based on complex or poorly quantitative measurements. Activation of ROCKs is not only a prerequisite for cells to develop contractility but is also considered a force-responsive process. Thus, visualizing ROCKs activity changes in response to different mechanical stimuli might provide us with a new tool to quantify force mechano-sensing in a more direct manner. For this reason, we develop a genetically encoded tool for interrogating the spatiotemporal regulation of this signaling pathway in response to different mechanical stresses. The sensor is a single-fluorophore excitation-ratiometric reporter that allows monitoring the activity of ROCKs in live cells.