Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus

Abstract

The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage processthat evolves at several temporal scales. An understanding of this complex process requires a precisemeasurement of forces and its correlation with protein responses in living cells. We present a methodto quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approachcombines atomic force microscopy with fluorescence imaging. Using this approach, we evaluatedthe recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxintriggered by applying forces in the nN regime to live cells. We observed in real time the developmentof nascent adhesion sites, evident from the accumulation of early adhesion proteins at the positionwhere the force was applied. We show that the method can be used to quantify the recruitmentcharacteristic times for adhesion proteins in the formation of focal complexes. We also found aspatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force.Our approach allows the study of a variety of complex biological processes involved in cellularmechanotransduction