Correlative Traction Force Microscopy and Fluorescence Fluctuation Analysis of Molecular Aggregation and Complex Formation in Cell Adhesions in Distinct Microenvironments

Abstract

The mechanical properties of the cellular microenvironment regulate processes that include migration, proliferation, and differentiation. These interactions occur at adhesions, which serve as both traction points and signaling hubs and mediate bi-directional sensing and responses to specific features of the surrounding extracellular matrix (ECM). Adhesions execute these activities through an intricate network of putative molecular interactions that largely remain to be demonstrated and characterized functionally in living cells. The challenge is to capture the highly localized and transient associations that characterize these activities in adhesions and determine how they respond to different microenvironments. In this study, we use high-resolution fluorescence fluctuation microscopy to map the formation and stoichiometry of integrin-associated complexes in the adhesions that populate the leading edge of migrating cells. We focus on putative integrin activating (kindlin and talin) and actin-linking (talin, vinculin and a-actinin) molecules and show that all molecules are present in adhesions as soon as they are visible; however, they form integrin containing complexes hierarchically, at different times, with variable stoichiometry within the adhesion itself and change as the adhesion matures into larger structures. To parse out the effects of the mechanical properties of the ECM on the numbers, aggregation states, and associations of these molecules, we extend these measurements to substrates with variable stiffness and correlate them with high-resolution traction force microscopy (TFM) measurements. We show that individual and newly formed adhesions at the leading edge of protruding cells transmit forces on soft as well as stiff substrates, with force magnitudes that correlate with the integrated intensity of the adhesions and the total number of individual adhesion molecules. These measurements provide novel information on complex formation as adhesions evolve and respond to substrate rigidity.