Integrin-mediated cell adhesion to extracellular matrices regulates the organization, maintenance and repair of numerous tissues, and abnormalities in adhesive interactions are often associated with pathological states. The adhesive process comprises integrin receptor binding to their extracellular ligand, integrin clustering, and assembly of discrete supramolecular structures containing cytoskeletal and signaling molecules. These focal adhesion complexes function as structural links and signal transduction elements between the cell and its extracellular environment. While significant progress has been attained in deciphering biochemical pathways regulating adhesion, the mechanical interactions between a cell and its environment remain poorly understood. The objective of this renewal application is to analyze the role of nanoscale organization and structure of focal adhesions in the generation of cell-extracellular matrix forces. Our central hypothesis is that the precise nanoscale organization of integrins and their interaction with specific cytoskeletal elements regulate adhesive interactions by controlling the distribution of mechanical forces within the adhesive interface. The rationale for this project is that it will provide new insights into how the structure of focal adhesion complexes regulates adhesive processes, such as adhesion strength and migration. Aim 1: Analyze the effects of nanoscale focal adhesion structure (cluster size, spacing) on cell adhesive force and migration. Aim 2: Elucidate the contributions of integrin-cytoskeleton interactions at the adhesion complex to adhesion strength and migration. The proposed research is innovative because it integrates robust quantitative assays, nanopatterning approaches, and unique cell biology reagents to precisely manipulate focal complex architecture and biomolecular interactions in order to analyze how these adhesive complexes generate adhesive forces. We will elucidate the contributions of nanoscale clustering to adhesive force generation. These rigorous analyses will yield new, sensitive measurements of the contributions of biomolecular interactions between focal complex components to adhesive force. Collectively, this research will generate a new understanding of the regulation of mechanical interactions between a cell and its extracellular matrix. PUBLIC HEALTH RELEVANCE The generation of adhesive forces is essential to cellular functions in physiological processes and pathological conditions. The objective of this research is to derive a fundamental understanding of the molecular events at the adhesive interface that regulate cell adhesive forces. This work will provide new insights in the molecular mechanisms controlling adhesive processes.