Understanding how cells sense and respond to the mechanical properties of their tissue microenvironment (mechanosense) would enhance our ability to control stem cell differentiation, enabling advances in regenerative medicine, and to develop drugs to disrupt tumor metastasis, the cause of the vast majority of cancer deaths. Despite the importance of extracellular matrix stiffness in controlling cell fate and behavior, the molecular mechanisms underlying mechanosensing remain unknown. Cells interact with the extracellular matrix (ECM) through focal adhesions and associated stress fibers, actin-rich structures which enable a cell to pull on its surroundings via myosin II motor protein mediated contractility. Focal adhesions contain more than 150 different proteins, forming a highly complex interaction network whose composition changes dramatically in response to contractile forces which are modulated by the stiffness of a cell's environment. The proposed studies will dissect a primary mechanism underlying this compositional remodeling: how dozens of proteins containing motifs known as LIM domains are recruited to focal adhesions and stress fibers when they are under tension. Although the role of most LIM proteins in focal adhesions and stress fibers has not been well characterized, substantial cytological evidence suggests that one family member, the protein zyxin, accumulates at sites where the actin cytoskeleton is deformed or damaged in a LIM domain-dependent manner. I therefore hypothesize that myosin motor activity, which is known to distort actin cytoskeletons in vitro, generates binding sites for LIM domains in cells when contractility is high on stiff ECMs. This proposal builds upon an innovative application of electron tomography, which I previously adapted to quantify the distributions of single molecules in reconstructed three-dimensional maps of heterogeneous networks of interacting proteins. I will employ this technology to characterize the distortions which myosin motor activity generates in reconstituted cytoskeletal networks. This will reveal for the first time the structural consequences of a physiological force generator on the actin cytoskeleton in molecular detail. I will then uncover which of these distortions act as binding sites for LIM domains using zyxin as a model, and how the interaction is formed. Finally, I will stringently establish which LIM proteins are mechanosensitively recruited to focal adhesions and stress fibers in cells, and how they also recognize deformed cytoskeletal structures. Developing pharmacological inhibitors of force-dependent LIM protein accumulation, a key aspect of a cell's mechanosensitive response to its microenvironment, will be accelerated by identifying and establishing the structural details of the interaction between force-sensitive LIM domains and their receptors on the actin cytoskeleton. My studies will also provide insight into how the prevalence of modular LIM domains in the focal adhesion and stress fiber protein-protein interaction network enables a large-scale mechanosensitive response by the alteration of a single class of interactions.