Project summary Cells exist in a physical world, and there is often a physical basis for human function and disease. Mechanotransduction is the molecular process by which cells sense and respond to mechanical signals in their environment. Abnormal mechanotransduction can contribute to many human diseases including asthma, heart failure, osteoporosis, and cancer. Thus, it is crucial to understand the molecular basis of mechanotransduction and how these signaling pathways are disrupted during disease. Integrin receptors are critical regulators of mechanotransduction at the plasma membrane that signal through the assembly supramolecular complexes termed “focal adhesions.” Focal adhesions physically connect the actin cytoskeleton to the extracellular environment, and forces generated in the actin cytoskeleton are transmitted across focal adhesions to drive tissue morphogenesis, cell movement, and extracellular matrix remodeling. Although the proper regulation of focal adhesions is essential for integrin-dependent mechanotransduction, important questions about their formation and function remain unanswered. We do not understand how focal adhesions form, how they grow, or how their molecular composition is regulated. Cell-based experiments have led to conflicting observations, and we have limited tools to understand how changing molecular composition can create focal adhesions with specific chemical or physical characteristics that alter downstream signaling. To address these important questions, Dr. Case has developed a novel biochemical reconstitution of focal adhesions using purified proteins on supported lipid bilayers. This work identified seven proteins that are sufficient to form focal adhesions through liquid-liquid phase separation. Studying integrin-dependent mechanotransduction through the lens of phase separation could drive significant advances in the field. The Case Lab will use a variety of experimental strategies to understand different aspects of integrin-dependent mechanotransduction. They will directly test different models of mechanotransduction with biochemical reconstitution and confirm the importance of any new in vitro observations with cell-based assays. They will investigate how focal adhesions mature, how forces are transmitted across focal adhesions, and how the biochemical composition of focal adhesions is regulated. This project will take advantage of a novel experimental approach to challenge the current dogma about integrin-dependent mechanotransduction, and will reveal how specific molecules regulate focal adhesion growth and composition.