Project Summary Mechanical interactions play a fundamental role in physiology, allowing cells to move, generate forces, and assemble into multicellular structures. Key to these processes is the ability of cells to turn mechanical signals into biochemical signals, an activity known as mechanotransduction. The search for mechanosensitive proteins that could facilitate mechanotransduction has primarily focused on proteins that undergo conformational changes in response to force or proteins that display changes in the bond kinetics under load. There exists another class of proteins, however, that recognize structures under strain. The canonical example of this class of proteins is the LIM domain protein zyxin, which recognizes strained actin stress fibers and recruits actin polymerization factors to repair them. Recent work has highlighted that the strain sensing mechanism of the LIM domains is not unique to zyxin and that numerous other members of the family of LIM domain proteins display a similar ability. This suggests LIM domain proteins could act as mechanotransducers, recognizing strain via their LIM domains and converting it to other biochemical signals via interactions with the other domains in the protein. To explore this hypothesis further it is crucial that we understand how LIM domains recognize strained actin filaments, and how those interactions propagate signals downstream of the strain sites. Here we propose to establish rigorous experimental strategies to decipher the mechanisms underlying LIM domain protein mechanotransduction. We employ a combination of biophysical techniques including laser ablations, optogenetics and cell stretching to quantitatively and repeatedly induce strain sites in the actin cytoskeleton. In Aim 1 we will test alternative mechanisms of LIM domain strain sensing by comparing proteins from the testin family of LIM domain proteins which require only a single LIM domain to recognize strain sites, compared to the three tandem LIM domains that zyxin requires. In Aim 2 we will test whether in addition to stretched actin filaments in stress fibers, LIM domain proteins can recognize other strained actin structures, such as compressed stress fibers or actin meshworks. Finally, in Aim 3 we will investigate how binding of LIM domain proteins to strain sites leads to a propagation of that mechanical signal to other parts of the stress fiber and the extracellular matrix. Together these studies will greatly expand our knowledge of mechanotransduction and provide insight into this fundamental signaling mechanism.