PROJECT SUMMARY For tissues to maintain a physical steady-state equilibrium with its dynamic surroundings (“mechanical homeostasis”), individual cells must be able to perceive mechanical cues in their local environment and respond accordingly. Mechanical homeostasis plays an essential role in morphogenesis, and its dysregulation can lead to disease states such as hypertension, fibrosis, and asthma. While there has been significant progress in understanding the physiological significance of mechanical homeostasis and cellular mechanosensation, the molecular mechanisms by which proteins convert mechanical stimuli into biochemical signals (“mechanotransduction”) are poorly understood, impeding the development of targeted therapeutics for dysregulated mechanotransduction and its disease states. The actin cytoskeleton plays a prominent role in mechanotransduction, notably actin-myosin cables known as stress fibers (SFs) which both actively generate contractile forces and transmit extracellular forces impinging on cell-cell and cell-matrix adhesions into the cytoplasm. Dynamic regulation of SF assembly, disassembly, and contractility are important for many physiological processes involving cellular mechanics and dynamic cell shape changes, such as epithelial tissue homeostasis and morphogenesis. Stochastic mechanical imbalance in SFs can result in mechanically-induced ruptures, termed stress fiber strain site (SFSS). While some SFSS proceed towards catastrophic breakage, the majority are repaired by zyxin, a mechanosensitive LIM (LIN- 11, Isl-1, & Mec-3) protein. Zyxin first localizes to strain sites through its three C-terminal tandem LIM domains, then recruits the cross-linking protein ɑ-actinin and polymerization factor VASP through its N-terminal domains to mediate SF repair in a matter of minutes. While there is evidence for this sequence of events at the cellular level, the biophysical mechanism of zyxin-mediated SF repair is not well understood. Furthermore, the architectural features of a SFSS which are recognized by zyxin’s LIM domains are unknown. Here I propose to determine the molecular and structural mechanism of zyxin-mediated SF repair. Through biophysical reconstitution and cellular assays, I will test the hypothesis that zyxin, α-actinin, and VASP directly co-assemble to repair mechanically damaged actin filaments and determine the biophysical mechanism of zyxin-mediated mechanical homeostasis (Aim 1). I will then apply cutting-edge correlative cryo-light electron microscopy to test the hypothesis that zyxin binds to a force-dependent actin conformation we have observed in vitro (Aim 2). In addition to providing specific insights into mechanical homeostasis of SFs, these studies are also likely to reveal general mechanisms of mechanotransduction through the cytoskeleton. In the longer term, this work will guide the development of therapeutics against dysregulated mechanotransduction pathways.