Project Summary/Abstract α-Actinin-4 (ACTN4) is a force-sensitive actin-binding protein that interacts with F-actin via a catch bond, which is defined as a protein-protein interaction that strengthens under tensile force. The K255E ACTN4 variant, which causes focal segmental glomerulosclerosis (FSGS), exhibits ultrahigh F-actin binding affinity and is rendered force-insensitive because it cannot catch bond with F-actin. Although actin-binding proteins have well- established mechanosensing functions, force-dependent conformational changes in actin-binding proteins have never been visualized. Our central hypothesis is that K255E ACTN4 binds F-actin in a constitutively strong state that structurally resembles the force-stabilized state of wild-type ACTN4. The long-term goal of this research is to elucidate structural mechanisms underlying deficient actin mechanosensing in hereditary podocytopathies. This project’s immediate objective is to determine the structural basis for actin mechanosensing by ACTN4 via in vitro force reconstitution assays, total internal reflection fluorescence microscopy, and cryo-electron microscopy (cryo-EM). In Specific Aim 1, a structural mechanism for deficient actin mechanosensing in FSGS caused by ACTN4 mutations will be revealed via a cryo-EM structure of the force-insensitive mutant K255E ACTN4 bound to F-actin. In Specific Aim 2, a structural mechanism for force-stabilized actin-binding by ACTN4 will be revealed via cryo-EM structures of wild-type ACTN4 bound to F-actin with and without mechanical force. Overall, this project will: (1) advance our insights into the structural basis for actin mechanosensing by ACTN4 and lack thereof in familial FSGS, (2) provide the first structure of a catch bonding protein under force, and (3) pioneer methods for introducing physiological mechanical forces in a manner amenable to cryo-EM. Dr. Gregory M. Alushin, PhD, an expert on cryo-EM studies of actin mechanobiology who practices active mentorship, and Dr. A. James Hudspeth, MD, PhD, an expert on molecular motors and mechanotransduction who has a proven track record of training successful physician-scientists, are co-sponsoring this proposal. The research will be conducted at The Rockefeller University within the deeply supportive Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. This fellowship constitutes an important career milestone for dual-degree students seeking to become independent investigators.