This project continues to build on our-long-term goal of identifying and understanding genes which when altered cause human kidney disease. Mutations in alpha-actinin-4 (ACTN4) cause a form of kidney disease characterized by progressive decline in function, proteinuria, and focal segmental glomerulosclerosis (FSGS). We have made substantial progress during the current award period in understanding the role of mutation-induced alterations in ACTN4-actin affinity on the biophysical properties of the actin network at in vitro and cellular levels. We demonstrated that genetic alterations in ACTN4 have profound effects on cell behavior including contractility, motility, response to stretch, and gene transcription, and ultimately produce glomerular pathology in the organism. We have generated strong evidence that phosphorylation events can reversibly modulate the ACTN4-actin affinity. These findings suggest a model where the global effects of ACTN4 disease-mutations on strain hardening and network brittleness are detrimental, and where ACTN4 phosphorylation allows for similar, but local and spatiotemporally regulated, changes to the actinin-actin network. Our working hypothesis is that a normally hidden actin-biding site (ABS1) is required for strain-dependent network hardening but at the expense of generating a more brittle cytoskeleton. Mutation or phosphorylation exposes this site. We will further define the role of ACTN4 S159 phosphorylation in regulating the cellular actin cytoskeleton, cellular adhesion and contractility, and lastly, in regulating in vivo podocyte function. We will use these studies as a springboard for identifying the signals, kinases and phosphatases regulating ACTN4 phosphorylation. Finally, we will elucidate the importance of ABS1-mediated strain hardening in cells and in vivo. This next set of studies extend and expand upon the aims of the original proposal, and will advance our understanding of not only ACTN4-mutation induced FSGS but also elucidate the importance of spatiotemporal regulation of the actinin-actin network through phosphorylation events. Specifically, we will: 1. Define the cellular role and regulation of ACTN4 by serine 159 phosphorylation; 2. Define the relationship between ACTN4-mediated, biophysical behavior of cells, alterations to the local microenvironment, and its regulation by phosphorylation; 3. Define the role of regulation of the ABD of ACTN4 by phosphorylation and mutation in the function of the kidney in vivo using new CRISPR-derived animal models we have developed; 4. Extend our understanding of the effects of ACTN4 alterations in regulating gene expression.