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.
Mutations in the human alpha-actinin-4 gene, ACTN4, cause kidney disease in humans. We are working to understand how the protein encoded by this gene works in the kidney, how alterations in its function cause kidney disease, and how its function is regulated. Better understanding how defects in this gene cause human disease will have significant and direct implications for understanding and ultimately treating both rare and common forms of kidney failure and kidney failure progression.
| Feng, Di; Notbohm, Jacob; Benjamin, Ava et al. (2018) Disease-causing mutation in ?-actinin-4 promotes podocyte detachment through maladaptation to periodic stretch. Proc Natl Acad Sci U S A 115:1517-1522 |
| Feng, Di; Steinke, Julia M; Krishnan, Ramaswamy et al. (2016) Functional Validation of an Alpha-Actinin-4 Mutation as a Potential Cause of an Aggressive Presentation of Adolescent Focal Segmental Glomerulosclerosis: Implications for Genetic Testing. PLoS One 11:e0167467 |
| Ehrlicher, Allen J; Krishnan, Ramaswamy; Guo, Ming et al. (2015) Alpha-actinin binding kinetics modulate cellular dynamics and force generation. Proc Natl Acad Sci U S A 112:6619-24 |
| Grgic, Ivica; Hofmeister, Andreas F; Genovese, Giulio et al. (2014) Discovery of new glomerular disease-relevant genes by translational profiling of podocytes in vivo. Kidney Int 86:1116-29 |
| Yao, Norman Y; Broedersz, Chase P; Depken, Martin et al. (2013) Stress-enhanced gelation: a dynamic nonlinearity of elasticity. Phys Rev Lett 110:018103 |
| Carrasquillo, Robert; Tian, Dequan; Krishna, Sneha et al. (2012) SNF8, a member of the ESCRT-II complex, interacts with TRPC6 and enhances its channel activity. BMC Cell Biol 13:33 |
| Wyss, Hans M; Henderson, Joel M; Byfield, Fitzroy J et al. (2011) Biophysical properties of normal and diseased renal glomeruli. Am J Physiol Cell Physiol 300:C397-405 |
| Yao, Norman Y; Becker, Daniel J; Broedersz, Chase P et al. (2011) Nonlinear viscoelasticity of actin transiently cross-linked with mutant ?-actinin-4. J Mol Biol 411:1062-71 |
| Broedersz, Chase P; Depken, Martin; Yao, Norman Y et al. (2010) Cross-link-governed dynamics of biopolymer networks. Phys Rev Lett 105:238101 |
| Gu, Changkyu; Yaddanapudi, Suma; Weins, Astrid et al. (2010) Direct dynamin-actin interactions regulate the actin cytoskeleton. EMBO J 29:3593-606 |
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