The goal of this proposal is to elucidate the molecular mechanism(s) that underlie pathophysiology of proteinuric kidney diseases. Work from this laboratory recently demonstrated direct regulation of actin assembly in vitro and in podocytes by dynamin, and suggested an intricate interplay between dynamin's oligomerization cycle and actin cytoskeleton dynamics. Our findings motivate the hypothesis that signaling at the slit diaphragm alters dynamin oligomerization cycle through direct dynamin-Nck interactions, which in turn initiates actin polymerization in the membrane vicinity.
In Specific Aim 1 we test whether clustering of Nck on the membrane promotes dynamin oligomerization into rings, which in turn drives actin polymerization in cultured podocytes. Using small molecules that specifically promote or inhibit dynamin oligomerization, as well as our novel actin binding domain mutants of dynamin, we test whether dynamin oligomerization cycle and actin-binding interactions are essential and/or sufficient for Nck-dependent polymerization of cortical actin cytoskeleton.
In Specific Aim 2 we test whether direct interactions between dynamin and Nck alter the dynamin oligomerization cycle and dynamin-dependent actin polymerization using in-vitro assays.
In Specific Aim 3, using TIR-FM we test whether direct dynamin-actin interactions are essential for clathrin mediated endocytosis.
In the United States alone, glomerular type of kidney diseases affects some 20 million people, and this number has roughly doubled within the last two decades. Here we propose to test the hypothesis that signaling at the slit diaphragm alters dynamin oligomerization cycle through direct dynamin-Nck interactions, which in turn initiates actin polymerization in the membrane vicinity. Positive data will identify the missing link between signaling at the slit diaphragm and organization of the actin cytoskeleton in podocytes.
|Sever, Sanja; Reiser, Jochen (2015) CD2AP, dendrin, and cathepsin L in the kidney. Am J Pathol 185:3129-30|
|Schiffer, Mario; Teng, Beina; Gu, Changkyu et al. (2015) Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models. Nat Med 21:601-9|
|Reiser, Jochen; Sever, Sanja; Faul, Christian (2014) Signal transduction in podocytes--spotlight on receptor tyrosine kinases. Nat Rev Nephrol 10:104-15|
|Gu, Changkyu; Chang, Joann; Shchedrina, Valentina A et al. (2014) Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity. Traffic 15:819-38|
|Sever, Sanja; Chang, Joann; Gu, Changkyu (2013) Dynamin rings: not just for fission. Traffic 14:1194-9|