Formation, maintenance and injury repair of podocyte foot processes is essential for slit diaphragm function in the glomerular filtration barrier. Coordinated morphological changes resulting in slit diaphragm formation require precise actin cytoskeletal arrangement. Nephrin is the key molecular component of the slit diaphragm; mutation of nephrin results in Finnish congenital nephrotic syndrome. Nephrin clusters at the base of the podocyte foot process and binds nephrin molecules on neighboring foot processes. The molecular mechanism by which nephrin clusters form and how these clusters persist in healthy podocytes is unknown. Moreover, because much of the complex biology regulating kidney podocyte architecture is also unknown, this system provides a unique opportunity to first understand biochemical and biophysical principles governing podocyte biology and then to use these principles in future studies to understand how they function in cellular systems. Artificial, antibody-induced clustering of nephrin on the plasma membrane induces phosphorylation on several tyrosine residues. Src homology (SH) 2/SH3 adaptor protein Nck, N-WASp and Arp2/3 complex are recruited to sites of nephrin tyrosine phosphorylation and induce localized actin cytoskeletal rearrangement. Addition of Nck and N-WASp to phosphorylated nephrin cytoplasmic tails in solution and on membranes induces oligomerization of nephrin, Nck and N-WASp through multivalent interactions. These phase-separated oligomers induce actin polymerization upon the addition of Arp2/3 complex and actin. Using in vitro reconstitution of nephrin, Nck, N-WASp, Src family kinase Fyn and tyrosine phosphatase PTP1B on supported lipid bilayers (SLBs) and giant unilamellar vesicles (GUVs), the dynamics of phosphorylation-dependent cluster formation, phosphatase-dependent cluster dissolution and molecular dynamics within clusters at steady state will be measured and analyzed using a combination of total internal reflection fluorescence (TIRF) microscopy and spinning disk confocal (SDC) microscopy. These experiments are designed to provide insight into the molecular mechanisms that govern nephrin cluster formation and persistence in foot processes during development and injury repair. Ultimately, this experimental system will be used to study actin dynamics downstream of nephrin. However, inclusion of actin in this experimental system is beyond the scope of this proposal. In addition to studying the dynamics of nephrin, Nck and N-WASp oligomerization, the effect of membrane composition on cluster formation will also be determined. Nephrin localizes to both liquid ordered (LO) and liquid disordered (LD) phases of the plasma membrane. However, it is unknown if nephrin-membrane interactions affect nephrin cluster formation. Using SLBs and GUVs composed of varying amounts of phospholipids, sphingomyelin, cholesterol and phosphoinositides, the dynamics of cluster formation and membrane domain segregation will be measured using TIRF and SDC microscopy. The results obtained from these experiments will provide insight into the potential lipid environment present in podocyte foot processes as well as provide information as to how lipid-protein interactions affect supramolecular cluster formation. This proposal will determine the dynamic nature of nephrin clusters and their surrounding membrane environment. The information obtained from the experiments described in this proposal will also provide a basis for understanding both cellular and organism-based experiments that investigate glomerular filtration barrier formation, maintenance and injury repair and inform future hypothesis-driven experimentation in the complex cellular environment.