Stability and dynamics of nephrin/Nck/N-WASp clusters on membranes

描述（由申请人提供）：足细胞足突的形成、维持和损伤修复对于肾小球滤过屏障中的狭缝隔膜功能至关重要。导致狭缝隔膜形成的协调形态变化需要精确的肌动蛋白细胞骨架排列。肾素是狭缝隔膜的关键分子成分；肾素突变导致芬兰先天性肾病综合征。肾素聚集在足细胞足突的底部，并结合邻近足突上的肾素分子。肾素簇形成的分子机制以及这些簇如何在健康足细胞中持续存在尚不清楚。此外，由于许多调节肾足细胞结构的复杂生物学也是未知的，该系统提供了一个独特的机会，首先了解控制足细胞生物学的生化和生物物理原理，然后在未来的研究中使用这些原理来了解它们如何在细胞系统中起作用。人造的,

DESCRIPTION (provided by applicant): 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.