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

描述（由申请方提供）：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。协调的形态学变化，导致裂缝隔膜的形成需要精确的肌动蛋白细胞骨架的安排。Nephrin是裂膈的关键分子成分; nephrin突变导致芬兰先天性肾病综合征。Nephrin聚集在足细胞足突的基部，并结合邻近足突上的nephrin分子。nephrin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，由于许多复杂的生物学调节肾脏足细胞的结构也是未知的，这个系统提供了一个独特的机会，首先了解生物化学和生物物理学原则，管理足细胞生物学，然后在未来的研究中使用这些原则，以了解它们如何在细胞系统中发挥作用。人造的， 抗体诱导的nephrin在质膜上的聚集诱导几个酪氨酸残基的磷酸化。Src同源（SH）2/SH 3衔接蛋白Nck、N-WASp和Arp 2/3复合物被募集到nephrin酪氨酸磷酸化位点并诱导局部肌动蛋白细胞骨架重排。将Nck和N-WASp添加到溶液中和膜上的磷酸化的nephrin胞质尾中通过多价相互作用诱导nephrin、Nck和N-WASp的寡聚化。这些相分离的寡聚体诱导肌动蛋白聚合后，添加Arp 2/3复合物和肌动蛋白。使用nephrin、Nck、N-WASp、Src家族激酶Fyn和酪氨酸磷酸酶PTP 1B在支持的脂质双层（SLB）和巨大单层囊泡（GUV）上的体外重建，磷酸化依赖的簇形成的动力学，磷酸酶依赖的簇溶解和稳定状态下簇内的分子动力学将被测量和分析，使用全内反射荧光（TIRF）显微镜和旋转盘共聚焦（SDC）显微镜。这些实验的目的是提供深入了解的分子机制，管理nephrin簇的形成和持久性在足过程中的发展和损伤修复。最终，这个实验系统将用于研究nephrin下游的肌动蛋白动力学。然而，将肌动蛋白纳入该实验系统超出了本提案的范围。除了研究nephrin，Nck和N-WASp寡聚化的动力学之外，还将确定膜组成对簇形成的影响。Nephrin定位于质膜的液体有序（LO）和液体无序（LD）相。然而，它是未知的，如果nephrin膜相互作用影响nephrin簇的形成。使用由不同量的磷脂、鞘磷脂、胆固醇和磷酸肌醇组成的SLB和GUV，将使用TIRF和SDC显微镜测量簇形成和膜结构域分离的动力学。从这些实验中获得的结果将提供深入了解潜在的脂质环境中存在的足细胞足的过程，以及提供信息，脂质-蛋白质相互作用如何影响超分子簇的形成。这一提议将确定nephrin簇及其周围膜环境的动态性质。从本提案中描述的实验中获得的信息也将为理解细胞和基于生物体的实验提供基础，这些实验研究肾小球滤过屏障的形成、维持和损伤修复，并为未来在复杂细胞环境中的假设驱动实验提供信息。

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.