DYNAMIC ORGANIZATION OF THE RENAL CELL MEMBRANE SKELETON IN VIVO

描述：(项目摘要)肾上皮细胞的载体转运功能的核心是肾上皮细胞表面膜蛋白的极化分布。最近的证据表明，以血影蛋白为基础的皮质细胞骨架，与许多完整的膜蛋白有关，也是高度极化的，可能在维持和引导地形膜组装方面发挥基础作用。最近的证据也表明，一个独立的但相关的高尔基光谱蛋白骨架(SAATS)调节某些膜蛋白的组装和运输，如Na，K-ATPase。拟议研究的总体目标将是了解皮质和囊泡细胞骨架如何实现其极化分布，以及这一过程与基底外侧完整膜蛋白Na，K-ATPase和E-cadherin的分类的关系。具体地说，研究将集中在针对肾上皮细胞侧缘的血影蛋白因子如何调节其与E-钙粘蛋白和Na，K-ATPase等其他整膜蛋白的相互作用，以及这些过程如何影响体内肾脏的发育、功能和肾脏对病理应激的反应。要检测的特定蛋白包括β1、βII和βIII，几种不同形式的锚蛋白，包括与囊泡状高尔基体膜蛋白骨架(AnkG119)相关的那些，以及与核相关的一种形式的锚蛋白。同样令人感兴趣的还有精选的接头蛋白，包括α和β-连环蛋白。这些蛋白质之间的相互作用将通过敏感的遗传和生化分析来衡量，它们在体内的作用将通过构建具有特定细胞骨架突变的转基因和基因敲除/敲入小鼠来衡量。与计划项目组的其他成员合作，还将探索基于血影蛋白的组装途径与上皮和内皮细胞肌动蛋白细胞骨架和黏附复合体的其他组件的交叉点。随着涉及血影蛋白-锚蛋白皮质和囊泡细胞骨架的新的调节级联被识别，这些级联在体内介导急性肾损伤中的作用将被探索。预计这些研究将指导我们寻找细胞膜骨架功能障碍在其中起病因学作用的肾脏疾病，并将有助于设计合理的临床反应，以应对伴随急性肾缺氧和缺氧性损伤的细胞事件。

Description: (Abstract of the project) Central to the vectorial transport function of renal epithelium is the polarized distribution of surface membrane proteins in renal epithelial cells. Recent evidence indicates that the spectrin based cortical cytoskeleton, associated with many integral membrane proteins, is also highly polarized and may play a fundamental role in maintaining and guiding topographic membrane assembly. Recent evidence also indicates that a separate but related Golgi spectrin skeleton (SAATS) modulates the assembly and trafficking of certain membrane proteins such as Na,K-ATPase. The overall goal of the proposed studies will be to understand how the cortical and vesicular cytoskeletons achieve their polarized distribution, and the relationship of this process to the sorting of the basolateral integral membrane proteins Na,K-ATPase and E-cadherin. Specifically, research will focus on how perturbation of the factors that target spectrin to the lateral margins of kidney epithelial cells regulate its interactions with other integral membrane proteins such as E-cadherin and Na,K-ATPase, and how these processes affect in vivo renal development, function, and the response of the kidney to pathologic stress. Specific proteins to be examined include beta 1, beta II, and beta III spectrin, several isoforms of ankyrin including those associated with the vesicular Golgi spectrin skeleton (AnkG119), and a form of ankyrin associated with the nucleus. Also of interest are selected adapter proteins including alpha and beta-catenin. The interaction between these proteins will be measured by sensitive genetic and biochemical assays, and their role in vivo gauged by constructing transgenic and gene knock-out/knock-in mice with specific cytoskeletal mutations. In collaboration with other members of the program project team, the intersection of the spectrin based assembly pathways with other components of the epithelial and endothelial cell actin cytoskeleton and adhesion complexes will also be explored. As novel regulatory cascades involving the spectrin-ankyrin cortical and vesicular cytoskeletons are identified, the role of these cascades in mediating acute renal injury in vivo will be explored. It is anticipated that these studies will guide our search for renal diseases in which membrane cytoskeletal dysfunction plays an etiologic role, and will aid in designing rationale clinical responses to the cellular events that accompany acute renal hypoxic and anoxic injury.