DYNAMIC ORGANIZATION OF THE RENAL CELL MEMBRANE SKELETON IN VIVO

（项目摘要）肾上皮的载体运输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，基于谱蛋白的皮质细胞骨架，与许多完整的膜蛋白相关，也是高度极化的，可能在维持和指导地形膜组装中起基本作用。最近的证据还表明，一个独立但相关的高尔基谱蛋白骨架（SAATS）调节某些膜蛋白（如Na， k - atp酶）的组装和运输。所提出的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整体膜蛋白Na， k - atp酶和E-cadherin分选的关系。具体而言，研究将集中在如何干扰靶谱蛋白到肾上皮细胞侧缘的因素，调节其与其他整体膜蛋白（如E-cadherin和Na， k - atp酶）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。要检查的特定蛋白质包括β 1、β II和β III谱蛋白，几种锚蛋白同工型，包括与水泡高尔基谱蛋白骨架（AnkG119）相关的锚蛋白，以及与细胞核相关的一种锚蛋白。同样感兴趣的是选择的适配蛋白，包括α和β -连环蛋白。这些蛋白之间的相互作用将通过敏感的遗传和生化分析来测量，并通过构建具有特定细胞骨架突变的转基因和基因敲除/敲入小鼠来测量它们在体内的作用。与项目团队的其他成员合作，还将探索基于谱蛋白的组装途径与上皮细胞和内皮细胞肌动蛋白细胞骨架和粘附复合物的其他成分的交集。随着涉及谱蛋白锚蛋白皮质和囊泡细胞骨架的新型调节级联的确定，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膜细胞骨架功能障碍起病因作用的肾脏疾病，并将有助于设计伴随急性肾缺氧和缺氧损伤的细胞事件的基本临床反应。

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.