Cytoskeleton's role in RPE's structure and function

描述(申请人提供)：视网膜色素上皮(RPE)是脊椎动物视网膜的一个多功能和不可或缺的组成部分。它的不对称性、特殊的膜结构和膜的运动性是其许多功能所必需的，严重依赖于高度有序的细胞骨架。目前，对细胞角蛋白介导的RPE功能的调控机制和分子机制知之甚少。本实验室发现，新近发现的肌动蛋白相关蛋白CLIC4在RPE顶端微绒毛中大量表达。在培养的RPE细胞中，CLIC4似乎是吞噬细胞杯的关键成分，吞噬细胞杯是吞噬光感受器外节的结构。为了研究Clic4在视网膜色素上皮中的S作用，我们通过将小干扰RNA(SiRNA)导入啮齿类动物的RPE中，实现了Clic4的沉默。CLIC4抑制的RPE细胞发生了几种形态变化，包括微绒毛缩短和细胞-细胞接触破裂。此外，这些动物出现了严重的视网膜脱离和光感受器萎缩，类似于之前描述的增殖性玻璃体视网膜病变的表型。提出了三个具体目标。目的1鉴定CLIC4直接参与RPE微绒毛和RPE-光感受器交错连接结构的发生。目的2研究CLIC4与肌动蛋白-质膜连接蛋白Ezrin之间的分子相互作用及其在体内CLIC4介导的RPE形态发生中的重要作用。目的3.为CLIC4的S参与视网膜色素上皮细胞外段吞噬作用提供功能证据，并剖析CLIC4参与的具体步骤(S)。我们相信，这些研究将为细胞骨架组织和调控在正常和疾病RPE中的关键作用提供新的见解。这些不仅是重要的细胞生物学问题，而且与我们对增殖性玻璃体视网膜病变以及其他退行性视网膜疾病的病因的理解高度相关。

DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) is a multifunctional and indispensable component of the vertebrate retina. Its asymmetry, specialized membrane structures, and membrane motility, which are essential for many of its functions, rely heavily on a highly ordered cytoskeleton. At present, relatively little is known about the machinery and the molecular mechanism regulating cytoskelton-mediated RPE functions. Our lab found that CLIC4, a recently identified actin-associated protein, was abundantly expressed in apical RPE microvilli. In cultured RPE cells, CLIC4 appears to be a key component in the phagocytic cup, the structure that engulfs the photoreceptor outer segment. To study CLIC4's role in RPE in vivo, we performed CLIC4 silencing by transfecting siRNA (small interfering RNA) into RPE of rodent eyes. The CLIC4-suppressed RPE cells developed several morphological changes including shortening of microvilli and breakdown of cell- cell contacts. Moreover, these animals developed profound retinal detachment and photoreceptor atrophy, resembling those phenotypes previously described for proliferative vitroretinopathy. Three specific aims are proposed. Aim 1 will identify the direct involvement of CLIC4 in the genesis of the microvillar and junctional structure of RPE and RPE-photoreceptor interdigitation. Aim 2 will investigate the molecular interactions between CLIC4 and Ezrin, an actin-plasma membrane linker protein, and the importance of such interactions in CLIC4-mediated RPE morphogenesis in vivo. Aim 3. will obtain functional evidence for CLIC4's involvement in outer segment phagocytosis by RPE and dissect the specific step(s) in which CLIC4 is involved. It is our belief that these studies will provide novel insights into the pivotal role of cytoskeletal organization and regulation in both normal and diseased RPE. These are not only important cell biological questions but also highly relevant for our understanding of the etiology of proliferative vitroretinopathy and perhaps also other degenerative retinal diseases.