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.
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