The long term objective of the proposed research is to determine the relationship between cell shape and cell function in the retinal pigment epithelium (RPE) of adult eyes. This information can be used to determine how RPE cells with altered phenotype contribute to ocular pathology, and to develop methods to maximize the restoration of normal epithelial phenotype in an RPE monolayer damaged by injury or disease. In the proposed project, subpopulations of human RPE cells will be used which express stable fusiform or epithelioid phenotypes in vitro. Experiments to address the first specific aim of the proposed research focus on the formation of the adherens junction during the process of in vitro morphogenesis which follows confluency. During morphogenesis epithelioid cells form a detergent-resistant and zonular cadherin- containing junction that is not formed in fusiform cells. The hypothesis to be tested states that epithelialization in a cell type like the RPE that expresses N-cadherin, which is also found in many non- epithelial cells, requires a specific time-dependent sequence of changes in the composition, tyrosine phosphorylation state or turnover ate of molecules comprising the cadherin/catenin complex. Cadherin/catenin complexes in extracts of epithelioid and fusiform RPE will be compared using the methods of immunoprecipitation, protein blotting, phosphatase and kinase inhibitor treatment, and confocal imaging. The second specific aim of the proposed project focusses on epithelioid and fusiform RPE cells late in the postconfluent time course when the phenotypes are well-developed. The goal of this aim is to identify differentially-expressed genes which may contribute of the maintenance of the distinct phenotypes, or which may identify distinct functions of RPE cells expressing different shapes. RNA extracts of the phenotypes will be compared using the method of mRNA differential display, which includes reverse transcription, the polymerase chain reaction and DNA sequence analysis.
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