IMMUNOLOGIAL APPROACHES TO OUTER SEGMENT DISASSEMBLY

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This research is directed toward a more precise understanding of the shedding of rod outer segment (ROS) discs and their phagocytosis by the retinal pigment epithelium (RPE). Together with the renewal of new discs, this process constitutes the turnover of photoreceptive membrane which is necessary to maintain proper visual function. Shedding-phagocytosis is a multistep process which is probably coordinated by regulation at several stages. These processes include the normal adhesion of visual cell outer segments to the RPE, as well as orderly disc detachment, phagocytic uptake and degradation. Although a collaboration between both cell types, the specific roles played by each are still in doubt. Using a procedure for separating epithelium and retina and reconstituting their functional interactions in vitro, we propose to begin analyzing various stages in shedding-phagocytosis. To complement this approach, highly sensitive and selective immunochemical techniques will be applied to this problem in order to identify and characterize macromolecules which function mechanistically. Heteroantisera directed towards epithelial and photoreceptor surfaces will be raised in rabbits and, after appropriate selection procedures, used as probes of retinal attachment and recognition of shed ROS discs. In addition, monoclonal antibodies will be generated to RPE cells actively engaged in disc uptake in order to identify antigens which function in the ingestion phase of phagocytosis. These immunoglobulin molecules will in turn be used as reagents for the biochemical isolation of cell constituents involved. It is hoped that with an arsenal of such immunochemical probes in hand and with a better description of the nature of cellular interactions, progress can be made in understanding the process of ROS disassembly.