Our long term objective is to analyze the cytoskeletal framework of the retinal pigment epithlium, the relationship between apical processes and the basal part of the cell, and the triggering of phagocytosis. Towards these goals, we propose to make a detailed study of the cytoskeletal components and their arrangement within the apical processes of the RPE of developing chick and of the rat. We propose to use immunofluorescence on single cells and on cryostat sections at the light level and negative staining and freeze fracture at the electron microscope level to localize the proteins and observe structural detail. We will couple these methods with the use of gel electrophoresis and immunoblats to identify the proteins which may be components of the cytoskeletons. A preliminary study has showm that rat RPE extend filopodia from the existing apical processess under the influence oif cyclic nucleotide. We will characterize this in vitro response abd determine what structural changes are involved. Other preliminary results show that several proteins which are components of the cytoskeleton are phosphorylated in vivo. We are anxious to know whether the phosphorylation of known proteins of the cytoskeleton such as myosin light or heavy chain, fodrin or vimentin, occur during the extension process, in relation to the dark/light cycle, or during active ingestion of rod outer segments. To study phagocytosis, we will develop a controlled stimulus for the uptake process, label the stimulus radioactively or with fluorescent labels and study the control of the uptake mechanism, especially with relationship to ionic dependence. Finally, we will attempt to identify the receptor on the RPE surface that interacts with rod outer segments, using affinity column techniques and cross-linking procedures. The results should add to the basic information on aspects of RPE structure and function and lay the ground work for the study of disease processes, such as those in the RCS rat model and in human retinitis pigmentosa.
