The retinal pigment epithelium (RPE) plays an essential role in eye function and is involved in many important retinal pathologies, such diabetic retinal detachment and macular degeneration. We propose to continue our efforts to elucidate the cellular and molecular bases of polarity of the retinal pigment epithelium (RPE). The relative roles of intracellular sorting in the Trans Golgi Network and the basolateral endosomal system will be initially established using two sets of exogenous apical-basolateral model proteins: (i) influenza HA and VSV G protein, introduced in RPE via infection with the respective viruses, and (ii) p75 neurotrophin receptor (NTR) and LDL receptor, introduced via replication- defective adenovirus mediated gene transfer. We will determine whether apical and basolateral proteins are segregated in the TGN or after reaching the cell surface in primary RPE cells, in a novel RPE cell line (RPE-J) and in RPE in situ. The intracellular pathways to the cell surface will be determined by laser confocal microscopy, immuno electron microscopy, and biotin targeting assays. We will investigate how the eye's microenvironment affects surface protein delivery in RPE by carrying comparative targeting studies in proteins that have different surface distributions in RPE in situ versus cultured RPE, such as the neural adhesion molecule (NCAM), RET-PE2 and Na,K-ATPase. To characterize the mechanisms of TGN sorting in RPE, we will study the effect of drugs that affect specifically the apical and basolateral pathways in kidney cells and characterize the cytosol-dependent assembly of apical and basolateral vesicles in the TGN in an in vitro assay. Finally, we will investigate whether the sorting signal(s) that target proteins to specific surface domains in RPE are similar or distinct from those operating in other simple epithelia, such as kidney or intestine. For this purpose, we will introduce by transfection or by adenovirus-mediated gene-transfer in a variety of NCAM and GPI-protein mutants into RPE cultures, and into the eye's RPE in situ, and analyze their distribution by morphological and by biotin targeting assays. It is expected that these studies will contribute key information on the cell biology of RPE that may help to elucidate its role in eye diseases.
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