The long term objectives of this work are to understand the construction and development of the cytoskeleton of the RPE cell, how it interacts to form the terminal web and the apical processes, and how it is linked to the membrane. In addition we aim to gain information about how the polarity of the RPE cell develops, including identification of membrane proteins in this cell which have a polar distribution and which may regulate cell-cell and cell-substratum interactions. Towards these goals we plan to use antibodies to identify components by immunofluorescence and immunoblotting in developing chick and rat RPE. We will use permeabilized cells which retain membrane-cytoskeletal attachments for studies of the structure of apical processes by electron microscopy including freeze-fracture. We will iodinate intact cells to determine which components of the external surface may interact with the cytoskeleton. We will study the development of the cells with a focus on the time of formation of apical processes to see if specific proteins become associated with the cytoskeleton at this time, and to determine if they are components of the apical processes. We will coculture the RPE from stages before process formation with neural retina of different aged embryos to determine if we can influence process formation. We will study the polarity of known membrane proteins in developing and cultured RPE. We will develop monoclonal antibodies to RPE cell surfaces which will be used to determine apical and basal membrane proteins, identify proteins involved in cell adhesion, characterize developmentally regulated membrane proteins and determine if any antibodies are capable of inhibiting phagocytosis. Our studies should shed light on the structure of the processes which support and phagocytose the photoreceptors, essential activities for normal vision. Additionally we hope to gain further understanding of the factors involved in maintaining RPE cell polarity and adhesion to Bruch's membrane.
