The elevated dark-adapted threshold of the hypopigmented, pearl mouse could be related to structural modifications of the retinal pigment epithelium (RPE) and retina of this mutant. Cytoskeletal interactions with the plasma membrane provide a functional interface between the cytosolic interior and extracellular surface of a cell. The objective of this study is to examine how this interface has been altered at the basal surface of the RPE of the pearl mouse. Examination of the dark-adapted rod terminals also may show cytoskeletal alterations that could be directly related to the functional deficit of the pearl retina. Electron microscopic directly related to the functional deficit of the pearl retina. Electron microscopic immunolocalization will be used to characterize the distribution of cytoskeletal and basement membrane components and to assess differences between pearl and wild-type in the structures examined. The composition of the basal surface of the RPE of wild-type and pearl mice will be characterized by localization of antibodies against cytoskeletal components: (1) clathrin, a major constituent of coated vesicles: (2) integrin, a plasma membrane link between the actin filament complex and ligands of the basement membrane: (3) spectrin and ankyrin, which have been associated with various ion channels of the plasma membrane, one of which is (4) band 3, a membrane protein with anion exchange properties. Functional aspects to be examined include (1) coated vesicle-mediated endocytosis (i.e. uptake of horseradish peroxidase, HRP) at the basal surface of the RPE: and (2) possible modulation of coated vesicle stability following perfusion with alkaline buffer. the composition of the basement membrane of the RPE will be analyzed following the application of antibodies against components of the basement membrane: fibronectin, laminin, type IV collagen, and heparin sulfate proteoglycan. Antibodies to the spectrin cytoskeletal complex will be applied to rod terminals. A quantitative estimate of coated vesicles associated with the plasma membrane of dark-adapted rod terminals will be made. Isolated retinas will be used to examine a possible pH effect on the endocytic activity (i.e. HRP uptake) of dark-adapted rod terminals. Structural anomalies of the RPe and retina are not unique characteristics of the pearl mouse but also are present in diseased and senescent humans, in diabetic rats, and in rats with inherited retinal dystrophy. These investigations should lead to an understanding of malfunctions of mammalian neural retina and RPE in general.
