MYOSIN-I IN RETINAL-PIGMENT EPITHELIAL-CELLS

Purpose. Myosin-I is a nonfilamentous motor protein associated with th e actin cytoskeleton and cellular membranes in several cell types. The occurrence and subcellular distribution of myosin-I in mammalian and fish RPE were investigated to examine the possible role of myosin-I in retinal pigment epithelium (RPE) motility processes. Methods. Antibod ies directed against myosin-I proteins from bovine adrenal medulla or chicken intestinal brush border were used to examine cultured fetal hu man RPE cells and freshly isolated bovine or green sunfish RPE by West ern immunoblots and immunocytochemistry, using both conventional and c onfocal fluorescence microscopy. Results. The monoclonal antibody dire cted against bovine adrenal myosin-I identified a single strong immuno reactive band at 116 kD in Western blots of homogenates of cultured hu man RPE cells and 114 kD in bovine RPE sheets. An immunoreactive band of similar molecular weight was also observed in bovine and rabbit ret ina. Cell fractionation studies of bovine RPE cells revealed that myos in-I was present in all fractions that included cell membranes. The po lyclonal antibody directed against chicken brush border myosin-I ident ified doublet immunoreactive bands at 115/110 kD in Western blots of h omogenates of fish retina but identified a strong predominant immunore active band at 140 kD in fish RPE and brain ho minor bands at 115/110 kD were identified in fish RPE homogenates. Immunocytochemistry of cul tured human RPE cells using the bovine adrenal myosin-I antibody revea led a broad distribution of myosin-I that appeared to be most concentr ated along the length of the lateral membranes; no colocalization was seen with actin-rich stress fibers. Proteins immunoreactive with myosi n-I antibodies are present in both RPE and retina of mammals and green sunfish. In confluent cultures of human RPE cells, myosin-I is concen trated along the lateral cell membranes of the cuboidal cells.