Surface-replica topography of retinal pigment epithelium during phagocytosis

The initial step in retinal phagocytosis may be mediated by complementary recognition molecules on the pigment epithelial microvilli and outer segment membrane surfaces. These molecules may display a surface morphology which is related topographically to phagocytic events. In order to explore this, we have developed a method for replicating the membrane surfaces of rat pigment epithelial explants during phagocytosis of latex beads. The explants are fixed in a mixed aldehyde and osmium fixative and the basal surface glued to a coverslip. After dehydration and critical point drying, the tissue is replicated with platinum and carbon in a freeze-fracture apparatus using either rotary or unidirectional coating and the replicas are examined by transmission electron microscopy. The membrane surfaces of the pigment epithelial cells and their microvilli are studded with numerous surface particles varying in size from 20 to 50 nm that are closely packed and give a cobblestone appearance to the membrane topography. During the early stages of phagocytosis, microvillous processes appear to spread over the beads and form branched processes as they contact bead surfaces. During bead engulfment the microvilli have shortened into flattened sheets with interdigitating processes overlapping the beads. These uptake sites resemble Venus' fly-traps, which as they close over the beads create craters in the membrane surfaces of the pigment epithelium. The crater-like uptake sites appear to flatten out in the later stages and form flattened membrane domains which are surrounded by crenulated membrane that displays an irregular particulate morphology.