Distribution of membrane proteins in mechanically dissociated retinal rods

Solitary rods were isolated from frog retinas by mechanical dissociation. Typically, the rods cleave sclerad to the nucleus and consist of outer segments with attached partial inner segments with either tapered or rounded profiles. Light and electron microscopy reveal that the outer and inner segments of rods with tapered inner segments, like rods in the intact retina, are joined by a single connecting cilium. In contrast, the outer and inner segments of rods with rounded inner segments are fused, with no extracellular cleft between the two segments. Opsin distribution was studied in both unfused and fused rods by light and electron microscopic immunocytochemistry. Extensive surface labeling is restricted to the outer segments of tapered rods, as observed in vivo. In contrast, both inner and outer segments of rods with rounded inner segments (fused) label heavily with anti-opsin. Thus opsin, a mobile membrane protein, diffuses from the outer to the inner segment of fused rods. Segregated distribution of opsin in unfused rods suggests that the connecting cilium and/or its associated structures may normally act as a diffusion barrier between the outer and inner segments to mobile membrane proteins such as opsin. Immunofluorescence studies demonstrate that Na+/K+ ATPase is restricted in distribution to the inner segment and calycal processes of both fused and unfused isolated rods, as observed in vivo. Maintenance of its restricted distribution in fused cells indicates that Na+/K+ ATPase is not mobile and may be tethered in the surface membrane of the inner segment.