A diurnal rhythm in opsin content of Rana pipiens rod inner segments

Quantitative electron microscope immunocytochemistry, employing an antibody specific to opsin, was used to evaluate the amount and location of opsin in Rana pipiens rod photoreceptors throughout a 24 hr light/dark cycle. We found a distinct diurnal rhythm in the density of anti-opsin labeling of the rough endoplasmic reticulum (RER) and Golgi apparatus in the myoid region of the rod inner segment. Opsin labeling of these organelles was lowest at light onset, increasing thereafter by three- to four-fold, and remained high until 2 hr into the dark phase. A fall in labeling density occurred within the following 4 hr, and remained low for the remainder of the dark phase. Our finding of a diurnal rhythm regulating inner segment opsin transport in Rana pipiens contrasts with published observations on outer segment membrane turnover, since it has been shown that the rates of disc formation and disc shedding are governed by environmental lighting alone in this species. These results imply that there is opsin pooling in the inner segment during the first 14 hr of a 24 hr light/dark cycle; thereafter the loss of inner segment opsin due to mobilization of this protein from the Golgi exceeds the rate of formation of new opsin. There was no evidence of accumulation of opsin-containing vesicles near the cilium or in the ellipsoid just prior to light onset. At light onset, prominent opsin labeling was identified at the proximal portion of the outer segment in regions separate from the disc stack. In two separate experiments, additional groups of frogs were killed around the time of light onset and were examined by conventional transmission electron microscopy. Disordered disc membranes were seen at the base of the outer segment which were not in register with the disc stack. These disordered membranes were observed as early as 2 hr before light onset, and were no longer observed by 1 hr after light onset. We suggest that these disordered membranes reflect a step in the biogenesis of new discs, serving as a pool of membrane that forms during the later part of the dark cycle. It appears that light onset triggers the ordering of neatly registered discs from this new membrane, rather than assembly of new membrane from pooled transport vesicles in the inner segment.