Retinal detachment prevents normal assembly of disk membranes in vitro.

The effects of retinal detachment upon disk membrane assembly in rod outer segments were assessed in Xenopus laevis retinas that had been maintained in eyecup cultures for up to 4 days. In these cultures, assembly of disk membranes occurred at a normal rate in regions of the retina that remained attached to the retinal pigment epithelium. In regions of the retina that were detached from the pigment epithelium, the assembly of new disk membranes either was abnormal or was inhibited. This result cannot be attributed to reduced access of cells in the detached retina to oxygen and metabolites. The experiments described here suggest that the apposition of the retina with the pigment epithelium is a necessary condition for normal disk membrane assembly in Xenopus retinas. This effect may be mediated by contact between the rod outer segments and the pigment epithelium, or by trophic factors in the subretinal space.

Temperature-dependent birefringence patterns in Xenopus rod outer segments.

Results reported here show that the birefringence of disk membranes in Xenopus rod photoreceptors depends upon the temperature at which the disks were assembled. Portions of the outer segments produced by the assembly of new disks while frogs are exposed to constant darkness and an ambient temperature of 25 degrees C have a higher birefringence than portions assembled at 18 degrees C. The intensity of birefringence reflects molecular-level structure and macromolecular-level organization of the disk membranes. It has been shown previously that increasing the ambient temperature enhances the rate of assembly of disk membranes in Xenopus rod outer segments. It also has been shown that disk membranes are assembled most frequently in the hours following light onset, and that disks assembled in the light have a lower birefringence than disks assembled in the dark. Therefore, although light and increased temperature both enhance the rate of disk membrane assembly, they have opposite effects upon the birefringence of Xenopus rod outer segments.

Lengths of immunolabeled ciliary microtubules in frog photoreceptor outer segments.

Monoclonal anti-tubulin antibodies were used to label microtubules in the connecting cilia and outer segments of retinal photoreceptors isolated from Rana pipiens. In paraformaldehyde-fixed rods from frogs maintained on diurnal light cycles, the anti-tubulin labeling of ciliary microtubules (mean length = 27 micron) typically extends to slightly over half the length of the outer segments (mean length = 46 micron). Rod outer segments from frogs kept in constant darkness for 3-4 weeks are longer (mean length = 53 micron) than rod outer segments from frogs maintained in cyclic lighting. However, the distribution of fractional lengths of anti-tubulin labeling of ciliary microtubules is the same for both lighting regimens. Incubating retinas in 1.0 mM colchicine prior to outer-segment fixation has no effect on the length of immunolabeling of ciliary microtubules, suggesting that post-mortem elongation artifacts are not significant. Incubating retinas in 10 microM taxol prior to fixation significantly increases the length of stained ciliary microtubules, suggesting that taxol either promotes post-mortem assembly of microtubules, or that taxol reduces post-mortem disassembly. The mean position of the end of anti-tubulin-labeled ciliary microtubules does not correspond to the position of disk shedding for any of the experimental conditions employed.