Dennis M. Defoe

Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors.

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cells plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles.

An improved method for isolation and culture of pigment epithelial cells from rat retina

An improved method for isolation and culture of pigment epithelial cells from normal rat retinas is described. Following a brief incubation in the neutral protease Dispase, large epithelial sheets can be harvested rapidly. The separation of the choroid from the pigment epithelium prior to retinal detachment greatly reduces the risk of contamination of the cultures with choroidal cells. Growth of pigment epithelial cells on Matrigel-coated microporous filters in hormonally-defined medium allows for development of high levels of transepithelial electrical resistance as well as for preservation of the differentiated pigment epithelial cell phenotype. This method should be useful for studies of pigment epithelial cell permeability and structural differentiation in vitro.

Reconstitution of the photoreceptor-pigment epithelium interface: L-glutamate stimulation of adhesive interactions and rod disc shedding after recombination of dissociated Xenopus laevis eyecups.

In order to investigate adhesive interactions between photoreceptor and pigment epithelial cells, we have mechanically separated neural retinas from Xenopus laevis eyecups and then recombined the tissues in vitro. When tissue pairs are incubated in a defined medium, cell-cell contact is achieved within 3 hr. However, the average proportion of reassembled eyecups in which photoreceptor outer segments interdigitate with epithelial microvilli is limited. Furthermore, rod disc shedding does not take place in these cultures, even following a dark to light transition. When recombined tissues are placed in medium supplemented with 12 mM L-glutamate, retinal reattachment is enhanced and there is a four-fold increase in epithelial phagosome content. The positive effect of excitatory amino acid exposure on shedding, however, is restricted to regions where visual and epithelial cells interdigitate. These results indicate that re-establishment of cell contact may be necessary for shedding of apical disc membranes prior to their engulfment by the epithelium. While reattachment is not affected by pre-incubation of separated tissues in normal medium, rod photoreceptors fail to undergo membrane turnover in response to L-glutamate if a delay of 1 hr or more is interposed between isolation of the retina and its recombination with the pigment epithelium. This is probably due to a decline in retinal function in culture, since a similar preincubation of the pigment epithelium prior to reassembly with a freshly isolated retina does not inhibit the shedding response.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochalasin D inhibits l-glutamate-induced disc shedding without altering l-glutamate-induced increase in adhesiveness

Excitatory amino acid-stimulated disc shedding is correlated with the appearance of microfilament-rich ensheathing processes of the retinal pigment epithelium (RPE) and increased apparent adhesiveness between photoreceptors and RPE in explanted eyecups of Xenopus laevis. We have compared the time course of disc shedding and increased retinal adherence during L-glutamate treatment. Increased adherence was measured on the basis of the tendency of the apical RPE domains to partition with isolated neural retinas. In medium supplemented with L-glutamate (12 mM) or kainate (100 microM), a glutamate analog, the time course of increased partitioning of melanin pigment-rich cell fragments which contain ensheathing processes differs, even though the kinetics of induced disc shedding is the same in either case. Co-treatment with cytochalasin D (5 microM) completely blocks L-glutamate-induced disc shedding, as well as formation of microfilament-rich ensheathing processes, even though it has little effect upon apparent adhesiveness. The virtually complete dissociation of the effects of L-glutamate on disc shedding from that on increased adhesiveness of photoreceptors to RPE suggests that increased retinal adherence and pseudopod formation may be unrelated causally.

Autoradiographic and biochemical assessment of rod outer segment renewal in the vitiligo (C57BL/6-mivit/mivit) mouse model of retinal degeneration

Rod outer segment renewal was assessed in vitiligo (C57BL/6-mivit/mivit) mice using autoradiographic and biochemical methods. This process was examined because the number of phagosomes is reduced in this mouse. Rod outer segment renewal was detectable in the mivit/mivit retina. Within 24 hr of intraperitoneal injection of 3H leucine, there was a distinct band of radioactivity present at the junction of the ROS and RIS in mutant mice that was similar to controls. The displacement of the radioactive band progressed normally in the peripheral regions of the vitiligo retina, but did not in the posterior retina. Morphometric analysis of the posterior region of mutant retinas, indicated that the band of radioactivity became less distinct between 1 and 3 days post-injection. In vitiligo retinas it remained at 2.23 microns, whereas in controls, the band migrated 6.16 microns from the ROS base. When posterior regions of retinas were evaluated 8 days post-injection, there was no band discernible in the vitiligo retinas, but a very dense band at the ROS apex in controls. Assessment of incorporation of radioactivity into rhodopsin using SDS-PAGE indicated a progressive displacement of radio-labeled rhodopsin through the RER, but not as complete a progression through the outer segments. The elongation of the outer segments in the posterior regions of the mutant retina suggests impaired shedding. This, plus the lack of attachment in the posterior retina of photoreceptor cells to RPE in this mouse, seem to be likely causes for the decreased number of phagosomes.