Rod Outer Segment Plasma Membrane Research Articles

Time-dependent cGMP-activated conductance of detached patches of ROS plasma membrane

cGMP markedly increases the cationic conductance of the ‘inside-out’ patches of rod outer segment plasma membrane when applied to the inner side. The cGMP-activated conductance of some patches was shown to be time-dependent. The data obtained suggest that the change of cGMP concentration in the near membrane layer underlies this phenomenon rather than the change in the channel's activity. The hydrolysis and, probably, the desorption of the nucleotide are responsible for this.

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of M r 230 000 and 110 000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.

Immunocytochemical binding of anti-opsin N-terminal-specific antibodies to the extracellular surface of rod outer segment plasma membranes. Fixation induces antibody binding.

We have examined the binding of anti-opsin antibodies to the plasma membrane of frog retinal rod outer segments (ROS) by fluorescence light microscopy and electron microscopy. Polyclonal and monoclonal antibodies specific for the N-terminal domain of opsin were observed to bind to the extracellular surface of ROS plasma membrane of aldehyde-fixed but not of unfixed retinas. This reaction was found regardless of whether purified ROS, rhodopsin, opsin, or an N-terminal peptide of opsin was used as the immunogen. The fixation-induced binding of these antibodies contrasts with the more frequently noted loss of antigenicity upon fixation. Concanavalin A, however, binds to unfixed ROS plasma membranes. Its binding sites in the plasma membrane may be oligosaccharides in the N-terminal region of opsin. These results suggest that the N-terminal domain of opsin is latent in the native membrane and that changes in conformation may account for its detectability in fixed membranes.

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylaiton of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca 2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca 2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca 2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca 2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na +-conductance of the plasma membrane.

Membrane assembly in retinal photoreceptors. II. Immunocytochemical analysis of freeze-fractured rod photoreceptor membranes using anti- opsin antibodies

We have used a cytochemical technique for labeling freeze-fractured tissues (Pinto da Silva, P., C. Parkison, and N. Dwyer (1981) Proc. Natl. Acad. Sci. U.S.A. 78: 343-347) to examine the distribution of immunoreactive opsin in rod photoreceptor membranes. Aldehyde-fixed retinas of African clawed frogs (Xenopus laevis) embedded in a cross-linked protein matrix were frozen and fractured at -196 degrees C, then thawed and labeled with biotinylated sheep anti-cow opsin IgG followed by avidin-ferritin. In thin sections of plastic-embedded retinas, rod outer segment (ROS) disc membranes exposed by fracturing bound specific antibody intensely and relatively uniformly. However, they differed from membranes of the inner segment as well as those of erythrocytes in that protoplasmic face leaflets did not assume an interrupted bilayer appearance and disc exoplasmic face leaflets were apparently lost during thawing. The disposition of opsin immunoreactivity in the cell membrane was highly asymmetric. Although ROS plasma membranes from which discs are elaborated labeled heavily with anti-opsin after cleavage, fractures passing along inner segment plasma membranes bound very little antibody. In cross-fractures exposing inner segment cytoplasm, we found specific labeling of Golgi complex elements, as well as both perimitochondrial and periciliary vesicles. The latter are presumed to be the vehicle shuttling newly synthesized membrane to the ROS for disc assembly. These results suggest that opsin-containing membrane is sorted out within the cell, being transported from synthetic sites to the immediate periciliary zone where localized insertion into the cell membrane takes place. Furthermore, the close correspondence of the present immunocytochemical analysis with the distribution of opsin deduced from prior quantitative freeze-fracture analysis (Besharse, J. C., and K. H. Pfenninger (1980) J. Cell Biol. 87: 451-463) offers the possibility that fracture-label may be generally useful for study of patterned membrane topography in neuronal cells.

Ultrastructural localization of S-antigen in retinal structures.

The localization of S-antigen, the soluble, uveitogenic, 51 kDa protein of the retina has been studied. Highly specific rabbit anti-bovine S-antigen antiserum reacted predominantly with the outer segment layer of the photoreceptor cells of rat retina in the indirect immunofluorescence technique, provided that the tissue was fixed by perfusion of the light adapted animal. More detailed information was obtained by immuno-electron microscopy using the same fixation technique and Protein A-coated gold particles for labeling. S-antigen was found to be predominantly bound to the rod outer segment plasma membranes and to the discs. The possible implication of this localization is discussed in view of the function of S-antigen.

Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors.

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin-ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.

Labeling of bovine rod outer segment surface proteins with 125I

Osmotically intact purified bovine rod outer segments (ROS) were labeled with 125I by lactoperoxidase-catalyzed iodination. Molecular weights of Coomassie-stained and 125I-labeled polypeptides were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Only four 125I-labeled proteins were consistently detected, although 13 Coomassie-stained bands were resolved. Rhodopsin was labeled, in addition to proteins having average apparent molecular weights of 226 000 (226 K), 110 000 (110 K) and 66 000 (66 K). The high molecular weight labeled proteins (226 K and 110 K) did not comigrate with Coomassie-stained bands. The 226 K band was much more heavily labeled than the 110 K and 66 K proteins or rhodopsin. Mild trypsin digestion of intact labeled ROS resulted in degradation of the 226 K labeled band, but did not alter the electrophoretic mobility of any of the other labeled bands, or of the Coomassie-stained bands. None of the labeled proteins were extracted from pelletable ROS membranes by 10 m m Tris-Cl, 0·5 m-KCl, 8 m urea or digitonin at 0·5 mg/mg of ROS protein. Sucrose density gradient analysis showed that the labeled proteins were not associated with gradient fractions other than those containing ROS. These results suggest that the 226 K and 110 K proteins are components of the ROS plasma membrane, and support previous reports that rhodopsin is a component of this membrane.

Cytochemical demonstration of guanylate cyclase activity in retinal photoreceptors with special reference to changes under light and dark adaptation.

The localization of guanylate cyclase activity was studied cytochemically in the rod outer segments of rats light and dark-adapted for various intervals. A brown colored reaction was detected in rod outer segments under a light microscope. Under an electron microscope, a significant change in the distribution of the reaction products under light and dark adaptation was observed. When rats were dark-adapted for longer than 90min, an intense activity was found on the cytoplasmic side of the plasma membrane, and most of the disc membranes showed lower activity than in light with the exception of a few disc membranes near the pigment epithelium which showed rather intense activity. At 1sec illumination, no significant change was found in the distribution of the guanylate cyclase. The first significant change was detected at 5sec of illumination. After a 15sec light exposure, the reaction products of guanylate cyclase activity were observed on all the disc membranes regularly, and the activity on the plasma membrane was very weak. The present results indicate that light affects the guanylate cyclase activity and its distribution in the rod outer segments, suggesting that guanylate cyclase or cyclic GMP plays an important role in the control of ion permeability in the rod outer segment plasma membrane and a possible role in the visual adaptation process.

Distribution of enzymes involved in nucleotide metabolism in the disk and other membranes.

Abstract— Intact disks and inverted disks were prepared from bovine retinal rods and the distribution in the disk membrane of such enzymes as guanyl cyclase, cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase), 5'‐nucleotidase and rhodopsin kinase was investigated. Guanyl cyclase was not detected in the disk; the enzyme activity was high in a membranous fraction containing the cilium or axoneme and the rod outer segment plasma membrane. Cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase) and rhodopsin kinase were associated on the external surface of disk in the presence of 2 mM Mg2+. The enzymes dissociated from the membrane when Mg2+ was depleted. Thus, magnesium ion seems to regulate the state of these enzymes in the outer segment. 5'‐Nucleotidase activity was low in intact disks but was significantly enhanced after inversion of the disk. The catalytic site of the enzyme, therefore, must be located on the internal (intradiscal) surface. Since the disks are known to be formed by invagination of the plasma membrane, 5'‐nucleotidase, by inference, would have its catalytic site exposed on the external surface of the plasma membrane. Preliminary experiments showed that the capability of light‐activated rhodopsin to activate cyclic nucleotide phosphodiesterase was inhibited by phosphorylation of the pigment. This supports the idea that rhodopsin kinase, cyclic nucleotide phosphodiesterase and GTPase exist as a functional complex on rod membranes.

Ultrastructural localization of lectin binding sites on the surface of retinal photoreceptors and pigment epithelium

The ferritin-lectin complexes of Ricinus communis agglutinin (RCA-120), wheat germ agglutinin (WGA), concanavalin A (con A), soybean agglutinin (SBA) and fucose binding protein (FBP) have been used to study the distribution of sugars along the plasma membranes of frog photoreceptor and pigment epithelial cells. The tissues were fixed prior to labeling in order to prevent the lectin-induced rearrangement of binding sites. The plasma membrane of different regions of the rod and cone photoreceptor cells bound each lectin to a different extent. In all cases, the outer segment plasma membrane bound more of the ferritin-lectin complex than did the inner segment plasma membrane. This might reflect the specific differentiation of the outer segment plasma membranes, related to their role in the visual process. The intensity of binding of each lectin to the pigment epithelium plasma membrane was similar to its binding to the rod outer segment plasma membrane. In cone outer segments, where one side of the disk membrane is accessible to the incubation medium, direct binding of lectin to the disk edges, as well as to the plasma membrane, was seen. Cone outer segments bound more of the α-D-N-acetylgalactosamine and D-galactose specific lectin, SBA, than did rod outer segments. No differences were observed in the binding of ferritin-lectin complexes to rod outer segments from dark or light adapted frogs.

Rhodopsin in the rod outer segment plasma membrane.

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.

ULTRASTRUCTURAL LOCALIZATION OF RHODOPSIN IN THE VERTEBRATE RETINA

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.