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D S Papermaster

Publications and source records attributed to D S Papermaster.

At least 37 records · Page 2Linked to original sources
PubMed1991

Polarized sorting of rhodopsin on post-Golgi membranes in frog retinal photoreceptor cells.

We have isolated a subcellular fraction of small vesicles (mean diameter, 300 nm) from frog photoreceptors, that accumulate newly synthesized rhodopsin with kinetics paralleling its appearance in post-Golgi membranes in vivo. This fraction is separated from other subcellular organelles including Golgi and plasma membranes and synaptic vesicles that are sorted to the opposite end of the photoreceptor cell. The vesicles have very low buoyant density in sucrose gradients (rho = 1.09 g/ml), a relatively simple protein content and an orientation of rhodopsin expected of transport membranes. Reversible inhibition of transport by brefeldin A provides evidence that these vesicles are exocytic carriers. Specific immunoadsorption bound vesicles whose protein composition was indistinguishable from the membranes sedimented from the subcellular fraction. Some of these proteins may be cotransported with rhodopsin to the rod outer segment; others may be involved in vectorial transport.

Animals↗
PubMed1990

Opsin gene expression during early and late phases of retinal degeneration in rds mice.

Opsin mRNA levels, opsin synthetic rates and localization of opsin were studied throughout the photoreceptor's life span in the rds mice. Mutant mice 11 days to 11 months old were investigated. Opsin mRNA levels were studied by means of northern blot analysis. Opsin synthesis was measured by incorporation of [35S]methionine into newly synthesized opsin in vitro. Distribution of opsin in the retina was determined by immunoelectron microscopy. Opsin mRNA was detected in young as well as old retinas, and opsin synthesis could be detected at early phases of degeneration but not in late phases. The absence of opsin synthesis in older rds mice might be due to translational down-regulation or some other defect in the capacity to synthesize opsin. In young mice, opsin was detected in the subretinal space in opsin-laden vesicular membranes: such membranes were absent from retinas of older mice. This disappearance parallels the cessation of opsin synthesis and the consequent failure to deliver opsin to the subretinal space in retinas from older mice. Immunochemical analysis revealed the presence of small amounts of opsin in all retinas up to 11 months of age. Immunoelectron microscopy localized the residual opsin, mostly to the plasma membrane which envelops the nuclei and synaptic terminals. These opsin molecules might be a consequence of very low levels of opsin synthesis, too low to be detected by our assays, or may have been synthesized at an earlier age and retained in the plasma membrane of the old mutant photoreceptors.

Animals↗
PubMed1990

Opsin synthesis and mRNA levels in dystrophic retinas devoid of outer segments in retinal degeneration slow (rds) mice.

Opsin gene regulation, as a function of outer segment structure, was studied in normal and mutant retinal degeneration slow (rds) mice. We investigated the level of expression of the opsin gene in the rds mutant to determine if the reduced opsin content observed in this mutation (around 3% of normal) is a consequence of lowered expression of its gene. Normal BALB/c and rds mice were analyzed for levels of opsin mRNA and opsin content by Northern and immunoblot analysis, respectively. The rate of opsin synthesis in isolated retinas was measured by 35S-methionine incorporation in vitro, followed by analysis of the radiolabeled opsin by SDS-gel electrophoresis and autoradiography. Photoreceptor cell loss at various stages of degeneration was determined by quantitation of surviving photoreceptor nuclei. Opsin was localized in the mutant photoreceptors by immunoelectron microscopy of LR gold-embedded retinas using anti-opsin and antibody gold conjugates. The results indicate that 11- and 30-d-old mutant mice have considerable levels of opsin mRNA (60-70% of normal) and opsin synthetic rates (76-92% of normal), after the data from mutant mice are corrected for photoreceptor cell loss. We conclude, therefore, that the very low level of opsin observed in rds mice (approximately 3%) is not a result of greatly reduced expression of the opsin gene. Rather, continuous turnover of newly synthesized opsin as a result of its failure to become sequestered into an intact outer segment appears to account for the low levels of opsin in the rds mutant.

Animals↗
PubMed1989

Immunocytochemical reactivity of Xenopus laevis retinal rods and cones with several monoclonal antibodies to visual pigments.

Immunocytochemical reactions with several antibodies to visual pigments were used to study visual cells of the Xenopus laevis retina. Monoclonal antibodies to bovine opsin "E," 1D4, and 4B4 (reactive with the N- and C-terminus and with the loop connecting transmembrane segments 5-6, respectively) and to chicken visual pigments COS-1 and OS-2 (binding to mammalian red/green and blue cones, respectively), as well as a rabbit antifrog opsin serum 11-7, were applied to semithin and thin sections of the retina. The bound antibodies were detected with the peroxidase technique at the light microscopic level; a three-stage immunogold procedure was used for electron microscopic immunocytochemistry. The overwhelming majority of rods were labeled by monoclonal antibodies "E," 4B4, 1D4, OS-2, and serum 11-7. A small fraction (2-3%) of rods did not bind monoclonal antibodies "E" and 4B4, but this minor population of rods was strongly reactive with 1D4 and to a lesser extent with OS-2, indicating the presence of different visual pigment. These rods differ in shape from the major rod type; they are thinner, shorter, and may be comparable to the blue-sensitive ("green") rods of other amphibia. Cones were morphologically heterogeneous: double cones, large single cones, and small single cones were found, and the large single and the double cones were occasionally duplicated. Double cones and large single cones (as well as their duplicated varieties) strongly bound monoclonal antibodies COS-1 and were unlabeled by all other monoclonal antibodies, except OS-2. The small single cone was remarkably unreactive with COS-1 and "E," weakly labeled by 1D4 and 4B4, and most reactive with OS-2 and 11-7. This unique pattern of immunocytochemical reactions in the small cone type indicates the uniqueness of its visual pigment from other cone types in the Xenopus retina. The present study shows the existence of two different opsins in morphologically distinct (thick and thin) rod types and at least two cone pigments in the heterogeneous cone population.

Animals↗
PubMed1989

Opsin distribution and synthesis in degenerating photoreceptors of rd mutant mice.

The distribution of opsin and the capacity of photoreceptors to synthesize opsin was studied in retinas of mice bearing the rd (retinal degeneration) mutation and compared to control normal mice. Opsin was localized by means of pre-embedding and post-embedding immunocytochemistry and electron microscopy. Cones were identified with anti-cone antibodies and peanut agglutinin lectin which labels cone matrix sheaths. Opsin synthesis was measured by incorporation of [35S]methionine into opsin which was detected by immunoblots. Immunocytochemistry revealed that degeneration of rod outer segments was accompanied by accumulation of opsin in the plasma membrane enveloping the inner segment, nuclei and synaptic terminals. Rod photoreceptors degenerated faster than cones. By post-natal day 19 (P19), 87% of the remaining inner segments were cones. Opsin synthesis in rd mice could no longer be measured after P15. However, opsin molecules could be detected both by immunocytochemistry and immunoblotting up to P30. Between P20 and P30 all detectable opsin was localized in the plasma membrane which envelopes nuclei and synaptic terminals. Unlabeled surviving nuclei after P30 are probably cones. The opsin which is detected in rod inner segment plasma membranes might be derived, by back diffusion, from degenerating outer segments and represent molecules synthesized at an earlier time. Alternatively opsin accumulation might be a result of low levels of opsin synthesis, undetectable by [35S]methionine incorporation, which continues in the absence of outer segments.

Animals↗
PubMed1989

Production of bovine rhodopsin by mammalian cell lines expressing cloned cDNA: spectrophotometry and subcellular localization.

Cloned cDNA encoding bovine rhodopsin has been recombined into an expression vector and cotransfected with an antibiotic resistance plasmid into cultured human embryonic kidney cells. The resulting cell lines produce 100-200 micrograms of bovine opsin per liter of saturated tissue culture medium (10(9) cells). Incubation in vitro with 11-cis retinal produces a photolabile pigment the absorbance spectrum of which is indistinguishable from that of bona fide bovine rhodopsin. Expressed rhodopsin accumulates in the plasma membrane as determined by immunoelectron microscopy.

Animals↗
PubMed1989

Immunocytochemical localization of opsin in degenerating photoreceptors of RCS rats and rd and rds mice.

Opsin is normally sequestered predominantly in the outer segment disc and plasma membranes of adult photoreceptors. Absence of opsin from the inner segment plasma membrane in normal photoreceptors is probably not due to the inability of the inner segment plasma membrane to retain opsin. Rather, in the adult mammalian retina, if opsin is inserted at sites in the apical inner segment plasma membrane, in a fashion comparable to the pathway in amphibians, it is rapidly transported predominantly to the outer segment by unknown mechanisms. Dystrophic rds retinas, lacking an outer segment, display newly synthesized opsin throughout the plasma membrane. If opsin is transported to the inner segment plasma membrane as a specific insertional site, diffusion in the plane of the membrane may redistribute opsin throughout the plasma membrane which encloses the nucleus and the synaptic terminal. Alternatively, opsin may be inserted randomly throughout the entire cell's plasmalemma beneath the cilium. Selective transport to the outer segment may preferentially clear the inner segment of most of its opsin and nearly clear the perikaryal and synaptic terminal's plasmalemma in normal cells. In dystrophic retinas, however, as outer segments degenerate or fail to form, opsin is detected readily in the remaining plasma membrane sites. In the rd mouse, some of the opsin molecules in the inner segment plasma membrane might be newly synthesized while others may arise from molecules which reached the inner segment by back-diffusion from the outer segment at least at early stages in the degeneration while outer segments survive. The opsin in the plasma membrane which envelopes the residual rod nuclei and synaptic terminals in dystrophic retinas may account for the persisting light perception in retinas which have lost both the rod outer and inner segments. Dystrophic retinas, such as the rd mouse and RCS rats and possibly human RP retinas, in which cone nuclei survive long after rods disappear, might retain light perception because of cone photo-pigments in the outer nuclear and outer plexiform layers. To explore these questions further, the localization of other components of the transduction cascade and the determination of the efficiency of their coupling in dystrophic cells is necessary. We need to know where the cyclic GMP-sensitive sodium channels lie in these dystrophic cells and the cellular requirements for proximity of these components to generate a signal. Outer segment-free photoreceptors, bearing opsin in their plasma membranes, resemble other cells which have receptor-mediated alterations in membrane permeability to ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Opsin accumulation in photoreceptor inner segment plasma membranes of dystrophic RCS rats.

Opsin localization in photoreceptor plasma membrane was studied in 10- to 30-day-old dystrophic RCS rats. Preembedding cytochemical procedures with antiopsin antibodies and electron microscopy were employed. In the second postnatal week, opsin was sequestered to the outer segment plasma membrane in affected rat retinas. This distribution resembled that observed in the photoreceptors of normal rats at this age. As unphagocytosed debris accumulated in the subretinal space, the outer segments degenerated, and the distribution of opsin in the plasma membrane changed. Opsin reappeared in the inner segment plasma membrane as the outer segments were lost. Loss of the regionalized distribution of opsin was not associated with visible ultrastructural changes in the inner segments or connecting cilium. Severely damaged cells invariably were labeled on their inner segments at high density. Thus, the presence of an outer segment was correlated with the clearance of opsin from the inner segment, while damage to the outer segment was followed by reappearance of opsin in the inner segment plasma membrane.

Animals↗
PubMed1986

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 cell's 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.

Animals↗
PubMed1986

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.

Amino Acid Sequence↗
PubMed1986

Immunocytochemical localization of opsin in the inner segment and ciliary plasma membrane of photoreceptors in retinas of rds mutant mice.

Homozygous 020/A mutant mice bearing the rds gene for slow inherited retinal degeneration have been observed to develop normal photoreceptor inner segments connecting cilia and synaptic contacts but fail to form outer segments. Their retinas are responsive to light, however. In order to assess the sources of these physiological responses we investigated the distribution of opsin in photoreceptors by means of immunoelectron microscopy. Opsin was detected in the inner segment plasma membrane and the distal ciliary plasma membrane. Antibody also bound to lamellar and vesicular membranes in the interphotoreceptor space and, in a small fraction of the photoreceptors, to membranes projecting from the distal cilium. These membranes may represent abortive formation of rod discs in this form of retinal degeneration. Failure to form an organized outer segment may contribute to the persistence of opsin in the inner segment plasma membranes of adult mutant mice.

Animals↗
PubMed1986

Electron microscopic immunocytochemistry of interstitial retinol-binding protein in vertebrate retinas.

Interstitial retinol binding protein (IRBP) is a soluble glycoprotein found in the interphotoreceptor matrix (IPM) and implicated in shuttling retinol between retina and pigment epithelium (PE) cells. The authors have studied the distribution of IRBP by EM immunocytochemistry. Thin sections of Lowicryl K4M embedded R. pipiens, X. laevis, bovine and human retinas were labeled sequentially with affinity purified rabbit antibovine IRBP, biotinyl-sheep antirabbit F(Ab')2, and avidin-ferritin, or with avidin and biotinyl-ferritin. Antigen was in the interphotoreceptor space and intercalated into the narrow spaces between PE cell microvilli. IRBP penetration between PE cells was delimited abruptly by the PE junctional complexes. IRBP was also observed in small vacuoles in the apical cytoplasm of PE cells and in PE cell phagosomes that contained IRBP surrounding ingested rod tips. IPM was heavily but inhomogeneously labeled. Antigen was usually deposited along the ROS and COS plasma membrane in a confluent layer, but sometimes it was distributed in large (ca. 0.2-micron thick) clumps. In bovine and human retinas, the connecting cilium was ensheathed by antigen at high density but an unlabeled halo surrounded its plasma membrane. The apical plasma membrane of the inner segment aligned along the connecting cilium was also densely coated by antigen. In both frog retinas, the ridges of the periciliary ridge complex (PRC) were coated with antigen. In none of the four species examined was Golgi labeling present. In bovine retinas, labeled vacuoles (granules) in the myoid region were found in very low numbers (15 vacuoles in 358 rod cells). Amphibian retinas also contained only small numbers of myoid vacuoles labeled by anti-IRBP. Absence of antibody binding to intracellular sites of synthesis in any of the cells that abut the interphotoreceptor matrix suggests that the antigen may be masked prior to its release from the synthetic cell(s) or that its level is below limits of detection.

Animals↗
PubMed1985

Correlation of Na+,K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings.

During salt-water adaptation, an increase occurs in Na+,K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to 1.9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+,K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+,K+-ATPase content and basolateral plasma membrane surface area in adapting and de-adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+,K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis.

4-Nitrophenylphosphatase↗
PubMed1985

Vesicular transport of newly synthesized opsin from the Golgi apparatus toward the rod outer segment. Ultrastructural immunocytochemical and autoradiographic evidence in Xenopus retinas.

Each day, rod photoreceptors of the vertebrate retina synthesize rhodopsin and insert it into new membranes of the rod outer segment (ROS). The authors determined which components of the rod cell transport opsin from the Golgi to the ROS by a combined EM autoradiographic and immunocytochemical study using radiolabeled amino acid precursors and antiopsin antibodies. Radiolabeled proteins in the ellipsoid region of Xenopus laevis retinal rods were localized by comparison of the distribution of silver grains with the predicted distribution generated by a hypothetical source: grain matrix. Sources of decay were not uniformly distributed. Small vesicles compressed between mitochondria and clustered beneath the connecting cilium that joins the inner to the outer segment contained more than 30% of the radiolabel and had a specific activity 17 times higher than the surrounding cytoplasm. Opsin was localized immunocytochemically on thin sections of retinas embedded in Lowicryl K4M (Polysciences; Warrington, PA) by reaction sequentially with biotinyl-rabbit antifrog opsin, biotinyl-sheep antirabbit F(ab')2, and avidin-ferritin. Golgi apparatus, intermitochondrial vesicles, and vesicles that clustered beneath the connecting cilium were prominently labeled. Subellipsoid smooth endoplasmic reticulum was labeled at background levels. These results demonstrate that intracellular vesicular membranes transport newly synthesized opsin from the Golgi to the base of the connecting cilium of X. laevis retinas. Antibody labeled the outer segment plasma membrane at a 10-fold greater density than the contiguous inner segment plasma membrane. The polarized distribution of opsin apparently involves not only vectorial transport of opsin in the inner segment but also restrictions to the randomization of opsin inserted into the inner and outer segment plasma membrane.

Animals↗
PubMed1984

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.

Age Factors↗
PubMed1984

Actin in the photoreceptor connecting cilium: immunocytochemical localization to the site of outer segment disk formation.

Actin has been localized in Rana pipiens retinas that were fixed and embedded in aldehyde cross-linked BSA. Thin sections were reacted sequentially with (a) affinity-purified antiactin antibodies induced in rabbits; (b) biotinyl-sheep anti-rabbit antibodies; and (c) avidin-ferritin conjugates. As expected, antiactin labeling density was high in the apical pigment epithelial cell processes and in the calycal processes of photoreceptors. Actin was also localized in a new site. The connecting cilium that joins the inner and outer segments of both rods and cones was heavily labeled by antiactin at its outer segment (OS), or distal, end. In this region of the cilium, the plasma membrane evaginates to form new OS disks and these basal disks were labeled in some instances. Below the new disks in rods, the cytoplasm of liplike expansions of the distal cilium was also heavily labeled. The plasma membrane and interior of the connecting cilium and the remainder of the OS were unlabeled. These findings suggest that actin may participate in the vectorial transport of opsin and other intrinsic membrane proteins that are incorporated into newly forming OS disks. The results also implicate actin in the membrane expansion involved with OS disk formation.

Actins↗