Tubulin in bovine retinal rod outer segments

1992 ◽  
Vol 103 (1) ◽  
pp. 157-166
Author(s):  
D.F. Matesic ◽  
N.J. Philp ◽  
J.M. Murray ◽  
P.A. Liebman
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Photoreceptor Cells ◽  
High Salt ◽  
Immunofluorescent Staining ◽  
Rod Outer Segments ◽  
Rod Outer Segment ◽  
Retinal Rod ◽  
Additional Role ◽  
Retinal Rod Outer Segments

Bovine rod outer segment (ROS) preparations contain a major 58 kDa protein doublet that was identified by immunoblot as tubulin. Quantification by gel densitometry showed that the total amount of tubulin was 5- to 10-fold higher than that attributable to the rod axoneme, suggesting additional role(s) for tubulin in photoreceptor cells. Approximately 20% of this nonaxonemal tubulin (15% of total tubulin) is tightly associated with outer segment membranes. This fraction remains membrane-associated after extensive low- or high-salt washing, requiring detergents or protein denaturants for release from ROS membranes. Unlike ROS soluble tubulin it associates tightly with liposomes upon detergent solubilization and reconstitution. The ROS membrane-associated tubulin is highly enriched in isolated ROS plasma membrane fractions compared to the total outer segment membrane pool and can be localized to the plasma membrane but not to disks by immunofluorescent staining, suggesting a possible role in the structure or electrophysiology of the rod outer segment plasma membrane.

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

1986 ◽  
Vol 34 (5) ◽  
pp. 659-664 ◽  
Author(s):  
A S Polans ◽  
L G Altman ◽  
D S Papermaster
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Outer Segment ◽  
Plasma Membranes ◽  
Rod Outer Segments ◽  
Terminal Domain ◽  
Retinal Rod ◽  
Fluorescence Light Microscopy ◽  
Fluorescence Light ◽  
Retinal Rod Outer Segments

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.

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

1984 ◽  
Vol 98 (5) ◽  
pp. 1788-1795 ◽  
Author(s):  
I Nir ◽  
D Cohen ◽  
D S Papermaster
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Plasma Membranes ◽  
Photoreceptor Cells ◽  
Rod Photoreceptor ◽  
Rod Photoreceptors ◽  
Retinal Photoreceptors ◽  
Retinal Rod ◽  
Ros Formation ◽  
Developing Rat

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.

Letter to the editor: Squalene is Localized to the Plasma Membrane in Bovine Retinal Rod Outer Segments

1997 ◽  
Vol 64 (2) ◽  
pp. 279-282 ◽  
Author(s):  
STEVEN J. FLIESLER ◽  
KATHLEEN BOESZE-BATTAGLIA ◽  
ZOPHIA PAW ◽  
R.KENNEDY KELLER ◽  
ARLENE D. ALBERT
Keyword(s):  
Plasma Membrane ◽  
Rod Outer Segments ◽  
Outer Segments ◽  
Letter To The Editor ◽  
Retinal Rod ◽  
Retinal Rod Outer Segments

scholarly journals Raman Spectroscopic Imaging of Cholesterol and Docosahexaenoic Acid Distribution in the Retinal Rod Outer Segment

2011 ◽  
Vol 64 (5) ◽  
pp. 611 ◽  
Author(s):  
Zachary D. Schultz
Keyword(s):  
Docosahexaenoic Acid ◽  
Outer Segment ◽  
Rana Catesbeiana ◽  
Spectroscopic Imaging ◽  
Photoreceptor Cells ◽  
Integral Membrane Protein ◽  
Rod Photoreceptor ◽  
Rod Outer Segment ◽  
Cellular Processes ◽  
Retinal Rod

Raman vibrational spectroscopic imaging was performed on retinal rod cells isolated from bullfrogs (Rana catesbeiana). The Raman spectra enable determination of the lipid and protein rich rod outer segment (ROS) from the nucleus and inner segment of the cell. Peak fitting analysis of spectra obtained from individual rod photoreceptor cells show characteristic vibrational modes that can be associated with cholesterol and docosahexaenoic acid-containing lipids. These results provide direct observations of biomolecular gradients in the rod photoreceptor cells, which, thus far, have been based on indirect detergent extracts and histochemical analysis with indicators such as filipin. The detected biomolecules are associated with regulation of the integral membrane protein rhodopsin, and methods capable of direct observation of these biomolecules offer new routes to exploring their role in the regulation of cellular processes.

scholarly journals Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration.

1992 ◽  
Vol 116 (3) ◽  
pp. 659-667 ◽  
Author(s):  
K Arikawa ◽  
L L Molday ◽  
R S Molday ◽  
D S Williams
Keyword(s):  
Plasma Membrane ◽  
Retinal Degeneration ◽  
Outer Segment ◽  
Photoreceptor Cells ◽  
Growth Stages ◽  
Rod Outer Segments ◽  
Rod Photoreceptor ◽  
Outer Segments ◽  
Disk Membrane ◽  
Membrane Morphogenesis

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.

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

1986 ◽  
Vol 34 (1) ◽  
pp. 5-16 ◽  
Author(s):  
D S Papermaster ◽  
B G Schneider ◽  
D DeFoe ◽  
J C Besharse
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Photoreceptor Cells ◽  
Disk Surface ◽  
Light Sensitivity ◽  
Apical Plasma Membrane ◽  
Rod Photoreceptor ◽  
Electron Microscopic ◽  
Retinal Rod ◽  
Membrane Bound

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.

Ca2+ influx into bovine retinal rod outer segments mediated by Na+/Ca2+/K+ exchange

1995 ◽  
Vol 269 (5) ◽  
pp. C1153-C1159 ◽  
Author(s):  
P. P. Schnetkamp ◽  
J. E. Tucker ◽  
R. T. Szerencsei
Keyword(s):  
Plasma Membrane ◽  
Light Adaptation ◽  
Rod Outer Segments ◽  
Site Model ◽  
Outer Segments ◽  
Retinal Rod ◽  
Absolute Requirement ◽  
Previous Loading ◽  
Retinal Rod Outer Segments ◽  
Ionophore Monensin

Ca(2+)-depleted rod outer segments (ROS) were purified from bovine retinal rod photoreceptors, and factors influencing Ca2+ influx into ROS via the plasma membrane Na+/Ca2+/K+ exchanger were analyzed. Intracellular alkali cation concentrations were manipulated by 1) previous loading via the ionophore monensin followed by removal of monensin and 2) addition of the channel ionophore gramicidin during Ca(2+)-influx measurements. Ca2+ influx was measured as a rise in cytosolic free Ca2+ with the Ca(2+)-indicating dye fluo 3. An absolute requirement for intracellular Na+ was observed with a Na+ dissociation constant of 30-40 mM, whereas intracellular K+ was a potent inhibitor of Ca2+ influx, apparently by competing with Na+ for a common site on the Na+/Ca2+/K+ exchanger. Half-maximal Ca2+ influx was observed at an external free Ca2+ concentration of 0.9 microM when no external Na+ was present and 3.5 microM when 10 mM external Na+ was present. Our observations are discussed in the context of 1) a three-site model for the Na+/Ca2+/K+ exchanger and 2) earlier experiments on light adaptation in rods, which depended on minimizing Ca2+ fluxes across the ROS plasma membrane.

scholarly journals Rhodopsin in the rod outer segment plasma membrane.

1976 ◽  
Vol 69 (1) ◽  
pp. 29-42 ◽  
Author(s):  
S Basinger ◽  
D Bok ◽  
M Hall
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Visual Pigment ◽  
Outer Segment ◽  
Gel Filtration ◽  
Acid Mixture ◽  
Rod Outer Segments ◽  
Gel Filtration Chromatography ◽  
Rod Outer Segment ◽  
Outer Segments

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.

Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment

Nature ◽  
1985 ◽  
Vol 313 (6000) ◽  
pp. 310-313 ◽  
Author(s):  
Evgeniy E. Fesenko ◽  
Stanislav S. Kolesnikov ◽  
Arkadiy L. Lyubarsky
Keyword(s):  
Plasma Membrane ◽  
Cyclic Gmp ◽  
Outer Segment ◽  
Rod Outer Segment ◽  
Retinal Rod
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