scholarly journals MEMBRANE CHARACTERISTICS AND OSMOTIC BEHAVIOR OF ISOLATED ROD OUTER SEGMENTS

1973 ◽  
Vol 56 (2) ◽  
pp. 389-398 ◽  
Author(s):  
Juan I. Korenbrot ◽  
Dennis T. Brown ◽  
Richard A. Cone
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Receptor Cell ◽  
Water Flux ◽  
Rod Outer Segments ◽  
Repeat Distance ◽  
Permeability Constant ◽  
Outer Segments ◽  
Length Reduction ◽  
Osmotic Behavior

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na+ influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na+ influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na+ permeability constant of about 2.8 x 10-6 cm/s and an osmotic permeability coefficient of greater than 2 x 10-3 cm/s.

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 Correlation of rhodopsin biogenesis with ultrastructural morphogenesis in the chick retina.

1975 ◽  
Vol 64 (1) ◽  
pp. 235-241 ◽  
Author(s):  
W T Mason ◽  
K J Bighouse
Keyword(s):  
Outer Segment ◽  
Receptor Cell ◽  
Rod Outer Segments ◽  
Membrane Biogenesis ◽  
Rod Outer Segment ◽  
Rods And Cones ◽  
Outer Segments ◽  
Chick Retina ◽  
Disk Membrane ◽  
Detergent Extraction

The developing chick retina from stages 39-45 has been examined by biochemical and electron microscope techniques. The levels of rhodopsin contained in the maturing chick retina were evaluated by detergent extraction and correlated with rod outer segment formation. It was found that the appearance of rhodopsin in significant levels preceded outer segment formation by at least 2 days, thus implying that rhodopsin is synthesized in the receptor cell inner segment and translocated to the outer limb when disk membrane biogenesis occurs. The level of rhodopsin continues to rise as the rod outer segment develops. Development of both rods and cones originates and proceeds most rapidly in the fundus or central region and proceeds toward the periphery. In general, rod outer segments were noted to develop far more rapidly than cone outer segments.

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.

The photoreceptors and pigment epithelium of the larval Xenopus retina: morphogenesis and outer segment renewal

1978 ◽  
Vol 201 (1143) ◽  
pp. 149-167 ◽  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Basal Body ◽  
Outer Segment ◽  
Pigment Epithelium ◽  
Ultrastructural Observation ◽  
Rod Outer Segments ◽  
Photoreceptor Outer Segments ◽  
Outer Segments ◽  
Constant Rate

Light microscopic autoradiography and electron microscopy were used to examine outer segment renewal and the development of photoreceptors and pigment epithelium in the larval Xenopus retina. Following the injection of [ 3 H]-leucine at stages 37/38–40 (when outer segments first develop) or 53–54 (when rod outer segments (r. o. s.) attain adult length), a band of label accumulated at the base of r. o. s. and was displaced sclerally with time, whereas label was diffusely distributed in cone outer segments (c. o. s.). By taking into account the change in shape of r. o. s. from conical to cylindrical around stage 46, and calculating outer segment growth (determined from the rate of band displacement) as volume of material added with time, we found a constant rate of membrane addition (1.59 μm/day) from the time of initial outer segment formation. The changes observed in r. o. s. length therefore indicate variations in the rate of disk shedding and phagocytosis, which is minimal before stage 46 and rises to 1.19 μm/day after stages 53–54. Ultrastructural observation showed that although all photoreceptor outer segments form by the repeated evagination of the plasma membrane of the connecting cilium, r. o. s. and c. o. s. are distinguishable by differences in membrane appearance even before they develop divergent membrane topologies. Fibrous granules near the basal body of young receptors may be precursors to the elongating ciliary microtubules. Clusters of cisternae observed near the ciliary base in photoreceptor inner segments may represent a stage in the transport of newly-synthesized opsin to the outer segment base.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF RHODOPSIN IN THE VERTEBRATE RETINA

1974 ◽  
Vol 62 (2) ◽  
pp. 257-273 ◽  
Author(s):  
Lily Yeh Jan ◽  
Jean-Paul Revel
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Sodium Dodecyl ◽  
Ultrastructural Localization ◽  
Rod Outer Segments ◽  
Membrane Particles ◽  
Outer Segments ◽  
Bovine Rhodopsin ◽  
Frog Retina ◽  
Extreme Care

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.

Cyclic AMP has no effect on the generation, recovery, or background adaptation of light responses in functionally intact rod outer segments: With implications about the function of phosducin

2000 ◽  
Vol 17 (6) ◽  
pp. 887-892 ◽  
Author(s):  
HANA JINDROVA ◽  
PETER B. DETWILER
Keyword(s):  
Outer Segment ◽  
Light Exposure ◽  
Cell Voltage ◽  
Light Sensitivity ◽  
Rod Outer Segments ◽  
Light Responses ◽  
Outer Segments ◽  
Sequence Of Events ◽  
Background Adaptation ◽  
Dependent Processes

In retinal rods, light exposure decreases the total outer segment content of both cGMP and cAMP by about 50%. The functional role of the light-evoked change in cAMP is not known. It is postulated to trigger changes in the phosphorylation state of phosducin, a phosphoprotein that is phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and dephosphorylated by basal phosphatase activity when PKA is inhibited by the light-evoked drop in cAMP. In biochemical studies, dephosphorylated phosducin binds to free βγ dimer of transducin (Tβγ) and prevents the regeneration of heterotrimeric transducin by blocking the re-association of the βγ and α subunits. Phosducin's interaction with Tβγ is blocked when it is phosphorylated on a single residue by PKA. To evaluate the effect of the light-evoked fall in cAMP, functionally intact isolated lizard rod outer segments were dialyzed in whole-cell voltage clamp with a standard internal solution and electrical light responses were recorded with and without adding cAMP to the dialysis solution. Since the total outer segment content of cAMP in darkness is ∼5 μM, internal dialysis with solution containing a much higher concentration (100 μM) of cAMP (or 8-bromo-cAMP) will overcome the effects of a light-evoked decrease in its concentration by keeping cAMP-dependent processes fully activated. Neither cyclic nucleotide had any influence on the generation, light sensitivity, recovery, or background adaptation of the flash response. These results also argue against the participation of phosducin in the sequence of events that are responsible for these aspects of rod function. This does not exclude the possibility of phosducin being involved in adaptation caused by higher light levels than used in the present study, that is, bleaching adaptation, or in light-dependent processes other than phototransduction.

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.

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