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Results for “Rod Cell Outer Segment”

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PubMed2004

An outer segment localization signal at the C terminus of the photoreceptor-specific retinol dehydrogenase.

Photoreceptor retinol dehydrogenase (prRDH) is a membrane-associated cytosolic protein that localizes to the outer segments (OS) of rods and cones. Here, we demonstrate that the C-terminal 16 amino acids of prRDH confer membrane association as well as cone and rod OS targeting on a linked green fluorescent protein. Membrane association in transfected 293 cells and in transgenic Xenopus photoreceptors is mediated by fatty acylation at one or more evolutionarily conserved cysteines within the prRDH C-terminal tail. In bovine OS, native prRDH is similarly acylated, and hydrolysis of this linkage releases the protein from the membrane. Efficient OS localization requires both membrane association and the prRDH sequence. (V/I)XPX at the extreme C terminus, which closely resembles the C-terminal sequence that targets opsin/rhodopsin to the OS. Taken together, these data imply that the C-terminal. (V/I)XPX sequence is a general OS localization signal that can function in the context of both integral and peripheral membrane proteins. This strategy for OS localization resembles those used for protein localization to mitochondria, peroxisomes, endosomes, and endoplasmic reticulum; in each case, a short N- or C-terminal sequence is shared among structurally diverse proteins that are targeted to the same subcellular destination.

Alcohol Oxidoreductases↗

PubMed1995

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.

Animals↗

PubMed1994

A newly discovered pathway of melanin formation in cultured retinal pigment epithelium of cattle.

According to a recent hypothesis the melanin granules in the retinal pigment epithelium of mammals originate from photosensory membrane degradation. To test this hypothesis the retinal pigment epithelium of cattle was kept in tissue culture and exposed to gold-labelled rod outer segments. Gold granules were later detected inside phagosomes, melanosomes and mature melanin granules. Tyrosinase, the key enzyme in melanogenesis, was additionally localized inside phagosomes. These results indicate that in cultured retinal pigment epithelium the matrix of the melanosome can originate from phagosomes. Therefore, the melanosome is a specialized lysosome.

Animals↗

PubMed1983

Methylation of proteins in photoreceptor rod outer segments.

A set of proteins in bovine rod outer segments is specifically methylated by S-adenosyl-L-methionine. The reaction can be demonstrated in the intact retina as well as in fragmented preparations of isolated rod outer segments. The apparent molecular weights of these proteins are 88,000, 61,000, and a subset between 21,000 and 26,000. The Mr = 88,000 protein is shown to be the alpha subunit of the rod outer segment cGMP phosphodiesterase by peptide mapping, two-dimensional gel electrophoresis, the ionic strength dependence of its interaction with the membrane, and immunoprecipitation by antiserum raised against purified phosphodiesterase. For each of these proteins, the incorporated methyl groups are hydrolyzed in alkali to yield methanol, indicating that the proteins are carboxymethylated.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1982

1H-NMR relaxation investigation of water bound to bovine rod outer segment disk membranes.

Spin-lattice relaxation times T(1) in deuterated aqueous dispersions of lecithin and rod outer segment disk membranes were measured at various concentrations and temperatures. Fast chemical exchange between two loosely defined phases of water molecules was shown to fit the data, allowing the dynamic features of "bound" water and the hydration of the biological membrane to be evaluated. The state of the water was shown to be also involved in vision physiology.

Animals↗

PubMed1984

The development of photoreceptors in the zebrafish, Brachydanio rerio. I. Structure.

Morphological development of photoreceptors in the central retina of the zebrafish, Brachydanio rerio, was studied by using light and electron microscopic techniques. Outer segments (OS) first appeared at 2.5 days postfertilization (d2.5). On d3, synaptic elaborations were seen. By d8, two OS types were present and were identified as cones. The first indication of rod formation was also evidenced at this time, when vitreally positioned nuclei were observed and rodlike cells were infrequently detected in electron micrographs. At d12, the full complement of zebrafish photoreceptors, rods and four cone types, was identified. From this time on cells grew until adult dimensions were reached at d24. These structural observations are consistent with results of functional studies which utilized physiological and behavioral techniques.

Animals↗

PubMed1987

A-protein catalyzes the ADP-ribosylation of G-protein from cow rod outer segments.

A 20-kilodalton adenosine nucleotide-binding protein (A-protein) extracted from rod outer segments is shown to catalyze the cholera toxin-mediated ADP-ribosylation of GTP-binding protein (G-protein) from the outer segment. Radiolabel from [adenylate-32P] NAD+ was associated specifically with both the alpha-subunit of G-protein and with A-protein in the presence of activated cholera toxin. In the absence of added A-protein, G-protein appears to undergo ADP-ribosylation at a slower rate. In the absence of G-protein, A-protein was found to be labeled following incubation with [adenylate-32P]NAD+ and cholera toxin. In the presence of G-protein, a light-dependent component of A-protein labeling was observed. A-protein is a labile component of rod outer segments and has an affinity for ADP. The findings suggest that A-protein may act as an ADP-ribosyltransferase in the cholera toxin-mediated ADP-ribosylation of G-protein.

ADP Ribose Transferases↗

PubMed1996

Regulation of the kinetics of phosducin phosphorylation in retinal rods.

Phosducin (Pd) is a widely expressed phosphoprotein that regulates G-protein (G) signaling. Unphosphorylated Pd binds to Gbetagamma subunits and blocks their interaction with Galpha. This binding sequesters Gbetagamma and inhibits both receptor-mediated activation of Galpha and direct interactions between Gbetagamma and effector enzymes. When phosphorylated by cAMP-dependent protein kinase, Pd does not affect these functions of Gbetagamma. To further understand the role of Pd in regulating G-protein signaling in retinal rod photoreceptor cells, we have measured the abundance of Pd in rods and examined factors that control the rate of Pd phosphorylation. Pd is expressed at a copy number comparable to that for the rod G-protein, transducin (Gt). The ratio of rhodopsin (Rho) to Pd is 15. 5 +/- 3.5 to 1. The rate of Pd phosphorylation in rod outer segment preparations was dependent on [cAMP]. K1/2 for cAMP was 0.56 +/- 0. 09 microM, and the maximal rate of phosphorylation was approximately 500 pmol PO4 incorporated/min/nmol Rho. In the presence of Gtbetagamma this rate was decreased approximately 50-fold. From these data, one can estimate a t1/2 of approximately 3 min for the rephosphorylation of Pd in rods during the recovery period after a light response. This relatively slow rephosphorylation of the Pd.Gtbetagamma complex may provide a period of molecular memory in which sensitivity to further light stimuli is reduced as a result of sequestration of Gtbetagamma by Pd.

Adaptation, Physiological↗

PubMed1988

Regulation of cyclic GMP binding to retinal rod membranes by calcium.

The apparently cooperative binding of 8-(5-thioacetamidofluorescein)-cGMP (SAF-cGMP) to cGMP-binding sites of the rod outer segments is regulated by Ca2+ in the 0.1-1 microM activity range. High Ca2+ reduces, and low Ca2+ increases the affinity of SAF-cGMP binding. This regulation involves only intrinsic membrane components. It is proposed that an allosteric regulation of cGMP binding by Ca2+ can contribute to photoreceptor potential adaptation.

Animals↗

PubMed2003

The role of subunit assembly in peripherin-2 targeting to rod photoreceptor disk membranes and retinitis pigmentosa.

Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.

Animals↗

PubMed1987

Retinal insulin receptors. 2. Characterization and insulin-induced tyrosine kinase activity in bovine retinal rod outer segments.

Bovine retinal rod outer segments (ROS) possess specific, high-affinity receptors for insulin. These receptors exhibit an insulin-stimulatable tyrosine-specific activity that is capable of phosphorylating the receptor's own beta-subunit and exogenous substrate. ROS insulin receptors exhibit heterogeneity in the apparent molecular weight of the receptor's alpha-subunit. In this regard, insulin receptors from this single cell type resemble insulin receptors obtained from whole retina, but are unlike receptors from brain and liver.

Animals↗

PubMed1991

Adaptive changes in visual cell transduction protein levels: effect of light.

Long-term environmental light-mediated changes in visual cell transduction proteins were studied to assess the influence of rearing environment on their levels and their potential effects on intense light-induced retinal damage. The levels of rhodopsin, S-antigen and the alpha subunit of transducin were measured in whole eye detergent extracts, retinal homogenates or rod outer segments isolated from rats reared in weak cyclic light or darkness, and following a change in rearing environment. Rats changed from weak cyclic light to darkness had 22% more rhodopsin per eye than cyclic-light rats after 12-14 days in the new environment. Western trans-blot analysis of retinal proteins from these dark-maintained animals contained 65% higher levels of immunologically detectable alpha transducin; S-antigen levels were approximately 45% lower than in cyclic-light rats. In rats changed from the dark environment to weak cyclic light, rhodopsin levels decreased by 18% during a comparable period; retinal alpha transducin was 35% lower, S-antigen was 30% higher. At various times after the change in rearing environment, some rats were exposed to intense visible light to determine their susceptibility to retinal damage. Two weeks after an 8-hr exposure, cyclic-light reared rats had rhodopsin levels only 10% lower than control (2.1 nmol per eye). However, rhodopsin was 75% lower when cyclic-light rats were maintained in darkness for 2 weeks before intense light. For animals originally reared in darkness, rhodopsin was 78% lower following 8 hr of intense light, whereas only 30% rhodopsin loss occurred in dark-reared rats after previous maintenance for 2 weeks in weak cyclic-light.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗

PubMed2000

Dietary 20:4n-6 and 22:6n-3 modulates the profile of long- and very-long-chain fatty acids, rhodopsin content, and kinetics in developing photoreceptor cells.

The objective of this study was to determine whether addition of dietary 20:4n-6 and 22:6n-3 to a conventional infant formula fat blend influences membrane long-chain and very-long-chain fatty acid composition, rhodopsin content, and rhodopsin kinetics in developing rat photoreceptor cells. The dietary fats were formulated based on the fat composition of a conventional infant formula providing an 18:2n-6/18:3n-3 ratio of 7:1 (SMA, Wyeth Nutritionals), which served as the control fat blend. This dietary fat blend was modified to contain 20:4n-6 [arachidonic acid (AA)], 22:6n-3 [docosahexaenoic acid (DHA)], AA + DHA, or an 18:2n-6/18:3n-3 ratio of 4:1 (alpha-linolenic acid). Dams were fed diets from birth, and rat pups were fed the same diet after weaning. Retinas and rod outer segments were prepared in the dark from pups at 2, 3, and 6 wk of age for fatty acid analysis of individual phospholipids, rhodopsin content, and rhodopsin disappearance kinetics after light exposure. Feeding AA + DHA in the diet increased 22:6n-3 levels in phosphatidylcholine and phosphatidylethanolamine. In phosphatidylcholine, total n-6 tetraenoic very-long-chain fatty acids and total n-3 pentaenoic and n-3 hexaenoic very-long-chain fatty acids increased after feeding AA and DHA, respectively. Developmental changes were characterized by a decrease in 20:4n-6 in the major phospholipids, whereas 22:6n-3 increased with age in rod outer segments. The highest rhodopsin content occurred in the retina of rats fed diets containing AA and/or DHA. The kinetics of rhodopsin disappearance after light exposure was highest in rats fed DHA at 6 wk of age. This study demonstrates that small manipulations of the dietary level of 20:4n-6 and 22:6n-3 are important determinants of fatty acid composition of membrane lipid and visual pigment content and kinetics in the developing photoreceptor cell.

Animals↗

PubMed1993

A comparison of phagocytosis by the retinal pigment epithelium in normal and delayed amelanotic chickens.

The retinal pigment epithelium (RPE) performs a critical role in maintaining the integrity and renewal of the retinal system by clearing the shed photoreceptor outer segment debris through the process of phagocytosis. If phagocytosis is impaired, accumulation of the debris occurs leading to the degeneration of the retina. Phagocytosis, measured by polyvinyl toluene bead uptake is a normal cell activity exhibited by the embryonic retinal pigment epithelium even before complete differentiation of the neuronal layers. It is significant that embryonic pigment epithelium from either normal or delayed amelanotic (DAM) strain is competent in the process of non-specific particle uptake. In normal chickens non-specific phagocytic activity is observable as early as embryonic day 9 and continues through the rest of the developmental period and throughout adult life. The retinal pigment epithelium in DAM chickens exhibits early phagocytic activity at embryonic day 9 and then through day 16, but by day 19 this function is lost. This loss of phagocytic capacity in RPE of the DAM strain precedes and may be instrumental in the gradual retinal degeneration exhibited by these animals. Specific phagocytosis of rod outer segments (ROS) was examined in 4-week-old post-hatch normal and DAM RPE. Phagocytosis of ROS was greatly reduced in DAM RPE implying that the defect in the DAM is expressed in non-specific and specific phagocytosis. This study indicates that the DAM retinal system can now serve as an alternative and comparative model for studying different aspects of retinal degeneration.

Animals↗

PubMed2003

BETA2/NeuroD1 null mice: a new model for transcription factor-dependent photoreceptor degeneration.

BETA2/NeuroD1 is a basic helix-loop-helix transcription factor that is expressed widely throughout the developing nervous system. Previous studies have shown that BETA2/NeuroD1 influences the fate of retinal cells in culture. To analyze the effect of BETA2/NeuroD1 on the structure and function of the retina, we examined a line of BETA2/NeuroD1 knock-out mice that survives until adulthood. At 2-3 months of age, homozygous null mice showed a 50% reduction in rod-driven electroretinograms (ERGs) and a 65% reduction in cone-driven ERGs. ERGs measured from knock-out mice that were >9 months of age were undetectable. At 2-3 months, the number of photoreceptors in the outer nuclear layer was reduced by 50%. In addition, electron microscopy showed that the surviving photoreceptors had shortened outer segments. The number of cones labeled by peanut agglutinin was decreased 50-60%. By 18 months, retinas from null mice were completely devoid of photoreceptors. There appeared to be few changes in the inner retina, although BETA2/NeuroD1 is expressed in this area. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining revealed a dramatic increase in cell death, peaking at approximately postnatal day 3 and continuing into adulthood. No defects in cell birth were detected using bromodeoxyuridine staining. Our results reveal that BETA2/NeuroD1 not only plays an important role in terminal differentiation of photoreceptors but also serves as a potential survival factor. Loss of BETA2/NeuroD1 results in an age-related degeneration of both rods and cones.

Animals↗

PubMed1994

Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase.

Using recombinant baculovirus vectors, the three subunits of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alpha beta gamma 2) have been expressed in insect cells. The recombinant alpha,beta subunits accumulate to 5 mg/liter culture, but most (98%) of the expressed polypeptides are insoluble. In the soluble fraction, individually expressed alpha and beta subunits showed insignificant PDE activity, but coexpression (by coinfection) of alpha beta subunits elevated PDE activity 7-fold and coexpression of alpha beta gamma up to 15-fold. The soluble expressed holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The Km of the soluble holoenzyme was 11-16 microM both for coexpressed alpha beta subunits and for alpha beta gamma subunits, similar to the Km (6-80 microM) of native PDE. Site-directed mutagenesis of cysteine to serine in the C-terminal CAAX box of both alpha and beta subunits substantially decreased the protein expression level, abolished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, however, showed a cGMP hydrolytic activity comparable with that of the normal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and that isoprenylation of the subunits is essential for membrane association and stability of PDE.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1989

Lipofuscin-like substances accumulate rapidly in brain, retina and internal organs with cysteine protease inhibition.

The protease inhibitor, leupeptin, has been previously shown to cause an accumulation of lysosomally associated intracytoplasmic dense bodies resembling lipofuscin when administered intraventricularly to the brains of young rats (Ivy et al., Science, 226:985, 1984). These findings support the idea that lipofuscin formation during normal aging involves perturbed proteolytic degradation. In the current study, we delineate more precisely the proteolytic mechanisms in question and examine the effects of protease inhibition on other tissues and species. Four sets of experiments were done. In the first, rats were administered leupeptin (L; an inhibitor of cysteine and some serine proteases), E-64C (a cysteine protease inhibitor), chloroquine (C; a lysosomal enzyme inhibitor), aprotinin (A; a serine protease inhibitor) or physiological saline (S) into the lateral ventricle of the brain at a constant rate for two weeks using an osmotic mini-pump. In the second set, beagle dogs were administered L or S intraventricularly in a similar fashion. In the third set, young rats received intraocular injections of L, E64C, C, A or S from 1 to 9 days at 24 hr intervals. In the fourth set, young rats and mice were administered L, E-64C or S intraperitoneally via an osmotic mini-pump for 2 or more weeks. The tissue of all animals was processed for light and electron microscopy as well as for fluorescence analysis. In rat brain cells (especially hippocampus and cerebellum), substances which by morphological, histochemical and fluorescence emission criteria resemble lipofuscin, accumulated following L, C or E64C, but not A or S treatment. A similar buildup of lipofuscin-like substance occurred in canine brain following L but not S infusion. In retinal pigment epithelial cells, undigested rod outer segment discs accumulated after L, C or E-64C and possibly A, but not after S injections. With increasing survival times, the accumulated substances became morphologically more similar to lipofuscin. Cells of liver (both hepatocytes and non-parenchymal cells) and kidney (mainly renal tubule cells) also accumulated substances resembling lipofuscin in response to L but not S infusion; other internal organs were differentially affected. These results demonstrate 1) that specific inhibition of cysteine proteases is sufficient to cause accumulation of lipofuscin-like substances in brain and retina and 2) that several species and organ systems are similarly affected by protease inhibition. Taken together, these findings are consistent with the idea that defective or decreased proteolysis by lysosomal cysteine proteases is responsible for lipofuscin accumulation during normal aging.

Aging↗

PubMed1995

Ultrastructural features of retinal dystrophy in mutant vitiligo mice.

Ultrastructural features of the retinal pigment epithelium and photoreceptor cells were studied in vitiligo (C57BL/6 mivit/mivit) mice. Eyes from 12-day- to 56-week-old animals were analysed. Abnormal photoreceptors were seen in 12-day-old mice. By 3 weeks malformed outer segments were evident in the posterior and equatorial retina, but normal photoreceptors were present in the periphery. By 28 weeks, a marked gradient in cell loss was evident, with a progressive increase in cell viability along the posterior-peripheral axis. Viable intact photoreceptors were still present in the peripheral retina of 56-week-old mice. Melanosome content varied between adjacent pigment epithelium cells in both the posterior and peripheral retina. In the choroid, however, a steep posterior-peripheral gradient in melanosome content was evident with highest pigmentation in the periphery. In the optic nerve head region abnormal development of photoreceptors was correlated with proliferation of abnormal pigment epithelium cells. Accumulation of rod outer segment debris in the posterior and peripheral subretinal space preceded photoreceptor cell death. Short pigment epithelial microvilli without proper attachment to photoreceptors are suggestive of alterations in pigment-epithelium-photoreceptor interaction, which might affect photoreceptor differentiation and phagocytosis of rod outer segments by pigment epithelium cells.

Animals↗