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PubMed1994

Transport of phosphatidylcholine to Xenopus photoreceptor rod outer segments in the presence of tunicamycin.

Study of the dynamics of membrane protein and phospholipid transport from the inner to the outer segment of vertebrate photoreceptors has shown an interesting dissociation of the two components under a number of experimental treatments which inhibit protein synthesis or transport. Under conditions which block the addition of opsin to outer segments, various lipids continue to be synthesized and transported to the outer segment in the presence of monensin, puromycin, brefeldin A, tunicamycin and several general metabolic inhibitors. In the current study, isolated retinas from adult Xenopus laevis were incubated with or without 20 micrograms mg-1 of tunicamycin in total darkness or light for 2-12 h in the presence of [3H]choline to study the dependence of phosphatidylcholine synthesis and transport on protein transport to the outer segment. Phosphatidylcholine is a major bulk lipid of outer segments, comprising close to one half of the phospholipid of outer segment phospholipids, and blocking choline uptake in retinas is known to cause photoreceptor degeneration. Biochemical analysis demonstrates that tunicamycin does not block the synthesis of phosphatidylcholine in photoreceptor inner segments or transport of radiolabelled phosphatidylcholine to outer segments during 6 h incubations with [3H]choline in light or total darkness. Light and electron microscopic autoradiography and morphometric analysis show that [3H]choline radiolabelled phospholipid does not accumulate in a band of newly formed basal discs in the outer segment or in the tubulo-vesicular structures which accumulate in the intersegmental space of tunicamycin-treated retinas. We conclude that transport of phosphatidylcholine can occur independently of opsin transport to the outer segment but whether this represents two separable components of a single pathway or involves two distinct routes of transport to the outer segment is still unresolved.

Animals↗
PubMed2006

Isolation and characterization of melanopsin and pinopsin expression within photoreceptive sites of reptiles.

Non-mammalian vertebrates have multiple extraocular photoreceptors, mainly localised in the pineal complex and the brain, to mediate irradiance detection. In this study, we report the full-length cDNA cloning of ruin lizard melanopsin and pinopsin. The high level of identity with opsins in both the transmembrane regions, where the chromophore binding site is located, and the intracellular loops, where the G-proteins interact, suggests that both melanopsin and pinopsin should be able to generate a stable photopigment, capable of triggering a transduction cascade mediated by G-proteins. Phylogenetic analysis showed that both opsins are located on the expected branches of the corresponding sequences of ortholog proteins. Subsequently, using RT-PCR and RPA analysis, we verified the expression of ruin lizard melanopsin and pinopsin in directly photosensitive organs, such as the lateral eye, brain, pineal gland and parietal eye. Melanopsin expression was detected in the lateral eye and all major regions of the brain. However, different from the situation in Xenopus and chicken, melanopsin is not expressed in the ruin lizard pineal. Pinopsin mRNA expression was only detected in the pineal complex. As a result of their phylogenetic position and ecology, reptiles provide the circadian field with some of the most interesting models for understanding the evolution of the vertebrate circadian timing system and its response to light. This characterization of melanopsin and pinopsin expression in the ruin lizard will be important for future studies aimed at understanding the molecular basis of circadian light detection in reptiles.

Amino Acid Sequence↗
PubMed2005

Physiological characterization of the compound eye in monarch butterflies with focus on the dorsal rim area.

The spectral, angular and polarization sensitivities of photoreceptors in the compound eye of the monarch butterfly (Danaus plexippus) are examined using electrophysiological methods. Intracellular recordings reveal a spectrally homogenous population of UV receptors with optical axes directed upwards and >or=10 degrees to the contralateral side. Based on optical considerations and on the opsin expression pattern (Sauman et al. 2005), we conclude that these UV receptors belong to the anatomically specialized dorsal rim area (DRA) of the eye. Photoreceptors in the main retina with optical axes <10 degrees contralateral or ipsilateral have maximal sensitivities in the UV (lambda(max)<or=340 nm), the blue (lambda(max)=435 nm) or in the long-wave range (green, lambda(max)=540 nm). The polarization sensitivity (PS) of the UV receptors in the DRA is much higher (PS=9.4) than in the UV cells (PS=2.9) or green cells (PS=2.8) of the main retina. The physiological properties of the photoreceptors in the DRA and in the main retina fit closely with the anatomy and the opsin expression patterns described in these eye regions. The data are discussed in the light of present knowledge about polarized skylight navigation in Lepidopterans.

Animals↗
PubMed1997

Vision in microalgae.

Flagellate green algae such as Chlamydomonas and related genera are guided by their eyes to places where light conditions are optimal for photosynthetic growth. These eyes constitute the simplest and most common visual system found in nature. The eyes contain optics, photoreceptors and the elementary components of a signal-transduction chain. Rhodopsin serves as the photoreceptor, as it does in animal vision. Upon light stimulation, its all-trans-retinal chromophore isomerizes into 13-cis and activates a photoreceptor channel which leads to a rapid Ca2+ influx into the eyespot region. At low light levels, the depolarization activates small flagellar currents which induce in both flagella small but slightly different beating changes resulting in distinct directional changes. In continuous light, Ca2+ fluxes serve as the molecular basis for phototaxis. In response to flashes of higher energy the larger photoreceptor currents trigger a massive Ca2+ influx into the flagella which causes the well-known phobic response. The identification of proteins contributing to this signalling system has just begun with the isolation and cloning of the opsins from Chlamydomonas and Volvox. These plant opsins are highly charged, are not typical seven-helix receptors, and are believed to form a protein complex with the photoreceptor channel. In Spermatozopsis, a G-protein has been found which interacts either directly with the rhodopsin or with the rhodopsin-ion channel complex. By using insertional mutagenesis, genes coding for proteins that are involved in signalling have been tagged. One of them is connected to the flagellar channel and crucial for the flagellar action potential. Elucidation of photoreception in flagellated algae will provide deeper insight into the development of visual systems, starting from single-celled organisms and moving up through higher animals.

Amino Acid Sequence↗
PubMed1984

Target size analysis of rhodopsin in retinal rod disk membranes.

Radiation inactivation of rhodopsin in situ using high-energy electrons gave a value for Mr of 20,200 by spectral assay, but 47,100 by assay of rhodopsin regeneration from opsin and 11-cis-retinal (sequence Mr = 38,840). No light/dark differences were seen. We conclude: (a) radiation inactivation measures the size of the functional unit, and the single hit hypothesis does not hold in our experiments; (b) 500 nm absorbance requires only about half the rhodopsin molecule to be intact, but reconstitution of rhodopsin from opsin requires the whole molecule; (c) we find no evidence for functional interactions between rhodopsin monomers in darkness or light.

Animals↗
PubMed1986

Developmental predetermination of the structural and molecular polarization of photoreceptor cells.

Through mechanisms still unknown, the apparently homogeneous neuroepithelium of the embryonic optic cup differentiates into such divergent cell types as photoreceptors, glia, and various subsets of neurons. Questions that still remain unanswered in this field include the timing and mechanism of action of the "instructive" events directing each neuroepithelial cell to undergo the sequence of phenotypic changes necessary to develop into a specific retinal cell type. This laboratory is investigating some of these questions using cultures in which dissociated neural retina cells, obtained before the onset of overt photoreceptor differentiation, develop at low density in the absence of glia and pigment epithelium. The cultures initially are a morphologically homogeneous population of process-free, round cells. Some cells retain this morphology throughout the first week in vitro, while others develop either as photoreceptors or as multipolar neurons. Photoreceptors elongate and become very asymmetric as they do in vivo, with characteristic compartments orderly arranged along their longitudinal axis (an outer segment-like process, inner segment, cell body, and a characteristically short, single neurite). Cell polarization can also be observed in the distribution of opsin immunoreactive materials and some cytoskeletal elements. Thus, certain precursor cells present in the embryonic retina seem to be programmed to differentiate into photoreceptors even when developing in the absence of contacts with other retinal cells. However, interactions with other constituents of the retina/pigment epithelium complex are probably necessary to ensure final photoreceptor maturation, including further growth of the opsin-rich outer segment process.

Animals↗
PubMed1986

Rod outer segment lipids in vitamin A-adequate and -deficient rats.

Weanling albino rats were fed a vitamin-A-adequate diet or vitamin-A-deficient diet and maintained in a cyclic light or dark environment for up to 14 weeks. One half of the rats were supplemented with additional dietary linolenic acid in the form of linseed oil. The lipid composition and rhodopsin-opsin contents of isolated rod outer segments were determined after 6-7 weeks or 12-14 weeks on diet. This study shows that feeding rats a standard vitamin A-adequate or -deficient diet results in an age-dependent loss of omega three docosahexaenoic acid and a concomitant increase in omega six docosapentanoic acid in the rod outer segments. The loss of docosahexaenoate appears to be caused by insufficient dietary omega three fatty acids. The increase in omega six docosapentanoic acid appears to arise from the high concentration of linoleic acid in standard diets containing either cottonseed, or peanut oil or supplemental corn oil. Feeding rats diets supplemented with linseed oil, however, results in a rod outer-segment lipid profile which is the same as for chow-fed animals. The same effects were seen in the fatty-acid profile of lipids from liver, although the content of polyunsaturates was much lower than in rod outer segments. Vitamin A deficiency, itself, does not lead to changes in the fatty-acid composition of either the rod outer segments or liver. After 6-7 weeks on A+ or A- diet, rhodopsin levels were, as expected, higher in dark-reared rats than in cyclic-light animals. Although the rhodopsin levels in dark-reared vitamin A-adequate rats were significantly higher than in vitamin A-deficient animals, measurements of the lipid to opsin ratio of rod outer segments indicate that the rods of vitamin A-deficient rats are not markedly different than those of vitamin A-adequate rats. It is concluded that these diets may be useful in providing a means for evaluating the role of docosahexaenoic acid in visual cell death from damaging light.

Age Factors↗
PubMed1989

Functional and structural characteristics of photoreceptor cells rescued in RPE-cell grafted retinas of RCS dystrophic rats.

In this study retinas of pink-eyed 26 day-old RCS dystrophic rats were injected with normal, melanotic retinal pigment epithelial (RPE) cells into the subretinal space. The areas containing underlying photoreceptor cells were subsequently investigated by electron microscopy at 60 days and light microscopy immunocytochemistry at 60 and 120 days. Retinal regions containing viable RPE-cell grafts exhibited melanin-contained RPE cell transplants attached to Bruch's membrane and containing shed rod outer segments (ROS). Also, the ROS of these photoreceptor cells exhibited a normal-appearing structural relationship with apical membrane projections of these RPE cells. The debris zone, prominent in retinas of 60 day-old RCS dystrophic rats, was much reduced in RPE-cell grafted regions. By at least 3 months after RPE transplantation, immunostaining for Na+,K+-ATPase was demonstrated along inner segments (IS) of photoreceptor cells in grafted regions, as was also shown in control retinas, but not in non-grafted regions. Opsin immunostaining in RPE-cell grafted regions and in control retinas was detected along ROS and rod inner segments (RIS) and at the periphery of cell bodies in the outer nuclear layer. This study has shown that transplanted normal RPE cells in retinas of RCS dystrophic rats appear to have normal functional and structural characteristics at least three months after transplantation. Furthermore, the rescued PRC's exhibited a normal distribution of and immunostaining density for the membrane-bound enzyme Na+,K+-ATPase and the photo-pigment opsin.

Animals↗
PubMed1990

Monoclonal antibody labels both rod and cone outer segments of turtle photoreceptors.

We have studied the immunoreactivity of turtle photoreceptors to a monoclonal antibody (MAb 15-18) which binds to the external loop connecting bovine rhodopsin helices IV-V. Three chromatic types of cone photoreceptors were identified by the presence and color of oil droplets. MAb 15-18 intensely labeled the outer segments of both rods and green cones. In addition, a weak cross-reactivity was also found in the outer segments of red cones having a pale-green oil droplet, and of blue cones. Other morphological subtypes of red cones, cones with a red oil droplet and both members of double cones, showed no labeling. Our results indicate that rhodopsin and green cone opsin have a similar antigenic determinant, and that two different structural forms of red cone opsin may be present in the turtle retina.

Animals↗
PubMed1991

Enzymatic deglycosylation of bovine rhodopsin.

We have investigated the action of three endo N-acetylglucosaminidases on rhodopsin. The oligosaccharide chains of native and denatured opsin and rhodopsin, both solubilized and membrane-bound, were shown to be cleaved by endohexosaminidase H, endohexosaminidase F, and peptide-N-glycosidase F (PNGase F) as revealed by SDS-PAGE. These enzymes were shown to be free of protease activity. Under correct conditions, the endoglycosidases could release one or both carbohydrate chains. Rhodopsin and opsin at concentrations between 2 and 65 nmol ml-1 were cleaved, with more complete deglycosylation occurring at the higher concentrations.

Amidohydrolases↗
PubMed1991

Levels of mRNA encoding proteins of the cGMP cascade as a function of light environment.

The levels of the retinal mRNAs encoding opsin, the alpha subunit of rod transducin (T alpha), S-antigen (S-ag) and the gamma subunit of rod-specific cGMP-phosphodiesterase (cGMP-PDE) were measured in rats reared in cyclic light or darkness and after adaptation for different periods of time to the opposite light environment. We found that rats changed from cyclic light to darkness, gradually increased their retinal content of opsin and T alpha mRNAs but decreased their levels of S-ag mRNA. The reverse results were obtained when rats were changed from darkness to cyclic light. In contrast, the levels of retinal cGMP-PDE gamma mRNA remained unchanged in animals adapted to either of the two rearing light conditions. Our results indicate that some mRNAs encoding proteins associated with the cGMP cascade are responsive to environmental lighting and may be involved in the long term light or dark adaptive processes.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

RPE conditioned medium stimulates photoreceptor cell survival, neurite outgrowth and differentiation in vitro.

In the present study we have investigated retinal pigment epithelium-photoreceptor cell interactions in vitro, and their contributions to photoreceptor cell survival and differentiation. Preparations enriched for intact photoreceptor cells from neonatal rat retina were grown in either serum-free medium supplemented with RPE-conditioned medium (RPE-CM) or in serum-free medium alone. A variety of substrate conditions were tested for the best neurite outgrowth. Cultures were monitored for 7 days by light and electron microscopy, as well as by opsin, vimentin and carbonic anhydrase-C immunocytochemistry. RPE-CM was found to stimulate both proliferation of flat cells and photoreceptor differentiation. The number of photoreceptors bearing neurites and their neurite length measurements showed significant differences between the RPE-CM group and the control group within 20 hr in culture. Elimination of contaminating flat cells by the addition of an antimitotic drug prevented photoreceptor cell morphological maturation; however, these cells survived as round cell bodies without processes for at least 10 days in the presence of RPE-CM and expressed opsin during this period. Conditioned medium from the flat-cell monolayers did not support photoreceptor differentiation or their survival. However, the presence of flat cells was a requisite to achieve any neurite outgrowth even in the presence of RPE-CM. In the absence of RPE-CM, neither photoreceptors nor flat cells survived or proliferated. Heat and trypsin treatment of the RPE-CM abolished all its growth-supporting activities which indicates its proteinaceous nature. This represents the first time in vitro that an RPE-derived factor(s) has been shown to be responsible for photoreceptor cell survival and differentiation.

Animals↗
PubMed1994

Sequence divergence, polymorphism and evolution of the middle-wave and long-wave visual pigment genes of great apes and Old World monkeys.

In man, the spectral shift between the middle-wave (MW) and long-wave (LW) visual pigments is largely achieved by amino acid substitution at two codons, both located in exon 5. A third amino acid site coded by exon 3 is polymorphic between pigments. We have studied the equivalent regions of the cone opsin genes in two members of the Hominidea (the gorilla, Gorilla gorilla and the chimpanzee, Pan troglodytes) and in three members of the Cercopithecoidea family of Old World primates (the diana monkey, Cercopithecus diana, the talapoin monkey, Miopithecus talapoin, and the crab-eating macaque, Macaca fascicularis). No variation in the codons that specify the amino acids involved in spectral tuning were found. We predict therefore that the MW and LW pigments of gorilla and chimpanzee have similar spectral characteristics to those of man. Multiple copies of the same opsin gene sequence were identified in the chimpanzee, talapoin and macaque and we also show that non-human Old World primates are similar to man in showing a bunching of polymorphic sites in exon 3. We discuss the ancestry of the separate MW and LW genes of Old World primates and the equivalent polymorphic gene of the marmoset, a New World primate.

Amino Acid Sequence↗
PubMed1983

Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin.

We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene. Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene. The mRNA homologous sequences in the 6.4 kb gene consist of a 96 bp 5' untranslated region, a 1044 bp coding region, and a surprisingly long approximately 1400 bp 3' untranslated region, and are divided into five exons by four introns that interrupt the coding region. Secondary structure analysis predicts that the bovine rhodopsin chain, like that of bacteriorhodopsin, contains seven transmembrane segments. Interestingly, three of the four introns are immediately distal to the codons for three of these segments, and one of these introns marks the boundary between the C-terminal domain and a transmembrane domain.

Amino Acid Sequence↗
PubMed1992

Constitutively active mutants of rhodopsin.

Two critical amino acids in the visual pigment rhodopsin are Lys-296, the site of attachment of retinal to the protein through a protonated Schiff base linkage, and Glu-113, the Schiff base counterion. Mutation of Lys-296 or Glu-113 results in constitutive activation of opsin, as assayed by its ability to activate transducin in the absence of added chromophore. We conclude that opsin is constrained to an inactive conformation by a salt bridge between Lys-296 and Glu-113. Recently, one of the mutants, K296E, was found in a family with retinitis pigmentosa, suggesting that degeneration of the photoreceptor cells in individuals with this mutation may result from persistent stimulation of the phototransduction pathway.

Amino Acid Sequence↗
PubMed1990

Norepinephrine suppresses both photoreceptor and neuron-like properties expressed by cultured rat pineal glands.

Our previous study showed that pineal glands of the newborn rat express properties of both photoreceptors and endocrine cells, when transferred to cell culture conditions, whereas opsin was not detected in cells of the intact pineal glands of the postnatally developing or mature rats (Araki et al., 1988). In the present study we asked a question why photoreceptor-like cells do not differentiate in vivo, although the pineal cells have the potency as revealed in cell culture conditions. Our hypothesis was that norepinephrine (NE) may suppress the differentiation of cells with photoreceptor properties. This was proved by adding NE into the culture medium, and only few opsin-like immunoreactive cells, if any, were differentiated in the presence of 10(-6) M NE. NE also suppresses the extension of neurite-like processes by serotonin-like immunoreactive (endocrine) cells, another property expressed in cultures. Other aminergic neurotransmitters, such as dopamine and serotonin, did not have such effects. These results imply that the massive innervations of the pineal gland by the sympathetic ganglion may suppress both photoreceptor and neuron-like properties of the mammalian pineal gland in vivo.

Animals↗
PubMed1991

Spectral sensitivity, structure and activation of eukaryotic rhodopsins: activation spectroscopy of rhodopsin analogs in Chlamydomonas.

Retinal normally binds opsin forming the chromophore of the visual pigment, rhodopsin. In this investigation synthetic analogs were bound by the opsin of living cells of the alga Chlamydomonas reinhardtii; the effect was assayed by phototaxis to give an activation spectrum for each rhodopsin analog. The results show the influence of different chromophores and the protein on the absorption of light. The maxima of the phototaxis action spectra shifted systematically with the number of double bonds conjugated with the imine (C = N+H) bond of the chromophore. Chromophores lacking a beta-ionone ring, methyl groups and all C = C double bonds photoactivated the rhodopsin of Chlamydomonas with normal efficiency. On the basis of a simple model involving one-electron transitions between occupied and virtual molecular orbitals, we estimate the charge distribution along the chromophore in the binding site. With this restraint we define a unique structural model for eukaryotic rhodopsins and explain the spectral clustering of pigments, the spectral differences between red and green rhodopsins and the molecular basis of color blindness. Our results are consistent with the triggering of the activation of rhodopsin by the light-mediated change in electric dipole moment rather than the steric cis-trans isomerization of the chromophore.

Chlamydomonas↗
PubMed2005

Patterns of cell proliferation and cell death in the developing retina and optic tectum of the brown trout.

We have analyzed the patterns of cell proliferation and cell death in the retina and optic tectum of the brown trout (Salmo trutta fario) throughout embryonic and postembryonic stages. Cell proliferation was detected by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), and apoptosis by means of the TUNEL method. Haematoxylin and DAPI staining were also used to demonstrate apoptotic cells. Photoreceptor cell differentiation was assessed by immunohistochemistry with antibodies against opsins. Throughout embryonic development, PCNA-immunoreactive (PCNA-ir) cells become progressively restricted to the peripheral growth zone of the retina, which appears to be the principal source of new retinal cells from late embryos to adults. However, some PCNA-ir cells are observed secondarily in the differentiated retina, first in the inner nuclear layer of 15-mm alevins and later in the outer nuclear layer of 16-mm alevins, after differentiation of the first rods in the central retina, as demonstrated with opsin immunocytochemistry. Our observations also support the view that the PCNA-ir cells observed secondarily in the INL of the central retina of alevins are photoreceptor precursors. The number and distribution of apoptotic cells in the retina and optic tectum of the trout change throughout development, allowing distinction of several waves of apoptosis. Cell death is detected in proliferating areas at early stages, then in postmitotic or differentiating areas, and later concurring temporal and spatially with the establishment of visual circuits, thus indicating a relationship between apoptosis and proliferation, differentiation and synaptogenesis.

Animals↗