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PubMed2003

In silico characterisation and chromosomal localisation of human RRH (peropsin)--implications for opsin evolution.

BACKGROUND: The vertebrate opsins are proteins which utilise a retinaldehyde chromophore in their photosensory or photoisomerase roles in the visual/irradiance detection cycle. The majority of the opsins, such as rod and cone opsins, have a very highly conserved gene structure suggesting a common lineage. Exceptions to this are RGR-opsin and melanopsin, whose genes have very different intron insertion positions. The gene structure of another opsin, peropsin (retinal pigment epithelium-derived rhodopsin homologue, RRH) is unknown. RESULTS: By in silico analysis of the GenBank database we have determined that the human RRH comprises 7 exons spanning approximately 16.5 kb and is localised to chromosome 4q25 in the following gene sequence: cen-EGF-RRH-IF-qter - a position that excludes this gene as a candidate for the RP29 autosomal recessive retinitis pigmentosa locus. A comparison of opsin gene structures reveals that RRH and RGR share two common intron (introns 1 and 4) insertion positions which may reflect a shared ancestral gene. CONCLUSION: The opsins comprise a diverse group of genes which appear to have arisen from three different lineages. These lineages comprise the "classical opsin superfamily" which includes the rod and cone opsins, pinopsin, VA-opsin, parapinopsin and encephalopsin; the RRH and RGR group; and the melanopsin line. A common lineage for RRH and RGR, together with their sites of expression in the RPE, indicates that peropsin may act as a retinal isomerase.

Amino Acid Sequence↗
PubMed2005

The opsins.

The photosensitive molecule rhodopsin and its relatives consist of a protein moiety - an opsin - and a non-protein moiety - the chromophore retinal. Opsins, which are G-protein-coupled receptors (GPCRs), are found in animals, and more than a thousand have been identified so far. Detailed molecular phylogenetic analyses show that the opsin family is divided into seven subfamilies, which correspond well to functional classifications within the family: the vertebrate visual (transducin-coupled) and non-visual opsin subfamily, the encephalopsin/tmt-opsin subfamily, the Gq-coupled opsin/melanopsin subfamily, the Go-coupled opsin subfamily, the neuropsin subfamily, the peropsin subfamily and the retinal photoisomerase subfamily. The subfamilies diversified before the deuterostomes (including vertebrates) split from the protostomes (most invertebrates), suggesting that a common animal ancestor had multiple opsin genes. Opsins have a seven-transmembrane structure similar to that of other GPCRs, but are distinguished by a lysine residue that is a retinal-binding site in the seventh helix. Accumulated evidence suggests that most opsins act as pigments that activate G proteins in a light-dependent manner in both visual and non-visual systems, whereas a few serve as retinal photoisomerases, generating the chromophore used by other opsins, and some opsins have unknown functions.

Amino Acid Sequence↗
PubMed2005

Circadian rhythms of behavioral cone sensitivity and long wavelength opsin mRNA expression: a correlation study in zebrafish.

Using a behavioral assay based on visually mediated escape responses, we measured long-wavelength-sensitive red cone (LC) sensitivities in zebrafish. In a 24 h period, the zebrafish were least sensitive to red light in the early morning and most sensitive in the late afternoon. To investigate if the fluctuation of behavioral cone sensitivity correlates with opsin gene expression, we measured LC opsin mRNA expression at different times in the day and night under different lighting conditions. Under a normal light-dark cycle, the expression of LC opsin mRNA determined by real-time RT-PCR was low in the early morning and high in the late afternoon, similar to the fluctuation of behavioral cone sensitivity. This rhythm of LC opsin mRNA expression, however, dampened out gradually in constant conditions. After 24 h of constant light (LL), the expression of LC opsin mRNA dropped to levels similar to those determined in the early morning in control animals. By contrast, when the zebrafish were kept in constant dark (DD), the expression of LC opsin mRNA increased, to levels about 30-fold higher than the expression in the early morning in control animals. This day-night fluctuation in LC opsin mRNA expression was correlated to changes in opsin density in the outer segment of cone photoreceptor cells. Microspectrophotometry (MSP) measurements found significant differences in red cone outer segment optical density with a rhythm following the behavioral sensitivity. Furthermore, dopamine modulated the circadian rhythms in expression of LC opsin mRNA. Administration of dopamine increased LC opsin mRNA expression, but only in the early morning.

Animals↗
PubMed2000

Vertebrate ancient-long opsin: a green-sensitive photoreceptive molecule present in zebrafish deep brain and retinal horizontal cells.

Nonretinal/nonpineal photosensitivity has been found in the brain of vertebrates, but the molecular basis for such a "deep brain" photoreception system remains unclear. We conducted an extensive search for brain opsin cDNAs of the zebrafish (Danio rerio), a useful animal model for genetic studies, and we have isolated a partial cDNA clone encoding an ortholog of vertebrate ancient (VA) opsin, the function of which is unknown. Subsequent characterization revealed the occurrence of two kinds of mRNAs encoding putative splicing variants, VA and VA-Long (VAL) opsin, the latter of which is a novel variant of the former. Both opsins shared a common core sequence in the membrane-spanning domains, but VAL-opsin had a C-terminal tail much longer than that of VA-opsin. Functional reconstitution experiments on the recombinant proteins showed that VAL-opsin with bound 11-cis-retinal is a green-sensitive pigment (lambdamax approximately 500 nm), whereas VA-opsin exhibited no photosensitivity even in the presence of 11-cis-retinal. Immunoreactivity specific to this functionally active VAL-opsin was localized at a limited number of cells surrounding the diencephalic ventricle of central thalamus, and these cells were distributed over approximately 200 micrometer along the rostrocaudal axis. Taken together with the previous study on the locus of the teleost brain photosensitivity (von Frisch K, 1911), it is strongly suggested that the VAL-positive cells in the zebrafish brain represent the deep brain photoreceptors. The VAL-specific immunoreactivity was also detected in a subset of non-GABAergic horizontal cells in the zebrafish retina. The existence of VAL-opsin, a new member of the rhodopsin superfamily, in these tissues may indicate its multiple roles in visual and nonvisual photosensory physiology.

Animals↗
PubMed1988

A diurnal rhythm in opsin content of Rana pipiens rod inner segments.

Quantitative electron microscope immunocytochemistry, employing an antibody specific to opsin, was used to evaluate the amount and location of opsin in Rana pipiens rod photoreceptors throughout a 24 hr light/dark cycle. We found a distinct diurnal rhythm in the density of anti-opsin labeling of the rough endoplasmic reticulum (RER) and Golgi apparatus in the myoid region of the rod inner segment. Opsin labeling of these organelles was lowest at light onset, increasing thereafter by three- to four-fold, and remained high until 2 hr into the dark phase. A fall in labeling density occurred within the following 4 hr, and remained low for the remainder of the dark phase. Our finding of a diurnal rhythm regulating inner segment opsin transport in Rana pipiens contrasts with published observations on outer segment membrane turnover, since it has been shown that the rates of disc formation and disc shedding are governed by environmental lighting alone in this species. These results imply that there is opsin pooling in the inner segment during the first 14 hr of a 24 hr light/dark cycle; thereafter the loss of inner segment opsin due to mobilization of this protein from the Golgi exceeds the rate of formation of new opsin. There was no evidence of accumulation of opsin-containing vesicles near the cilium or in the ellipsoid just prior to light onset. At light onset, prominent opsin labeling was identified at the proximal portion of the outer segment in regions separate from the disc stack. In two separate experiments, additional groups of frogs were killed around the time of light onset and were examined by conventional transmission electron microscopy. Disordered disc membranes were seen at the base of the outer segment which were not in register with the disc stack. These disordered membranes were observed as early as 2 hr before light onset, and were no longer observed by 1 hr after light onset. We suggest that these disordered membranes reflect a step in the biogenesis of new discs, serving as a pool of membrane that forms during the later part of the dark cycle. It appears that light onset triggers the ordering of neatly registered discs from this new membrane, rather than assembly of new membrane from pooled transport vesicles in the inner segment.

Animals↗
PubMed1988

A specific ELISA using purified opsin, for studying autoimmunity in retinal diseases.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed to measure nanogram quantities of rhodopsin or its apoprotein, opsin, in bovine retinal rod outer segment (ROS) preparations. Anti-opsin anti-sera could detect as little as 4 ng of purified opsin or of opsin in ROS preparations. The purified opsin was prepared by quantitative elution from a preparative polyacrylamide gel, and showed higher immunoreactivity with anti-opsin than did ROS when the same amount (per weight) of protein was allowed to bind in the wells of the ELISA plates. The effect of the ionic detergent SDS (sodium dodecyl sulphate) on the immunoreactivity and antigen binding to the ELISA wells was studied. Concentrations of 0.1% SDS and above reduced the apparent binding of opsin with anti-opsin when examined by ELISA. This may have been because the negatively charged SDS reduced the efficiency of the antigen coating process, or because changes in the epitopes' conformations made them less recognisable by the corresponding antibodies. A similar ELISA system using a specific anti-S-antigen anti-serum allowed the detection of even very small amounts (nanograms) of S-antigen in ROS preparations. The presence of S-antigen in ROS preparations was confirmed by immunoblotting. Thus purified opsin is preferable to ROS for ELISA tests of autoimmunity to rhodopsin in retinal diseases. These sensitive ELISA techniques could be used to examine the presence of minute amounts of rhodopsin, opsin or S-antigen in different retinal preparations.

Animals↗
PubMed1994

Relationships of G-protein-coupled receptors. A survey with the photoreceptor opsin subfamily.

Relationships among the G-protein-coupled receptors were evaluated using several distance matrices with the neighbor-joining method of Saitou and Nei (1987). The relationships generated vary depending upon alignment, length, or region of sequence compared, and the distance matrix used to score similarity. To provide a statistical level of confidence, bootstrap resampling was applied to the analysis of a selection of G-protein-coupled receptors and the subfamily photoreceptor opsins. A general consensus indicates that the opsins behave as a discrete subfamily among the superfamily of G-protein-coupled receptors. Their relationship to other subfamilies remains unresolved. Within the opsin subfamily, the retinochromelike opsins segregate as a discrete group, but are more closely related to the invertebrate than vertebrate opsins. Among vertebrate opsins, the long wavelength cone opsins, the blue/violet opsins, and the rod opsins (including a class of green cone opsins) form distinct subgroups, but their relationships to one another remain unresolved. For this superfamily of receptors, the confidence levels for many branch pairings are low. The application of methods complimentary to those used in this preliminary study will be necessary to resolve questions about appropriate pairing and evolutionary relationships.

Amino Acid Sequence↗
PubMed1996

The zebrafish ultraviolet cone opsin reported previously is expressed in rods.

PURPOSE: To examine expression of the zebrafish ultraviolet cone opsin pigment in goldfish and zebrafish retinas. METHODS: Digoxigenin-labeled cRNA probes were prepared by run-off transcription from plasmids containing cDNAs for zebrafish ultraviolet opsin, goldfish ultraviolet cone opsin, and goldfish rod opsin. Probes were hybridized to cryosections of retina and visualized with immunocytochemistry. RESULTS: The zebrafish ultraviolet opsin probe hybridized selectively to rod photoreceptors, but not to ultraviolet cones or any other cone type, in both zebrafish and goldfish retinas, and the pattern of expression was identical to that of the goldfish rod opsin probe. The goldfish ultraviolet opsin, in contrast, hybridized to ultraviolet cone photoreceptors in both goldfish and zebrafish. CONCLUSIONS: The cDNA previously identified by Robinson et al as zebrafish ultraviolet opsin is not a cone opsin but is likely to be a rod opsin.

Animals↗
PubMed1996

Light perception in the vertebrate brain: an ultrastructural analysis of opsin- and vasoactive intestinal polypeptide-immunoreactive neurons in iguanid lizards.

Recent biochemical and immunocytochemical evidence indicates that a population of circadian and reproductive rhythm-entraining photoreceptors lies in the basal diencephalon of iguanid lizards. Here, we report the results of correlated light and electron microscopy of opsin-immunoreactive cells in the basal brain, and we discuss their ultrastructural relationship to known photoreceptors. Cerebrospinal fluid (CSF)-contacting bipolar neurons in the lizards Anolis carolinensis and Iguana iguana were immunolabeled with antisera generated against vertebrate retinal opsins and vasoactive intestinal polypeptide (VIP). Within the brain, opsin-immunoreactive cells were found exclusively in the ependyma of the basal region of the lateral ventricles (adjacent to nucleus paraolfactorius/nucleus ventromedialis and neostriatum/paleostriatum). Cells in the same anatomical location and with the same morphology were labeled with anti-VIP antisera. These cells possessed a dendritic process that extended toward the lateral ventricle, ending in a bulbous terminal that protruded into the ventricle. Axonal processes travelled ventrally and caudally. The entire cell, including the axonal process, exhibited opsin-like and VIP-like immunoreactivity. By light microscopy, opsin-like immunostaining appeared punctate, with immunoreactivity greatest in the bulbous terminal. Opsin- and VIP-immunostained thick sections were resectioned, and individual cells observed by light microscopy were then characterized using electron microscopy. We found that all immunostained cells were morphologically similar and that they were morphologically distinct from neighboring nonimmunoreactive cells. CSF-contacting opsin- and VIP-immunoreactive cells lacked the membranous stacks characteristic of retinal photoreceptors but were ciliated and contained numerous large electron-dense vesicles. Multiple synaptic contacts were made on the soma and putative dendritic processes of opsin- and VIP-immunoreactive CSF-contacting neurons. Our results provide the first ultrastructural characterization of opsin-immunostained encephalic CSF-contacting neurons in a vertebrate animal, and they indicate that these putative photoreceptors share structural features with pineal photoreceptors and with certain invertebrate extraretinal photoreceptors, but they are morphologically and biochemically distinct from visual photoreceptors of the retina.

Animals↗
PubMed1996

Positional information and opsin identity in retinal cones.

To test the hypothesis that local environmental cues regulate the expression of middle wavelength-sensitive (MWS) and short wavelength-sensitive (SWS) opsins in cone photoreceptors, we examined the development of the neonatal mouse retina in an organotypic culture system. The segregation of MWS and SWS cones into dorsal and ventral fields in the mouse retina offers an opportunity to isolate a phenotypically homogeneous population of immature cones prior to opsin expression. Retinae were harvested from mice ranging in age from birth (P0) to P18 and maintained in vitro for up to 4 weeks. Cones from newborn mice were first immunoreactive to SWS opsin-specific antibodies (OS-2 and JH455) after 5 days in vitro, which corresponds to a time course similar to that in vivo. The topographic separation of SWS cones into distinct dorsal and ventral fields was also obvious in retinal explants from newborn mice. However, the MWS opsin, identified by polyclonal antibody JH492, was expressed only in vitro when dorsal explants were harvested from P3 or older pups. Despite the absence of MWS opsin expression in newborn retinal cultures, there was no evidence of an increase in the numbers of SWS cones. To test if local diffusable cues could induce immature cones to express an aberrant opsin, dorsal and ventral retinal explants at different stages of maturation were cocultured during the incubation period. Neither the emergence of the cone fields nor the difference in the regional and temporal development of the MWS and SWS opsins was affected in these experiments. These results suggest that positional information in the retina and the opsin identity of cones is determined prior to birth and argue against the hypothesis that postnatal cones can be induced to express an aberrant opsin.

Animals↗
PubMed1997

Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal. Quantitative-competitive PCR analysis of a stable yeast transformant showed that one copy of the opsin gene was integrated into the yeast genome. The expression level in this transformant corresponded to approximately 0.3 mg of opsin per liter of cell culture (A600 = 1.0). Sucrose density sedimentation analysis indicated that the opsin was associated exclusively with the membrane fraction. Similar to retinal opsin, P. pastoris-expressed opsin migrated as a single band of approximately 37 kDa on SDS-PAGE and showed high mannose N-glycosylation. A portion of the expressed opsin (approximately 4-15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (lambda max 500 nm), and after purification showed ground and excited state spectral characteristics indistinguishable from those of the native pigment. Further, the metarhodopsin-II-mediated G-protein-activating potential of yeast expressed rhodopsin was similar to that of native rhodopsin. These results show that P. pastoris cells have the capacity to functionally express bovine opsin.

Alcohol Oxidoreductases↗
PubMed2005

Pteropsin: a vertebrate-like non-visual opsin expressed in the honey bee brain.

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes (AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis, and therefore represents the presence of this vertebrate-like light-detecting system in insects.

Animals↗
PubMed2000

The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.

Mice express S and M opsins that form visual pigments for the detection of light and visual signaling in cones. Here, we show that S opsin transcription is higher than that of M opsin, which supports ultraviolet (UV) sensitivity greater than midwavelength sensitivity. Surprisingly, most cones coexpress both S and M opsins in a common cone cell type throughout the retina. All cones express M opsin, but the levels are graded from dorsal to ventral. The levels of S opsin are relatively constant. However, in the far dorsal retina, S opsin is repressed stochastically, such that some cones express M opsin only. These observations indicate that two different mechanisms control M and S opsin expression. We suggest that a common cone type is patterned across the retinal surface to produce phenotypic cone subtypes.

Animals↗
PubMed1995

Transducin activation by the bovine opsin apoprotein.

The interaction of the bovine opsin apoprotein with transducin in rod outer segment membranes was investigated using a guanyl nucleotide exchange assay. In exhaustive binding experiments, opsin activates transducin, with half-maximal exchange activity occurring at 0.8 mol of opsin/mol of transducin. The opsin activity was light-insensitive, hydroxylamine-resistant, unaffected by stoichiometric concentrations of retinaloxime, and more heat-labile than rhodopsin. The t1/2 of transducin activation in the presence of excess opsin was 8.5 min, compared with 0.7 min for metarhodopsin (II). The second-order rate constants were determined to be 0.012 pmol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) bound per min/nM opsin and 0.35 pmol of GTP gamma S bound per min/nM metarhodopsin (II). Opsin was able to activate more than one transducin, although there appeared to be a turnover-dependent inactivation of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for optimal activity, with no activity in buffers of pH > 9, whereas metarhodopsin (II) exhibited activity at pH > 9. Regulation of opsin activity by stoichiometric amounts of retinal was observed, with inhibition by 11-cis-retinal and stimulation by all-trans-retinal. A model for opsin activity is proposed.

Animals↗
PubMed1996

Mechanisms of opsin activation.

Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu113 and cytoplasmic acidic residues, including Glu134, lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff base-derivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.

Animals↗
PubMed1998

Light-dependent activation of rod transducin by pineal opsin.

The pineal gland expresses a unique member of the opsin family (P-opsin; Max, M., McKinnon, P. J., Seidenman, K. J., Barrett, R. K., Applebury, M. L., Takahashi, J. S., and Margolskee, R. F. (1995) Science 267, 1502-1506) that may play a role in circadian entrainment and photo-regulation of melatonin synthesis. To study the function of this protein, an epitope-tagged P-opsin was stably expressed in an embryonic chicken pineal cell line. When incubated with 11-cis-retinal, a light-sensitive pigment was formed with a lambdamax at 462 +/- 2 nm. P-opsin bleached slowly in the dark (t1/2 = 2 h) in the presence of 50 mM hydroxylamine. Purified P-opsin in dodecyl maltoside activated rod transducin in a light-dependent manner, catalyzing the exchange of more than 300 mol of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate))/mol of P-opsin. The initial rate for activation (75 mol of GTPgammaS bound/mol of P-opsin/min at 7 microM) increased with increasing concentrations of transducin. The addition of egg phosphatidylcholine to P-opsin had little effect on the activation kinetics; however, the intrinsic rate of decay in the absence of transducin was accelerated. These results demonstrate that P-opsin is an efficient catalyst for activation of rod transducin and suggest that the pineal gland may contain a rodlike phototransduction cascade.

Animals↗
PubMed2006

Autosomal recessive retinitis pigmentosa and E150K mutation in the opsin gene.

Retinitis pigmentosa (RP) is a heterogeneous group of hereditary disorders of the retina caused by mutation in genes of the photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Although there are over 100 identified mutations in the opsin gene associated with RP, only a few of them are inherited with the arRP pattern. E150K is the first reported missense mutation associated with arRP. This opsin mutation is located in the second cytoplasmic loop of this G protein-coupled receptor. E150K opsin expressed in HEK293 cells and reconstituted with 11-cis-retinal displayed an absorption spectrum similar to the wild type (WT) counterpart and activated G protein transducin slightly faster than WT receptor. However, the majority of E150K opsin showed a higher apparent molecular mass in SDS-PAGE and was resistant to endoglycosidase H deglycosidase. Instead of being transported to the plasma membrane, E150K opsin is partially colocalized with the cis/medial Golgi compartment markers such as GM130 and Vti1b but not with the trans-Golgi network. In contrast to the endoplasmic reticulum-retained adRP mutant, P23H opsin, Golgi-retained E150K opsin did not influence the proper transport of the WT opsin when coexpressed in HEK293 cells. This result is consistent with the recessive pattern of inheritance of this mutation. Thus, our study reveals a novel molecular mechanism for retinal degeneration that results from deficient export of opsin from the Golgi apparatus.

Cell Line↗
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↗