A Diurnal Rhythm in Opsin Content of RQDQ Pipiens Rod Inner Segments Alon C

A Diurnal Rhythm in Opsin Content of RQDQ pipiens Rod Inner Segments Alon C. Bird,* John G. Flannery.f ond Dean Bokft

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. Invest Ophthalmol Vis Sci 29:1028-1039, 1988

Two different mechanisms have been demon- amined in two amphibian species. Opsin biosynthesis strated to regulate disc shedding from rod outer seg- was reported to be constant throughout the light/dark ments in vertebrates. Both of these mechanisms are cycle in Xenopus laevis eyecups maintained in vitro cyclic, being regulated by light, or an intrinsic circa- by Hollyfield et al.1819 By contrast, opsin synthesis dian rhythm.1"" The assembly of new discs at the was found to vary in Rana pipiens by Matsumoto and base of the outer segment12"16 has been shown to Bok.20 In the latter study, the specific activity of occur with similar periodic characteristics.17"18 newly synthesized protein changed by as much as The cyclic regulation of biosynthesis of opsin, the 13-fold, although changes in outer segment opsin- major disc membrane intrinsic protein, has been ex- specific activity suggested only a two-fold increase in synthesis. The disparity in results between R. pipiens and X. From the *Institute of Ophthalmology, Moorfields Eye Hospital, laevis may reflect major differences in the regulatory London, England, and the jJules Stein Eye Institute and tDepart- mechanisms controlling opsin metabolism in these ment of Anatomy, UCLA School of Medicine, Los Angeles, Cali- two species. Alternatively, the changes in nascent fornia. opsin-specific activity in R. pipiens could be attrib- Supported by National Eye Institute (Bethesda, Maryland) uted to fluctuations in either opsin biosynthesis or the grants EY-00444 and EY-00331 to DB and EY-05624 to JGF. ACB was a Visiting International Scholar at UCLA, in receipt of a pool size of inner segment opsin during the diurnal grant from Fight for Sight, London. DB is the Dolly Green Profes- cycle. Enhanced levels of messenger RNA controlling sor of Ophthalmology at UCLA and a Research to Prevent Blind- opsin synthesis following light onset have been re- ness, Inc. Senior Scientific Investigator. ported recently in fish and toads.21 Taken together Submitted for publication: October 6, 1987; accepted January 8, with the observed variation in the specific activity of 1988. 20 Reprint requests: Dean Bok, Jules Stein Eye Institute, UCLA outer segment opsin during the diurnal cycle, this School of Medicine, Los Angeles, CA 90024. suggests strongly that part of the change in nascent

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opsin-specific activity is due to fluctuations in synthe- In all experiments, the enucleation and initial pro- sis. The results would also be more compatible if cessing were undertaken in deep red safelight condi- there were large diurnal changes in the opsin pool in tions when frogs were killed during the dark phase. the inner segments of R. pipiens at certain times in the light/dark cycle but not in X. laevis. The possibil- Preparation of Affinity-Purified Rabbit Antibodies ity of inner segment opsin pooling in R. pipiens has to Frog Opsin been suggested,20 but has not been examined to date. Rod outer segments were isolated from 40 fresh In order to test this hypothesis, we used electron mi- frog eyes according to the method of Papermaster croscope immunocytochemical localization to quan- and Dreyer.22 Opsin was separated from other pro- titate the synthesis and delivery of opsin from the teins on a 4 mm thick sodium dodecyl sulfate poly- rough endoplasmic reticulum (RER) to the outer acrylamide gel (SDS-PAGE) according to the method segment. of Laemmli,23 and the position of the opsin band The results of this initial study caused us to under- determined by scanning the gel at 280 nm. The re- take a second series of observations on the relation- gion corresponding to opsin was excised from the gel, ship of outer segment membrane assembly to disc and the protein was extracted and further purified by production around the time of light onset. hydroxyl apatite chromatbgraphy according to previously published methods.24 Subsequent SDS- Materials and Methods PAGE analysis of the extracted opsin and silver Experimental Animals For Immunocytochemical staining of the gel showed no contaminants other Analysis and Standard Electron Microscopy than opsin multimers. One hundred micrograms of purified opsin in complete Freund's adjuvant was in- Two separate and complete groups of animals were jected intradermally into each of two rabbits on a prepared for immunocytochemical examination. In weekly basis for 6 weeks. Animals were bled from the each experiment, adult R. pipiens were entrained to a marginal ear vein on the fifth, sixth and seventh 12 hr light/12 hr dark cycle for a 3 week period, with weeks. The presence and specificity of opsin antibod- light onset occurring at 7 AM. The frogs were main- ies were determined by labeled antibody staining of tained under cyclic illumination of 32 foot-candles nitrocellulose replicas (Western blots) obtained by provided by incandescent bulbs, and were kept at a electrophoretic transfer of solubilized outer segment constant temperature of 20°C. The exact time of light proteins from SDS gels.25 onset and offset were confirmed by a separate photo- cell and chart recorder. The frogs were fed live meal- Staining of outer segment proteins on Western worms weekly, and were treated in accordance with blots was limited to opsin and its multimers. Specific the ARVO Resolution on the Use of Animals in Re- anti-opsin IgG was affinity-purified from immune search. serum by coupling purified bovine opsin to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine In the first experiment, one frog was killed at each Chemicals, Piscataway, NJ) following instructions of six time points during the light dark cycle: 7 AM, provided by the manufacturer, except that NaCl was 7:30 AM, 8:30 AM, 11 AM, 3 PM and 9 PM, which are 0, 0.5, 1.5, 4, 8, and 14 hr after the onset of light, omitted from the buffer that was used to bind the respectively. One eye of each frog was processed for antigen. After the serum sample was loaded onto the electron microscopic immunocytochemistry. affinity column and washed to remove unbound proteins, specific IgG was eluted with 4 M MgC^ in In the second experiment, two frogs were killed at 7 phosphate-buffered saline (PBS; 10 mM sodium AM, one at 7:30 AM, two at 8:30 AM, one at 11 AM, phosphate in 0.9% NaCl). One milliliter fractions one at 3 PM, one at 9 PM, two at 11:30 PM, and two were eluted into 5 ml volumes of PBS in order to at 2:30 PM, which are 0, 0.5, 1.5, 4, 8, 14, 17, 20, hr achieve rapid dilution of the MgCl . The specific IgG after light onset, respectively. In this experiment both 2 eyes were used from each animal. was dialyzed against PBS and concentrated prior to use. In the final series of observations, two groups of frogs were entrained in an identical manner. In the first group two frogs were killed at 2 and 1 hr before, Tissue Preparation and Immunocytochemistry and 0.5 and 1.5 hr after light onset. In the second, one At the times specified, frogs were decapitated, the animal was killed at each of the following time points: eyes were enucleated and opened, and the tissue was 2 hr and 1 hr before light onset, at light onset and 1 fixed in 2.5% glutaraldehyde/2% formaldehyde for 2 and 2 hr after light onset. Both eyes of each animal hr, and transferred to 2% formaldehyde for 4 days to were processed for standard transmission electron enhance antigenicity before standard infiltration and microscopy. embedding in LR White epoxy resin (Polysciences,

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Fig. 1. Electron micrographs of longitudinal sections of Rana rod photoreceptor inner segments labeled with rabbit antibody to frog opsin from a frog sacrificed at light onset (a), and 14 hr into the light/dark cycle (b). The density of labeling of Golgi apparatus (arrows) and rough endoplasmic reticulum is much smaller at light onset than at 14 hr. The inner segments are short at light onset. They become progressively longer and narrower during the following 14 hr due to photo- mechanical movement, after which time they return to the light onset form XI 2,500).

Inc., Warrington, PA). Polymerization of LR White copper grids, and incubated on drops of 0.1 M Na was performed at 60°C for 12 hr. Ultrathin sections phosphate (pH 7.4) containing 5% bovine serum al- were mounted on carbon-stabilized form var-coa ted bumin (BSA) for 5 min to block nonspecific binding.

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Grids were then labeled for 1 hr using affinity-purified rabbit antibody to frog opsin diluted to 0.1 o.d. in the first experiment, and 0.06 o.d. in the second (A28o, 1 cm light path) in 0.1 M Na phosphate containing 0.1% BSA. Grids were washed and transferred for an additional hour to 20 mM Tris buffered saline, (pH 8.2) containing goat anti-rabbit IgG absorbed to 10 nm gold colloid (Janssen Pharmaceuticals, Beerse, Belgium), 0.1% BSA and 2 X 10"2 M Na azide. Lastly, grids were contrasted with 5% aqueous uranyl acetate. Grids from all sacrifice times were labeled

simultaneously in each experiment. 8 12 16 Ultrathin sections were examined with a JEOL Time (hour s > 100C electron microscope (JEOL Inc., Tokyo, Japan) and micrographs were printed to a final magnifica- tion of XI 5,000. A minimum of two 1 mm2 areas of retina were examined for each sacrifice time. The

Time (hours ) Fig. 3. Plot of the mean density of anti-opsin labeling over the area of the Golgi apparatus throughout a 24 hr cycle in the first (a) and second (b) experiments. In both series the highest level of density was recorded at 14 hr after light onset and the lowest at light 8 12 16 onset. A series of t-tests at the a = 0.01 level of significance con- T ime (hours ) firmed that there are discrete shifts represented in both experiments: 0, 0.5, 1.5 hr are labeled at an equivalent density, there is a significant rise in density from 1.5 to 4 hr, and a fall in density after 14 hr (14-24 hr in the 1st experiment and 14-17 in the second).

appropriate rod inner segment regions were identified on electron micrographs, their areas measured using a digital planimeter (Laboratory Computer Systems, Cambridge, MA) and the gold particles counted. The colloidal gold particle density over specific organelles was derived from these measurements. Grating rep- lica grids (Ernest Fullam, Inc., Schenectady, NY) were photographed, printed and measured identically 0 4 8 12 16 20 24 ** T ime ( hour s ) in order to calibrate the area measurements. Quantitative data were analyzed using a VAX Fig. 2. Plot of the mean density of anti-opsin labeling of RER in the first (a) and second (b) experiments. The light onset was at time 11/780 computer (Digital Equipment Corp., Mayn- 0, and offset at 12 hr. The 24 hr datum points are duplicates of the ard, MA) with the SAS (Statistical Analysis System) time 0 points. In both experiments the highest level was recorded at and SAS/Graph software packages (SAS Institute, 14 hr into the light/dark cycle, and the lowest at light onset. The Cary, NC). The labeling densities were compared by major rise occurred during the period 1.5 to 4 hr in the first experi- pairwise t-tests between each of the sample times. ment and at 8 to 14 hr in the second. The major fall in both was Errors between each sample time were minimized during the period after 14 hr in both series. These changes in den- 26 27 28 sity were significant at the 0.01 level. Error bars represent ±2 stan- using K-ratio t-test ' and the multiple range test dard deviations from the mean in all figures. for analysis of variance to the a = 0.01 stringency.

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Fig. 4. Electron micrograph of a longitudinal section of the ciliary region from a frog fixed 14 hr after light onset (a) and at light onset (b). Vesictes labeled with rabbit antibody to frog opsin antibody (arrows) are observed at both timepoints (X21,000).

Tissue Preparation For Ultrastructural Examination course of the light/dark cycle (Fig. 2), being lowest at In groups of frogs for standard ultrastructural ex- the time of light onset and rising rapidly soon thereaf- amination the eyes were fixed in 2% formaldehyde ter to high levels at 4 hr. Highest levels were reached and 2.5% glutaraldehyde in 0.1 M Na phosphate at 14 hr into the L/D cycle before falling again. The exact timing of the downturn in labeling density was buffer at pH 7.4 for 2 hr, post-fixed in 4% OsO4, dehydrated in a graded series of ethanol, infiltrated in not resolved in the first experiment due to our choice propylene oxide and embedded in Araldite 502 epoxy of sacrifice times (Fig. 2a). In the second experiment resin (Ciba Products Co., Summit, NJ). At each time we were able to make an informed choice of sampling point the number of rods with disordered basal times, and the data indicate that the reduction in membrane and the number of phagosomes/rod was labeling occurs early in the dark phase of the light/ counted. dark cycle (Fig. 2b). Labeling of the Golgi Apparatus Results The Golgi apparatus appeared as a longitudinal la- Immunocytochemical Studies beled membrane system in the myoid region at those The appearance of the inner segments changed time points at which the inner segments were elon- during the light/dark cycle. At light onset the inner gated (4 to 17 hr into the cycle) (Fig. lb). At times segment was short and broad (Fig. la), and became when the inner segments were short and squat, part of more elongated as the light cycle progressed (Fig. lb). the Golgi was identified as a transverse structure par- Return to the light onset form occurred progressively allel to the border of the nucleus (Fig. la). The Golgi as the dark phase progressed. These changes were ac- was most heavily labeled 14 hr into the L/D cycle in companied by movement of pigment granules into both experiments, and thereafter the density of label epithelial processes after light onset, and their with- fell. The major fall was between 14 hr and light onset drawal into the cell body after light offset. This acted in the first experiment (Fig. 3a), and between 14 and as an internal control for the light and dark adapted 17 hr in the second (Fig. 3b). The lowest levels of state. density were at light onset, and the main rise at 1.5 to 4hr. Labeling of the Rough Endoplasmic Reticulum (RER) Labeling of the Ellipsoid Region In both groups of Rana, we found the density of Our observation of a rapid reduction in labeling labeling over the RER to vary significantly over the density in the second half of the light/dark cycle for

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both Golgi and RER led us to search for the new locus of the mobilized protein. It was considered that the protein might be found in high concentration in opsin transport vesicles in the ellipsoid region, partic- ularly near the cilium.29 Labeled vesicles were found in this region at all times in the cycle (Fig. 4a, b), but . . ; jj were most numerous at the time of high labeling . • -A5'.; concentration in the Golgi rather than corresponding ' , . with the fall in Golgi labeling density. The labeled A vesicles appeared to be restricted to a specific linear ' \— A pathway rather than being evenly distributed with the • E ellipsoid (Fig. 5). 9 We measured antibody density over the ellipsoid in order to quantitate this observation (Fig. 6). In both groups of frogs these results showed highest labeling " \ at 14 hr, which declined thereafter, being lowest at light onset, before rising again.

Labeling of the Outer Segment Membrane A novel phenomenon was observed concerning receptor membrane at the base of the outer segment at the time of light onset in both experiments. Linear membrane-associated labeling was observed which was separate from the registered disc stack (Fig. 7). In some rods this membrane appeared to form pouches which protruded away from the long axis of the outer segment, and in others it appeared to be invaginated into the inner segment. This membrane configura- tion was seen in a more restricted form 30 min after light onset, but not thereafter.

Standard Electron Microscopy In order to investigate the significance of this ectopic labeled membrane, two additional groups of frogs were killed around light onset and examined by standard transmission electron microscopy. In some *f rods, apparently redundant disc membrane was seen at the proximal outer segment which was parallel to, and separated from, the outer segment in some receptors, while in others the membrane appeared to be invaginated into the distal portion of the inner segment (Fig. 8). The number of receptors with ectopic membrane was counted around the time of light onset, and phagosomes were counted as an internal control (Fig. 9). The number of receptors with ectopic

Fig. 5. Electron micrograph of the longitudinal section of a receptor from the time point 4 hr into the light phase labeled with rabbit antibody to frog opsin. The Golgi is labeled (solid arrows), and a track of labeled vesicles (open arrow) extends from the myoid to the cilium (X 17,500).

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of the amount of opsin in individual organelles, since the techniques available do not allow an accurate measure of the absolute number of antigens in a given area of a section. Estimates by Griffiths and Hop- peler30 suggest that the immunogold labeling effi- ciency over Lowicryl sections of RER and Golgi was 18.4% and 6.6%, respectively. As suggested by Grif- fiths and Hoppeler, this effect may be due to a difference in affinity of the antibody for the antigen in the different organelles or a differential effect of fixation on the two organelles. However, this does not invali- 8 12 16 date measurements of the relative density of an anti- Time (hour s ) gen in the same organelle at different time points.

Comparison of Autoradiographic and Immunocytochemical Results in R. pipiens The observation that the opsin pool in the RER and Golgi apparatus was small in the early stages of the light/dark cycle is compatible with predictions derived from the results of Matsumoto and Bok.20 These investigators observed a 13-fold increase in inner segment opsin specific activity following light onset. A small opsin pool at light onset which in- creases with time would have caused the specific ac- D.0 4 6 12 16 20 24 tivity to be high at early points in the cycle, and Time (hour s) would account for a fall thereafter, even if incorpora- Fig. 6. Plot of the ellipsoid anti-opsin labeling density in the first tion of labeled amino acids into opsin were constant. (a) and second (b) experiments. In both experiments, t-tests con- Our conclusions agree qualitatively with those of firm a single significant increment in labeling during the light phase (between 1.5 and 4 hr in the first, and 8 and 14 hr in the second Matsumoto and Bok in reference to changes in the experiments), and decrement after 14 hr. concentration of opsin in the inner segment. In their experiments the change in specific activity during the course of the diurnal cycle was 13-fold, whereas our disc membrane was high just before light onset and measurements only showed changes of three- to fell rapidly soon thereafter. In the first experiment the four-fold. The quantitative disparity may reflect dif- percentage of rods with dysmorphic membranes ferences in techniques. It may not be justified to reached 67 at 0.5 hr prior to light onset, and in the compare immunocytochemical techniques, which second was 40 at light onset. The abnormal mem- analyze only the surface of the tissue (a two-dimen- brane complexes were larger as well as more numer- sional display), with a technique which allows access ous as the time of light onset approached. As ex- to three dimensions, such as autoradiography or pected, the number of phagosomes was negligible 2 hr chemical extraction. before light onset and high soon afterwards. How- Other factors may be relevant. At the hours when ever, in both experiments the number of phagosomes the tissue was only lightly labeled by the antibody in rose perceptibly before light onset. our study, it was more difficult to define the limits of the Golgi apparatus and it is likely that some unla- Discussion belled Golgi was not recognized in these sections. The absence of this unlabelled area would decrease the Changes in Opsin Concentration measured area in comparison with the true area and cause a falsely high calculated density. Overestima- Our results indicate that regular changes in the tion of the density at the low points in the L/D cycle concentration of opsin in the RER and Golgi appa- would reduce the identified difference between the ratus occur during the 24 hr light/dark cycle. Our two observed minimum and maximum densities. For this experiments are in close agreement on this principle. reason it is possible that our results understate the We were not able to make a quantitative estimate magnitude of variation in the quantity of opsin stored

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*'« k~ •*••

Fig. 7. Electron micrographs of longitudinal sections of the basal region of outer segments at light onset labeled with rabbit anti-frog opsin. Some show linear la- )0 -• beling of the plasma membrane (arrows) separated .^ ',' from the label on the disc (Jb) •' ' stack by a clear space (a-c); in one instance the bal- looned plasma membrane of a neighboring receptor appeared in the section (b, open arrow). The redundant labeled membrane appeared to be invaginated into the distal inner segment in some receptors (d) (X 13,500).

in the Golgi apparatus throughout the light/dark the previously reported low specific activity occurring cycle. at this time, could only be explained by a lower rate of It is only at the times later than 14 hr into the light incorporation of protein into the inner segment dur- cycle that our results do not agree with the predicted ing the dark period. The density of label in the RER behavior of the opsin pool in the Golgi apparatus. We may give some indication of the rate of opsin forma- did not expect the level of opsin in the Golgi and tion, although it is clear that this density will repre- RER to fall until much later in the light/dark cycle, sent a balance between rates of formation and mobi- perhaps close to the time of light onset. The reduction lization. If the marked lowering of RER labeling ob- of opsin pool size identified in this experiment, and served after 14 hr implies a reduction in opsin

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.?•

•

*.«r *

Fig. 8. Electron micrographs of longitudinal sections of the junction of inner and outer segment of osmicated, Araldite-embedded tissue at light onset. Ectopic membrane appears to be invaginating the inner segment (a, c, d) or parallel to the outer segment disc stack (b) (X21,000).

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synthesis, this would help explain the dilemma. It is also consistent with lower levels of opsin messenger RNA reported recently for toads and fish.21

Comparison With Autoradiographic Results in X. laevis Our results support the primary conclusion of Holly field and coworkers18 that the increased rate of membrane assembly at light onset is not dependent upon an enhanced rate of opsin synthesis. There is, -15 -10 -0.5 0.0 0.5 however, some conflict with the conclusion that pro- Hour s after light onse1 tein synthesis is constant during the 12 hr light/dark cycle.19 The results of our study together with the specific activity measurements of Matsumoto and Bok21 imply that the rate of opsin incorporation in the inner segment varies over the course of the light/ dark cycle in R. pipiens. A plausible explanation for the different results is that there is a major difference in opsin metabolism between R. pipiens, on which our observations were made, and X. laevis with which Hollyfield et al,19 worked. These two species differ in their regulatory

mechanisms of outer segment shedding and mem- -2.0 -1.5 -to -0.5 0.0 brane synthesis; in R. pipiens it is believed to be gov- D Hours alter light onset erned by light alone,29 whereas in X. laevis there is a 310 circle • evaginalions / 100 rods well established intrinsic circadian rhythm. square • phagosomes / 100 rods However, a major difference exists between the Fig. 9. Plot of the percentage of rods showing ectopic outer seg- studies in that much of the work on X. laevis was ment membranes, and the ratio of phagosomes to rods expressed as undertaken in vitro. The in vitro situation differed a percentage during the time around light onset in the first (a) and from the in vivo studies in that the 3H-labeled pre- second (b) experiments. The number of receptors showing disor- cursors added to the culture medium were contin- dered membrane is high before and at light onset, and falls soon thereafter. The ratio of rods to phagosomes is low before light onset ually available for protein synthesis throughout the and rises progressively. incubation. This is in contrast to the "pulse" of 3H- precursor which occurs when the 3H-label is injected into whole animal. It is also possible that there is some damping of the intrinsic circadian characteris- were unable to demonstrate any such accumulation. tics in vitro. In addition, examination of the ellipsoid region of the That prolonged alteration of lighting conditions rod at the time when the density of labeling in the may affect the rate of opsin synthesis is not in dispute; Golgi was decreasing did not show any increase of several days of prolonged darkness have been shown labeled material. The presence of labeled vesicles at to cause reduced opsin synthesis in X. laevis,X9 and all sample times suggests that movement of opsin to light may enhance opsin incorporation after pro- the outer segment is continuous, although the rate longed darkness in R. pipiens.3] may vary during the light/dark cycle. The path of delivery through the myoid appears to be along well The Destination of the Mobilized Opsin defined channels. It seems credible to us that opsin, once transported We sought the site of opsin pooling immediately to the ciliary region, becomes incorporated into outer prior to light onset. There is evidence that opsin is segment membrane some hours prior to formation of transported in vesicles from the Golgi, and that those completely ordered discs. Support for this concept vesicles fuse with the apical inner segment plasma was given by our observation of ectopic and misor- membrane prior to being incorporated in the outer ientated plasma membrane associated with the prox- segment.29 We therefore expected that the apical imal portion of the outer segment, which was seen inner segment would be a likely pooling site for opsin. only in retinas fixed just prior to light onset or slightly We searched for collections of labeled vesicles near afterward. The appearance of this region in both im- the cilium during the later part of the dark phase, but munocytochemical and conventional electron mi-

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croscopy implies the presence of "redundant" mem-. disc morphogenesis. Nonetheless such observations brane, which is then taken up by the formation of facilitate the formulation of concepts concerning the new discs soon after light onset. This membrane ap- cyclic cellular activity. pears to consist of double thicknesses of membrane Key words: rhodopsin biosynthesis, electron microscope which is continuous with the membrane within the immunocytochemistry, diurnal rhythm, retina, photore- disc stack. It is likely that it serves as a reservoir of ceptors membrane for the "burst" of new disc formation which occurs at light onset. If this interpretation is Acknowledgments correct, the ordering of disc membrane into its final The authors would like to express their thanks to: Dr. form is a process distinct from the assembly of disc Brian Matsumoto of the Institute of Environmental Stress, membrane, the latter occurring during several hours University of California, Santa Barbara for preparation of prior to light onset whereas the former is light-acti- the opsin antiserum while he was a graduate student in Dr. vated. Bok's laboratory; to Ms. Caryl Lightfoot and Ms. Marcia Lloyd for their invaluable technical assistance, and Ms. The implication that the mechanism initiating disc Alice Van Dyke for her excellence in the darkroom. ordering is light-activated and resides in the outer segment is in close accord with the results reported by References Hollyfield et al.18 In their experiments with X. laevis, 1. La Vail MM: Rod outer segment disk shedding in the rat retina: an enhanced rate of outer segment membrane assem- Relationship to cyclic lighting. Science 194:1071, 1976. bly occurred during the early light phase of the diur- 2. Basinger S, Hoffman R, and Matthes M: Photoreceptor shed- nal cycle in the absence of an increase in the rate of ding is initiated by light in the frog retina. Science 194:1074, opsin synthesis. Their observations imply that disc 1976. formation does not require the biosynthesis of new 3. Besharse JC, Hollyfield JG, and Rayborn ME: Turnover of rod photoreceptor outer segments: II. Membrane addition and loss protein, and are compatible with the notion that the in relationship to light. J Cell Biol 75:507, 1977. newly formed discs are constructed from protein al- 4. Young RW: The daily rhythm of shedding and degradation of ready assimilated by the outer segment. Furthermore, cone outer segment membranes in the lizard retina. J Ultra- other investigators have observed that incorporation struct Res 61:172, 1977. of opsin by rod outer segments is only moderately 5. La Vail MM and Ward PA: Studies on the hormonal control of reduced in R. pipiens maintained for 24 hr in dark- circadian outer segment disc shedding in the rat retina. Science 3 20 17:1189, 1978. ness. ' The lack of a light cue apparently does not 6. Hollyfield JG and Basinger SF: Photoreceptor shedding can be adversely affect the movement of protein to the outer initiated within the eye. Nature 274:794, 1978. segment. 7. Young RW: The daily rhythm of shedding and degradation of A process of disc morphogenesis by evagination of rod and cone outer segment membranes in the chick retina. the ciliary membrane, as proposed by Steinberg et Invest Ophthalmol Vis Sci 17:105, 1978. 32 8. Flannery JG and Fisher SK: Light-triggered rod disc shedding al, is compatible with our observations and suggests in Xenopus retina in vitro. Invest Ophthalmol Vis Sci 18:638, that evaginations proceed beyond the physical limits 1979. of ordered discs formed at an earlier time. The unor- 9. Basinger SF and Hollyfield JG: Control of rod shedding in the dered membrane may be "reeled in" subsequently by frog retina. In Neurochemistry of the Retina, Bazan NG and Lolley RN, editors. Oxford, Pergamon Press Ltd., 1980, p. 81. some contractile mechanism as part of a process of 10. Flannery JG and Fisher SK: Circadian disc shedding in Xen- placing new discs in the proper register. In support of opus retina in vitro. Invest Ophthalmol Vis Sci 25:229, 1984. 3334 this, Chaitin and coworkers have localized actin 11. Besharse JC: Photoreceptor disc shedding and phagocytosis: to the site of disc evagination. Furthermore, Williams Mechanism and regulation. J Cell Biol 99:115, 1984. et al35 have shown that cytochalasin D produces dis- 12. Young RW and Droz B: The renewal of protein in retinal rods and cones. 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