Distribution of Membrane Proteins in Mechanically Dissociated Retinal Rods

M. 5pencer,*t P- B- Derwiler,* and A. H. Bunr-Milamj-

Solitary rods were isolated from frog retinas by mechanical dissociation. Typically, the rods cleave sclerad to the nucleus and consist of outer segments with attached partial inner segments with either tapered or rounded profiles. Light and electron microscopy reveal that the outer and inner segments of rods with tapered inner segments, like rods in the intact retina, are joined by a single connecting cilium. In contrast, the outer and inner segments of rods with rounded inner segments are fused, with no extracellular cleft between the two segments. Opsin distribution was studied in both unfused and fused rods by light and electron microscopic immunocytochemistry. Extensive surface labeling is restricted to the outer segments of tapered rods, as observed in vivo. In contrast, both inner and outer segments of rods with rounded inner segments (fused) label heavily with anti-opsin. Thus opsin, a mobile membrane protein, diffuses from the outer to the inner segment of fused rods. Segregated distribution of opsin in unfused rods suggests that the connecting cilium and/or its associated structures may normally act as a diffusion barrier between the outer and inner segments to mobile membrane proteins such as opsin. Immunofluorescence studies demonstrate that Na+/K+ ATPase is restricted in distribution to the inner segment and calycal processes of both fused and unfused isolated rods, as observed in vivo. Maintenance of its restricted distribution in fused cells indicates that Na+/K+ ATPase is not mobile and may be tethered in the surface membrane of the inner segment. Invest Ophthalmol Vis Sci 29:1012-1020,1988

In the 1970s, several laboratories showed that rec- the inner segment. The mechanism of fusion was not ognizable cell types could be isolated from the verte- known nor was it clear if this would occur with disso- brate retina by mechanical and/or enzymatic dissoci- ciation procedures lacking proteolytic enzymes. ation.1"4 Photoreceptors isolated by these techniques The retinal rod is a highly polarized cell. From the are currently used for electrophysiology, biochemis- outer segment to the synaptic terminal, well defined try and cell biology studies of rods and cones. In spite regional differences in morphology and biochemical of widespread use of dissociated photoreceptors, only composition underlie the specialized physiology of one study describes their fine structure. Townes-An- different parts of the cell. Fusion of the outer and derson et al5 compared salamander rod ultrastructure inner segments could result in reorganization of mo- before and after enzymatic dissociation. They noted lecular components and create significant functional that the narrow extracellular cleft that normally sepa- differences between rods in the intact retina and fused rates the outer and inner segments of rods in the isolated rods. intact retina was absent in isolated rods. With dissoci- This paper further characterizes fusion of the outer ation, the surface membrane of the basal outer seg- and inner segments of isolated rods and describes en- ment appeared to fuse with the apical membrane of suing changes on a molecular scale by use of immunocytochemistry to monitor the distribution of two membrane proteins, opsin and Na+/K+ ATPase, that From the Departments of *Physiology and Biophysics and are normally restricted to the outer and inner seg- fOphthalmology, University of Washington, Seattle, Washington. ments, respectively. In addition to providing new Supported by NIH (Bethesda, Maryland) grants EYO-2748 to morphologic observations of dissociated rods, data PBD, EYO-1311 to AB-M, EYO-7031, an unrestricted award from from this study also provoke questions about mecha- Research to Prevent Blindness, Inc., and a special fellowship from the Retinitis Pigmentosa Foundation, Inc. to MS. AB-M is a Wil- nisms that normally regulate regional distribution of liam and Mary Greve International Research Scholar of Research membrane proteins in rods of the retina. to Prevent Blindness, Inc. Submitted for publication: October 1, 1987; accepted November Materials and Methods 24, 1987. Reprint requests: Maribeth Spencer, PhD, Department of Oph- This investigation adhered to the ARVO Resolu- thalmology, RJ-10, University of Washington, Seattle, WA 98195. tion on the Use of Animals in Research.

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Mechanical Dissociation of Photoreceptors to dissociation, or rods were dissociated into Ringer's solution containing 50 nM DDC or placed into this Rana pipiens or Rana catesbiana were obtained solution after dissociation. Random fields of cells from Kons (Germantown, WI) and maintained in the were photographed by fluorescence microscopy (ex- University of Washington Health Sciences Vivarium citation 395 nm, emission > 450 nm), and rephoto- on a 12 hr light-dark cycle. For all experiments, frogs graphed using bright field illumination and phase were dark-adapted a minimum of 30 min before de- contrast optics. capitation and double pithing, to facilitate separation For protein localization at the light microscopic of the neural retina from the retinal pigment epithe- level, dissociated rods were fixed in 4% paraformalde- lium. The eyes were removed under infrared or dim hyde in buffer (130 mM NaH2PO4, pH 7.2) for a red illumination and sliced in coronal section poste- minimum of 2 hr. After rinsing six to ten times in rior to the ciliary body. The eyecups were then quar- buffer with 10% sucrose and 0.5% BSA, the cells were tered in oxygenated frog Ringer's solution (100 mM bathed for 1 hr in 1:500 anti-opsin or 1:100 anti- NaCl, 1 mM KC1, 1.2 mM MgSO4, 1 mM CaCl2, 2.8 Na+/K+ ATPase monoclonal antibody diluted in mM Hepes, 10 mM glucose, pH 7.55). Wells formed buffer with 0.5% BSA, and rinsed six to ten times in by rubber O-rings (9 mm ID) glued to coverslips were buffer. Under dim red illumination, rods were im- filled with 100 n\ of Ringer's solution. Each quarter of mersed for 1 hr in anti-mouse IgG conjugated to FITC a dark-adapted retina was peeled from the retinal (ICN Immunobiologicals, Lisle, IL) or phycoerythrin pigment epithelium and gently swirled in a well of (Biomeda, Foster City, CA) diluted 1:50 in buffer. Ringer's solution to release dissociated rods. For the Na+/K+ ATPase experiments, 0.1% Triton X-100 was included in the primary and secondary Antibodies antibody buffers to improve penetration, since the + + Monoclonal antibodies directed against the N-ter- a-subunit of Na /K ATPase lies on the cytoplasmic minal region of opsin were provided by R. S. Molday side of the plasma membrane. After six to ten rinses (4D2),6 P. A. Hargrave (R2-15),7 and V. P. Gaur in buffer, rods were photographed as above by fluo- (14-8, 15-3, 15-18).8 D. M. Fambrough provided a rescence and bright field microscopy. monoclonal antibody (#5) against purified avian kid- Video: Freshly dissociated rods in O-ring wells were ney Na+/K+ ATPase that recognizes the a-subunit in viewed through the inverted microscope using infra- immunoblots and immunoprecipitations (K. Ren- red (>800 nm) illumination and a RCA Ultracon aud, B. Wolitzky, K. French and D. M. Fambrough, camera. Cells were observed for up to 30 min in ei- Johns Hopkins University, personal communica- ther Ringer's solution or a dissociation solution con- tion). taining 14 U/ml papain (Cooper Biomedical, Mal- vern, PA) used by Townes-Anderson et al.5 Light Microscopy Electron Microscopy: Pre-embedment Immunogold Labeling Nomarski: Freshly dissociated rods were diluted with an equal volume of 2% glutaraldehyde/2% para- Dissociated cells were transferred to BEEM cap- formaldehyde buffered in 65 mM NaH2PO4 (pH 7.2), sules (Ted Pella, Tustin, CA), diluted to the top with placed between two coverslips, viewed with a Nikon fixative, and centrifuged at 60 g for 5 min. The su- Optiphot microscope with NDIC optics and photo- pernatant was removed and the capsules refilled with graphed using Kodak Technical Pan 2415 film. fixative. All succeeding solutions were exchanged Fluorescence and phase contrast: A nylon mesh after gentle centrifugation as above. disc (20 /im square grid; Pharmacia, Piscataway, NJ) Optimal fixation and labeling were obtained when was fitted to the inside diameter of the O-ring to re- rods were fixed with 2% glutaraldehyde/2% para- tain the dissociated cells. The mesh allowed solution formaldehyde in 65 mM NaH2PO4 buffer (pH 7.2) changes, and rods were viewed continuously and for 3 hr. An alternate fixative used was 1.25% glutar- photographed using a Nikon Diaphot inverted micro- aldehyde in 65 mM cacodylate buffer (pH 7.4). Fixed scope with epifluorescence. The cells suffered no dis- cells were then rinsed in buffer for 20 min and im- cernible damage from this method and few were lost mersed overnight at 4°C in 1:500 4D2 anti-opsin during processing. Kodak Ektachrome 400 film was monoclonal antibody in PBS with 0.5% BSA. The used for fluorescence and bright field photography. rods were rinsed in PBS-BSA for 30 min and im- The integrity of the rod plasma membranes was mersed in 1:10 goat anti-mouse IgG-15 nm colloidal assessed using the fluorochrome N,N' didansyl-L- gold (Janssen, Piscataway, NJ) in PBS-BSA for 1 hr. cystine (DDC) (Sigma, St. Louis, MO). Under dim The cells were next treated with 1% OsO4 with 1.5% red illumination, eyecups were filled with DDC prior potassium ferrocyanide in buffer for 1 hr, rinsed in

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detached outer segments, or between the myoid and the nucleus, yielding partially intact rods referred to as RIS-ROS.9 We have observed10 that RIS-ROS have inner segment profiles that fall into two distinct categories: tapered or rounded (Fig. 1). Phase or differential-interference contrast microscopy reveals a prominent extracellular cleft between the outer and inner segments of RIS-ROS with tapered but not rounded inner segments (Fig. 1). We detect no other morphologic differences between the two cell types by light microscopy. Both tapered and rounded RIS-ROS can exclude DDC (Fig. 2). The fluorescent efficiency of DDC in- creases approximately 20-fold when it is membrane- bound.11 Thus, the high density of rod intracellular membranes causes RIS-ROS with ruptured surface membranes to fluoresce brilliantly, while RIS-ROS with intact membranes are only weakly fluorescent. Both cell types are typically weakly fluorescent but heavily stained cells of both types are observed occa- sionally, which we attribute to nonselective cell damage. These results indicate that there is no systematic difference in the integrity of the surface membranes of tapered and rounded cells. The same results are obtained when the eyecup is incubated in DDC Ringer's solution during the dissociation procedure, indicating that the surface membrane of neither cell type ruptures during dissociation. When examined by bright field illumination, ruptured RIS-ROS (brightly fluorescent cells) have curved outer segments with prominent disc striations and the inner segment plasma membranes have a granular, vesicular appearance. Conversely, intact Fig. 1. Mechanically dissociated rods (RIS-ROS) with partial RIS-ROS that exclude DDC have straight, tubular inner segments (RIS) and intact outer segments (ROS), viewed with outer segments with little definition of disc mem- Nomarski optics, (a) RIS-ROS with a tapered inner segment has a branes and smooth inner segments (Fig. 2). prominent cleft (arrow) between the inner and outer segments, as observed in rods in vivo, (b) RIS-ROS with a rounded inner seg- By electron microscopy, tapered RIS-ROS resem- ment lacks a pronounced cleft between the inner and outer seg- ble rods in intact retina (Fig. 3a). The outer and inner ments. Bar, 2 nm. segments are separated by an extracellular cleft and are joined by a connecting cilium. The long, tapered distilled H O for 15 min, stained en bloc with 0.5% inner segments have dense ellipsoid mitochondria 2 and extensive rough endoplasmic reticulum, Golgi uranyl acetate in distilled H2O for 1 hr, dehydrated through a graded ethanol series, and embedded in membranes, and vesicles in the myoid. Calyces Medcast (Ted Pella). Thin sections (1000 A) were project apically in a regularly spaced pattern from the collected on parlodion and carbon coated grids, inner segment plasma membrane and traverse the stained with uranyl acetate and lead citrate, and extracellular cleft to lie adjacent to the longitudinal viewed with a JEOL 100S (Tokyo, Japan) electron grooves of the outer segment incisures. The connect- microscope. ing cilium, axonemal microtubules and basal bodies appear normal (Fig. 3b). In most cases, basal evagin- Results ations of the outer segment are spaced uniformly, confluent with the plasma membrane and open to the Mechanically Dissociated Rods: Morphology extracellular space. Some outer segments lack basal and Integrity evaginations or, rarely, the basal evaginations are re- Mechanical dissociation of the retina typically placed by rows of vesicles (Fig. 3c). shears rods at either the connecting cilium, yielding Rounded RIS-ROS lack the extracellular cleft that

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Fig. 2. Both fused and unfused RIS-ROS can have intact surface membranes, (a) Phase microscopy of a random field of mechanically dissociated RIS-ROS treated with the fluorochrome DDC. (b) Fluorescence micrograph of the same field of cells. Damaged RIS-ROS fluoresce brightly, demonstrating patency of surface membranes to the fluorochrome. Fused and unfused RIS-ROS with intact plasma membranes fluoresce faintly. The extracellular cleft (arrow) of unfused RIS-ROS and the distal tips (arrowheads) of some outer segments are the most fluorescentregion s of intact RIS-ROS. Bar, 10 pm.

separates the segments of rods in vivo and in tapered rods14 are easily identified because they do not label RIS-ROS, in agreement with our light microscopic with the anti-opsin antibodies used here (see below). observations (Fig. 4). The outer and inner segments Most red rod RIS-ROS are rounded (fused) and most of rounded RIS-ROS are fused; the plasma mem- green rod RIS-ROS have tapered (unfused) inner seg- brane is continuous around the circumference of the ments.

rod. Inner segment organdies abut the basal most + + discs, which are closed to the extracellular space (Fig. Distribution of Opsin and Na /K ATPase 4b). Most rounded RIS-ROS have dense mitochon- The distribution of surface membrane proteins in dria in the ellipsoid but fewer myoid organelles than fixed fused and unfused RIS-ROS was studied using tapered RIS-ROS (Fig. 4a). The plasma membrane five monoclonal antibodies to opsin and a mono- that normally separates the calyces from the outer clonal antibody to Na+/K+ ATPase, followed by sec- segment in an intact rod may be complete, partial or ondary antibodies conjugated to FITC or phycoer- lacking in fused RIS-ROS (Fig. 4c-e). Partial calyces ythrin for fluorescence microscopy. This method include those with only distal tips intact, and those in allows the distribution of each protein to be surveyed which the plasma membranes separating the calyces rapidly in many cells. Labeled opsin is found in only from the outer segment are incomplete and replaced the outer segments of unfused cells (Fig. 5a, b) but is by flattened vesicles. The connecting cilium and basal found in both the outer and inner segments of fused bodies of rounded RIS-ROS appear normal morpho- RIS-ROS (Fig. 5c, d). This result was obtained with logically (Fig. 4f). Inner segment cytoplasmic micro- all anti-opsin monoclonal antibodies tested. In con- tubules insert into lateral feet that extend radially trast, Na+/K+ ATPase is localized to the inner seg- from the basal body of the connecting cilium.1213 The ment but never to the outer segment of either fused or plasma membrane in the region of the ciliary neck- unfused RIS-ROS (Fig. 5e-h).15 The same results are lace does not fuse, creating a tunnel adjacent to the obtained when cells are incubated in Ringer's solu- cilium that appears in sections as 1-6 nm diameter tion for 30 min before fixation and labeling. The vesicles. maintained localization of Na+/K+ ATPase in the The proportions of unfused and fused RIS-ROS inner segments of fused cells suggests that this protein vary in each dissociation. RIS-ROS of frog green has restricted mobility in the rod plasma membrane.

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11 \ \ *• • •*•••'

Fig. 3. Unfused RIS-ROS. (a) Unfused RIS-ROS have long tapered inner segments with ellipsoid and myoid organelles. The inner and outer segments are separated by an extracellular cleft (*). This red rod RIS-ROS was labeled with anti-opsin antibody followed by anti-mouse IgG-colloidal gold before embedment. Surface label is restricted primarily to the outer segment. Bar, 1 nm. (b) Connecting cilium of an unfused RIS-ROS from a green rod. Bar, 0.5 ftm. (c) Extensive reorganization of unstable basal evaginating membranes results in rows of vesicles in a green rod RIS-ROS. Bar, 0.5 ^m. (d) Unfused red rod RIS-ROS exposed to anti-opsin antibody and anti-mouse IgG-colloidal gold before embedment. Label is present on the outer segment membrane but little label occurs on the inner segment plasma membrane. Bar, 0.5

Downloaded from iovs.arvojournals.org on 09/27/2021 Fig. 4. Fused RIS-ROS. (a) A fused red rod RIS-ROS, labeled as in Figure 3a, has no extracellular cleft between the segments and has a rounded inner segment containing ellipsoid mitochondria. Anti-opsin antibody labels the plasma membrane of the entire outer and inner segment except for the periciliary region (arrows). Bar, 1 ^m. (b) The plasma membrane is continuous between the outer and inner segments at the circumference of fused RIS-ROS. No open basal discs are present. Bar, 0.5 jtm, Calyces of fused RIS-ROS may be complete (c) or lacking (e). Broad regions of cytoplasm interrupted by occasional vesicles (arrowheads) separate the outer segment discs from remnants of partially fused inner segment calyces (d). Bar 0.5 **m. (f) The connecting cilium of a fused RIS-ROS appears normal. Cytoplasmic microtubules (MT) insert into a lateral foot (LF) of the basal body (BB). No anti-opsin label occurs on the plasma membrane of the proximal connecting cilium, which exhibits a fuzzy outer coat. A vesicle {•) is found adjacent to the connecting cilium. SR, striated rootlet. Bar, 0.5 jim. (g) The surface of the periciliary ridge complex of a fused red rod RIS-ROS does not label with anti-opsin. Membrane vesicles are associated with the plasma membrane at the periciliary ridge complex. Bar, 0.5 ^m.

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Fig. 5. Immunofluores- cence labeling of opsin and Na+/K+ ATPase. (a-d) Sur- face anti-opsin labeling of unfused and fused red rod RIS-ROS. An unfused RIS- ROS (a, b) and a fused RIS- ROS (c, d) were bathed in anti-opsin antibody followed by anti-mouse IgG- FITC and photographed by brightfield (a, c) or epifluorescence (b, d) microscopy. Only the outer segments of unfused RIS-ROS fluoresce, while both the outer and inner segments of fused RIS-ROS fluoresce. (e-h) Surface anti-Na+/K+ ATP- ase labeling of unfused and fused RIS-ROS. An unfused RIS-ROS (e, f) and a fused RIS-ROS

Opsin distribution was studied by electron micros- processed by pre- and post-embedment immunogold copy using secondary antibodies conjugated to col- techniques.616 Thus, opsin redistribution probably loidal gold. In unfused cells, anti-opsin labels the sur- does not occur prior to mechanical dissociation. face membrane of the outer segment and distal ciliary Infrared video microscopy used to monitor fresh, shaft but not the plasma membrane of the proximal unfixed cells immediately after dissociation reveals connecting cilium, penciliary ridge complex, or inner that over a period of 30 min (starting less than 1-2 segment (Fig. 3a, d). As expected from immunofluo- min after dissociation), no morphologic changes rescence, fused RIS-ROS show extensive surface la- occur in either tapered or rounded RIS-ROS. Also, beling with anti-opsin on both the outer and inner fusion cannot be provoked in tapered cells by me- segment plasma membranes (Fig. 4a, f, g). Only the chanical prodding, treatment with papain or steady plasma membrane of the periciliary ridge and the exposure to dim white light. proximal connecting cilium (in the region of the axonemal-plasma membrane crosslinkers) is unlabeled Discussion with anti-opsin (Fig. 4f, g). Mechanical dissociation of the frog retina yields Time of Fusion rods with partially intact inner segments having ei- Fusion of rod outer and inner segments probably ther tapered or rounded profiles. Rods with tapered occurs during the dissociation procedure. We have inner segments resemble their counterparts in the in- not observed fused rods in intact R. pipiens or R. tact retina; outer and inner segments are joined by a catesbiana retinas prepared for conventional electron single connecting cilium. Rods with rounded inner microscopy. Furthermore, anti-opsin is localized to segments are fused; the plasma membranes of the rod outer but not inner segments of intact retina by basal outer segment and apical inner segment are indirect immunofluorescence, and to the plasma joined and continuous around the full circumference membrane of in situ rod outer but not inner segments of the rod.

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Time and Mechanism of Fusion Retention of the segregated distribution of Na+/K+ Tapered, unfused rods are stable over time. Once ATPase in fused cells suggests that the molecule is anchored in the inner segment membrane. This is dissociated, they do not convert into fused rods either + + spontaneously or after further mechanical manipula- consistent with the limited mobility of Na /K ATP- ase measured in cultured kidney epithelial cells.27 In tion or treatment with papain. Fusion probably + + occurs during the dissociation procedure. Despite re- these cells, Na /K ATPase colocalizes with spectrin and actin,28 suggesting that its mobility may be re- ports of cytoplasmic bridges between outer and inner + + segments in intact mammalian rods17"19 or occa- stricted by cytoskeletal elements. Since Na /K ATP- ase colocalizes with F-actin in the inner segments and sional fusion between the segments of intact Xenopus 29 30 20 calyces of rods, ' it is possible that similar cytoskel- laevis rods, we have not observed fused rods in R. + + pipiens or R. catesbiana retinas. etal complexes tether Na /K ATPase in the inner Typically, RIS-ROS are formed by cleavage of the segment membrane. 31 34 myoid. This region is just sclerad to the nucleus, Opsin is a mobile surface membrane protein " where the inner segments are anchored to adjacent that normally does not diffuse into the inner segment. Mueller cells by continuous zonulae adherentes that The appearance of opsin in the inner segment of form the external limiting membrane.21 There are fused rods suggests that the diffusion barrier in un- fewer myoid organelles in fused than in unfused RIS- fused rods is not a general property of the inner seg- ROS, suggesting that the cleavage site is more sclerad ment membrane. While outer and inner segment in the former. Thus, the fusion of a RIS-ROS appears plasma membranes differ in protein and lipid com- to depend on where the cell is severed during dissoci- position,35 these differences clearly do not prevent ation. This could explain the observation that most opsin diffusion. Redistribution of opsin in fused cells red rod RIS-ROS are fused, while most green rod occurs within the 60-100 seconds required to dissoci- RIS-ROS are unfused. The myoid of a green rod is ate and fix cells for immunofluorescence. This is narrow and elongated compared to that of a red rod consistent with the time required for a molecule with and occupies a more sclerad position relative to the the diffusion constant of rhodopsin (D = 3-5 X 10"9 external limiting membrane.14 A shear force directed cm2 s"1)36 to diffuse 8 fxm, the approximate distance along the external limiting membrane would tend to between the basal margin of the outer segment and cleave the myoid closer to the ellipsoid of a red rod the rounded end of the inner segment of a fused rod. than a green rod, and hence favor production of fused This suggests that rhodopsin can diffuse equally well red rod RIS-ROS and unfused green rod RIS-ROS. in outer and inner segment membranes and supports The position of the cleavage site may influence in- previous proposals that transegmental diffusion of tersegmental fusion by causing differential disruption opsin in intact cells is blocked by a barrier located in of cytoskeletal elements required for mechanical the connecting cilium.15'23'37"39 maintenance of the extracellular cleft. Without suffi- How the connecting cilium limits diffusion is not cient cytoskeletal restraint, the outer and inner seg- known. The barrier could be a discrete fence of teth- ment membranes on opposing sides of the cleft may ered membrane proteins or it might be a more diffuse come into contact and fuse spontaneously. Mem- region of low membrane fluidity. While our results brane fusion is a necessary step in the formation of do not address this issue directly, they do provide new outer segment discs from basal evaginations of 22 information about the location and extent of the dif- the surface membrane. Thus, the cleft membrane fusion barrier. In unfused cells, anti-opsin labeling is may be inherently unstable as evidenced by its ten- lacking from the proximal cilium and the inner seg- dency to vesiculate (Fig. 3c), and prone to fusion. ment surface membrane (see also1540). This would locate the diffusion barrier in the distal portion of the Compartmentalization of Membrane Proteins connecting cilium. In fused cells, anti-opsin label Spatial differences in the distribution of molecular does not appear on the connecting cilium membrane; components are necessary for the functional special- label does not penetrate beyond the periciliary ridge ization of different regions of the photoreceptor. The complex from the inner segment side. This suggests distribution of rhodopsin and Na+/K+ ATPase is re- that either a second discrete barrier is positioned at stricted, respectively, to the outer6'1523 and inner24"26 the periciliary ridge or that the entire region extend- segments of intact photoreceptors. While the basis of ing from the distal cilium through the periciliary compartmentalization of these two membrane pro- complex is one continuous diffusion barrier. The two teins is not understood, it is clear from our results alternatives have different implications for the move- showing redistribution of opsin but not Na+/K+ ment of opsin past the connecting cilium in that the ATPase in fused rods, that two different mechanisms latter would require a mechanism other than diffu- are responsible. sion in the surface membrane.

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Key words: dissociated frog rods, immunocytochemistry, 20. Besharse JC: Photosensitive membrane turnover: Differen- opsin, Na+/K+ ATPase, protein distribution tiated membrane domains and cell-cell interaction. In The Retina: A Model for Cell Biological Studies, Volume 1, Adler Acknowledgments R and Farber D, editors. New York, Academic Press, 1986, pp. The authors are grateful to D. Possin, I. Klock, and F. 297-352. Dahlan for technical assistance, B. Clifton, R. Jones, and C. 21. Bunt-Milam AH, Saari JC, Klock IB, and Garwin GG: Zonu- Stephens for photographic help, K. Chin for secretarial as- lae adherentes pore size in the external limiting membrane of sistance, and Drs. J. Saari, V. Sarthy, and W. Sather for the rabbit retina. Invest Ophthalmol Vis Sci 26:1377, 1985. scientific input. 22. Steinberg RH, Fisher SK, and Anderson DH: Disk morphogenesis in vertebrate photoreceptors. J Comp Neurol 190:501, References 1980. 1. Anctil M, Ali MA, and Couillard P: Isolated retinal cells of 23. Defoe DM and Besharse JC: Membrane assembly in retinal some lower vertebrates. Review of Canadian Biology 32:107, photoreceptors: II. Immunocytochemical analysis of freeze- 1973. fractured rod photoreceptor membranes using anti-opsin antibodies. J Neurosci 5:1023, 1985. 2. Kaneko A, Lam DMK, and Wiesel TN: Isolated horizontal 3 cells of elasmobranch retinae. Brain Res 105:567, 1976. 24. Stirling CE and Lee A: [ H]Ouabain autoradiography of frog 3. Bader CR, MacLeish P, and Schwartz EA: Responses to light retina. J Cell Biol 85:313, 1980. of solitary rod photoreceptors isolated from tiger salamander 25. Stirling CE and Sarthy PV: Localization of the Na-K pump in retina. Proc Natl Acad Sci (Wash) 75:3507, 1978. turtle retina. J Neurocytol 14:33, 1986. 26. Schneider BG, Papermaster DS, and Sweadner KJ: Polarized 4. Sarthy PV and Lam DMK: Biochemical studies of isolated + + + glial (Mueller) cells from turtle retina. J Cell Biol 78:675, 1978. distribution of a subunit of (Na ,K )ATPase in the inner 5. Townes-Anderson E, MacLeish PR, and Raviola E: Rod cells segment and axonal plasma membrane of rat retinal rod inner dissociated from mature salamander retina: Ultrastructure and segments. J Cell Biol 103:1743a, 1986. 27. Jesaitis AJ and Yguerabide J: The lateral mobility of the uptake of horseradish peroxidase. J Cell Biol 100:175, 1985. + + 6. Hicks D and Molday RS: Differential immunogold-dextran (Na ,K )-dependant ATPase in Madin-Darby canine kidney labeling of bovine and frog rod and cone cells using mono- cells. J Cell Biol 102:1256, 1986. clonal antibodies against bovine rhodopsin. Exp Eye Res 28. Nelson WJ and Veshnock PJ: Dynamics of membrane-skele- 42:55, 1986. ton (fodrin) organization during development of polarity in 7. Hargrave PA, Adamus G, Arendt A, McDowell JH, Wang J, Madin-Darby canine kidney epithelial cells. J Cell Biol Szary A, Curtis D, and Jackson RW: Rhodopsin's amino ter- 103:1751, 1986. minus is a principal antigenic site. Exp Eye Res 42:363, 1986. 29. Del Priore LV, Lewis A, Tan S, Carley WW, and Webb WW: 8. Bunt-Milam AH, Gaur VP, Saari JC, and Sarthy PV: Immu- Fluorescence light microscopy of F-actin in retinal rods and nocytochemical study of rod and cone outer segments using glial cells. Invest Ophthalmol Vis Sci 28:633, 1987. monoclonal antibodies. ARVO Abstracts. Invest Ophthalmol 30. Vaughan DK and Fisher SK: The distribution of F-actin in Vis Sci 27(Suppl):235, 1986. cells isolated from vertebrate retinas. Exp Eye Res 44:393, 9. Biernbaum MS and Bownds MD: Frog rod outer segments 1987. with attached inner segment ellipsoids as an in vitro model for 31. Poo M-M and Cone RA: Lateral diffusion of rhodopsin in photoreceptors of the retina. J Gen Physiol 85:83, 1985. Necturus rods. Exp Eye Res 17:503, 1973. 10. Spencer M, Detwiler PB, and Bunt-Milam AH: Opsin redistri- 32. Poo M-M and Cone RA: Lateral diffusion of rhodopsin in the bution in rods with fused inner and outer segments. ARVO photoreceptor membrane. Nature 247:438, 1974. Abstracts. Invest Ophthalmol Vis Sci 27(Suppl):235, 1986. 33. Liebman PA and Entine G: Lateral diffusion of visual pigment 11. Yoshikami S, Robinson WE, and Hagins WA: Topology of the in photoreceptor disk membranes. Science 185:457, 1974. outer segment membranes of retinal rods and cones revealed 34. Govardovskii V: The early receptor potential, its mode of gen- by a fluorescent probe. Science 185:1176, 1974. eration, application to study of photoreceptors, and functional 12. Greiner JV, Weidman TA, Bodley HD, and Greiner CAM: significance. In Photoreceptors, Borsellino A and Cervetto L, Ciliogenesis in photoreceptor cells of the retina. Exp Eye Res editors. New York, London, Plenum Press, 1984, pp. 129-146. 33:433, 1981. 35. Fliesler SJ and Anderson RE: Chemistry and metabolism of 13. Greiner JV, Bodley HD, Weidman TA, and Peace DG: Peri- lipids in the vertebrate retina. Prog Lipid Res 22:79, 1983. centriolar processes of photoreceptor cell basal bodies in the 36. Wey CL, Cone RA, and Edidin MA: Lateral diffusion of rho- mammalian retina. Z Mikrosk Anat Forsch 97:309, 1983. dopsin in photoreceptor cells measured by fluorescencepho - 14. Nilsson SEG: An electron microscopic classification of the tobleaching and recovery. Biophys J 33:225, 1981. retinal receptors of the leopard frog (Rana pipiens). J Ultra- 37. Papermaster DS and Schneider BG: Biosynthesis and mor- struct Res 10:390, 1964. phogenesis of outer segment membranes in vertebrate photore- 15. Spencer M, Detwiler PB, Bunt-Milam AH, and Fambrough ceptor cells. In Cell Biology of the Eye, McDevitt DS, editor. DM: Localization of the Na+/K+ ATPase in retina and disso- New York, Academic Press, 1982, pp. 475-531. ciated photoreceptors. ARVO Abstracts. Invest Ophthalmol 38. Besharse JC, Forestner DM, and Defoe DM: Membrane as- VisSci28(Suppl):341, 1987. sembly in retinal photoreceptors: III. Distinct membrane do- 16. Nir I and Papermaster DS. Differential distribution of opsin in mains of the connecting cilium of developing rods. J Neurosci the plasma membrane of frog photoreceptors: An immunocy- 5:1035, 1985. tochemical study. Invest Ophthalmol Vis Sci 24:868, 1983. 39. Papermaster DS, Schneider BG, and Besharse JC: Vesicular 17. Pedler C and Tilly R: Ultrastructural variations in the photore- transport of newly synthesized opsin from the Golgi apparatus ceptors of the macaque. Exp Eye Res 4:370, 1965. toward the rod outer segment: Ultrastructural immunocyto- 18. Richardson TM: Cytoplasmic and ciliary connections between chemical and autoradiographic evidence in Xenopus retina. the inner and outer segments of mammalian visual receptors. Invest Ophthalmol Vis Sci 26:1386, 1985. Vision Res 9:727, 1969. 40. Peters K-R, Palade GE, Schneider BG, and Papermaster DS: 19. Tokuyasu K and Yamada E: The fine structure of the retina: Fine structure of a periciliary ridge complex of frog retinal rod V. Abnormal retinal rods and their morphogenesis. J Biophys cells revealed by ultrahigh resolution scanning electron micros- BiochemCytol 7:187, 1960. copy. J Cell Biol 96:265, 1983.

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