The retinal pigment epithelium (RPE) differs from other epithelia in that the apical surface is not free; instead, it interacts with both photoreceptors and a specialized extracellular material, the interphotoreceptor matrix. Biochemical characterization of the apical and basolateral surfaces of RPE in adult rat eye cups, using a novel in situ biotinylation assay, revealed very different protein compositions and identified a major surface antigen, RET-PE2, with a predominantly apical distribution (∼74%). The apical polarity of RET-PE2 was confirmed by immunofluorescence and laser scanning confocal microscopy. In striking contrast, RET-PE2 antigen was preferentially basolateral in primary cultures derived from adult rat RPE and in an immortalized RPE cell line (RPE-J). Under all conditions, RET-PE2 was highly soluble in Triton X-100 (>81% at 4°C), suggesting that its redistribution was not dependent on changes in cytoskeletal interactions. Analysis of the localization of RET-PE2 in normal rats at postnatal (PN) days 1, 7, and 14 indicated that RET-PE2 redistributes from predominantly basolateral to predominantly apical during that time. Since photoreceptors develop during the first two weeks after birth in the rat, our results suggest that the apical redistribution of RET-PE2 is dependent on the establishment of adult interactions between the RPE and the neural retina and/or the interphotoreceptor matrix, either via direct contacts or through alterations in the intracellular sorting patterns of RPE cells.

The retinal pigment epithelium (RPE), a highly specialized derivative of the embryonic neural tube (Zinn and Marmor, 1979), combines the barrier functions of a simple transporting epithelium with the support role of glia and the phagocytic activity of macrophages. The RPE acts as a selective permeability barrier between the choroidal bloodvessels and the subretinal space and performs a variety of essential support functions for the neural retina, such as the uptake, transport, and processing of vitamin A and the phagocytosis and digestion of photoreceptor outer segments (Bok, 1993). The essential support functions of RPE require a characteristic distribution of some surface proteins. N-CAM-140, an isoform of the calcium independent neural cell adhesion molecule that localizes basolaterally in the kidney cell line MDCK (Powell et al., 1991), is observed on the apical surface of RPE in young (one-week-old) rats (Gundersen et al., 1993). Unlike most other epithelia, RPE cells display Na+,K+-ATPase on their apical surface (Bok, 1993; Caldwell and McLaughlin, 1984; Gundersen et al., 1991; Okami et al., 1990; Rizzolo, 1990).

It is likely that the reversed polarity of certain apical RPE proteins depends on their unique apical microenvironment. Indeed, N-CAM-140 changes its distribution to basolateral in primary cultures of RPE (Gundersen et al., 1993). Furthermore, Na+,K+-ATPase in rats becomes nonpolar under the same experimental conditions (Marrs et al., 1995; Nabi et al., 1993). It has been shown that interaction with the basement (Bruch’s) membrane alone is insufficient to induce apical polarity of Na+,K+-ATPase in cultured chicken RPE (Rizzolo, 1991). The distribution of Na+,K+-ATPase in a given cell may be determined by two mechanisms acting alone or in combination: intracellular sorting into defined post-Golgi vesicles or selective stabilization by a domain-specific ankyrin-fodrin submembrane cytoskeleton (Caplan et al., 1986; Gottardi and Caplan, 1993; Hammerton et al., 1991; Zurzolo and Rodriguez-Boulan, 1993). In MDCK cells, Na+,K+-ATPase is stabilized by a lateral ankyrin-fodrin cytoskeleton (Hammerton et al., 1991), probably induced by E-cadherin-mediated intercellular adhesion (Nelson, 1991; Marrs et al., 1995). In contrast, the RPE in the eye expresses both ankyrin-fodrin and Na+,K+-ATPase on the apical surface, probably as a result of the absence of E-cadherin in RPE cells (Gundersen et al., 1991, 1993; Rizzolo, 1990), which express cadherins of the N-or P-type (Gundersen et al., 1993; Lagunowich and Grunwald, 1989; Marrs et al., 1995). Recent results suggest that ankyrin but not fodrin codistribute with Na+,K+-ATPase in the apical microvilli of avian RPE cells, implying that only ankyrin develops functional interactions with the enzyme (Rizzolo and Zhou, 1995).

In order to understand the mechanisms responsible for the unique polarity properties of the apical surface of RPE, it is crucial to identify components of that membrane that may interact with the interphotoreceptor matrix (IPM) or the photoreceptor outer segments. Here, we use a novel adaptation of the biotin polarity assay previously developed in our laboratory (Sargiacomo et al., 1989) to identify apical and basolateral surface proteins from RPE in situ. We show that the RET-PE2 antigen (Neill and Barnstable, 1990) is a major component of the apical membrane of RPE in situ, but undergoes a reversal of its polarity from apical to basolateral in primary cell culture. Developmental studies indicate that this marker polarizes to the apical surface exactly at the time when photoreceptor outer segments and the IPM mature. The control of RET-PE2 distribution by the maturation of photoreceptors constitutes a novel mechanism of apical polarization that depends on extracellular cues.

Reagents

All cell culture reagents were obtained from Gibco/BRL Life Technologies (Grand Island, NY). All other reagents were obtained from Sigma Chemical Co. (St Louis, MO) unless otherwise indicated.

Animals and primary cell cultures

Male and female Long-Evans rats of various ages were obtained from Charles River (Wilmington, MA). Animals were kept on 12 hour light/dark cycles and fed ad libitum.

Primary cultures of RPE were established from adult Long-Evans rats as follows: animals were sacrificed by CO2 asphyxiation, and the eyes enucleated and placed in 10 mM Hepes buffered Hank’s balanced salt solution (HBSS) at 4°C in the dark for 4 hours. A circumferential incission was made below the ora serrata and anterior segments and neural retinae were removed. After digestion for 1 hour with trypsin (Difco, Detroit, MH) in HBSS (2 mg/ml) at 37°C, RPE were teased from the eyecup with a 25 G needle. RPE cells were further dissociated in trypsin/EDTA, and plated on Matrigel® (Collaborative Research, Bedford, MA) coated Transwell® filters (Corning-Costar Corp., Cambridge, MA) (2 eyes/1.2 cm diameter filter) in DMEM containing 20% fetal bovine serum (FBS), glutamine, penicillin/streptomycin, and non-essential amino acids. Cells were kept at 37°C for 4-6 weeks without subculturing prior to use. Primary RPE cultures acquired transepithelial resistances of ∼60-80 Ω × cm2.

RPE-J cell culture

The SV-40 immortalized RPE-J cell line was maintained as previously described (Nabi et al., 1993) in Dulbecco’s minimum essential medium (DMEM) containing 4% Cellect Gold FBS (ICN, Costa Mesa, CA), supplemented with glutamine, non-essential amino acids and penicillin/streptomycin at 32°C. Cells were passaged with trypsin/EDTA. For biochemical studies RPE-J cells were plated on Matrigel® coated Transwell filters® at a density of 3.5×105 cells/cm2. Cells were grown for 6-7 days at 32°C and then allowed to differentiate for 2 days at 39.5°C in medium supplemented with 10−8 M retinoic acid. RPE-J cells grown in this fashion acquired transepithelial resistances in excess of 200 Ω ×cm2.

Preparation of eyecups and isolation of RPE

Eyes were enucleated and a circumferential incision was made above the ora serrata. The cornea, lens, iris, and vitreous body were removed. To remove the neural retina without damaging the apical surface of the RPE we used a modification of the protocol of Wang et al. (1993). Eyecups were incubated in 10 ml of HBSS containing 290 U/ml of bovine testicular hyaluronidase for 10-30 minutes at 37°C. A second incision was then made below the ora serrata and the neural retina was carefully peeled away and cut at the optic nerve. For detergent solubility studies, RPE was isolated from the eyecups after digestion with hyaluronidase and collagenase, as described by Wang et al. (1993).

Immunofluorescence and confocal microscopy

Eyecups or RPE-J monolayers were fixed for 30 minutes in 2% paraformaldehyde in phosphate buffered saline containing 0.1 mM CaCl2, 1.0 mM MgCl2 (PBS/CM), and quenched with 50 mM NH4Cl in PBS/CM. When necessary, RPE-J monolayers were permeabilized in absolute methanol for 4 minutes at -20°C. For cryosectioning, eyecups were infiltrated successively with 10% and 20% sucrose, and then with Tissuetek® 4583 (Miles Inc., Elkhart, IN). When it was desirable to maintain the RPE-neural retina interaction, rats were killed by CO2 asphyxiation, and subject to intracardiac perfusion with HBSS followed by 4% paraformaldehyde in PBS/CM. Perfusion fixed eyes were enucleated, the corneas incised, and then further immersion fixed overnight. Following immersion fixation, the eyes were infused with 30% sucrose in PBS/CM, and then with Tissuetek® 4583. After freezing in liquid nitrogen/isopentane, 10 µm cryosections were cut and the tissue allowed to adhere to SuperFrost slides (Fisher, Springfield, NJ). After blocking in PBS/CM containing 0.2% BSA (PBS/CM/BSA), monolayers or sections were stained with a rabbit polyclonal antisera raised against rat laminin (Gibco) and/or mouse monoclonal antibody RET-PE2 for 1 hour and then with FITC-conjugated goat anti-mouse and/or Texas red-conjugated donkey antirabbit IgG secondary antibodies (Cappel, Durham, NC) in PBS/CM/BSA containing a 1:200 dilution of DNase free RNase I (Boehringer Mannheim, Indianapolis, IN) for 1 hour. The cells were washed and in some cases stained with propidium iodide as described previously (Hanzel et al., 1991). After a final wash, filters were excised and mounted in vectashield (Vector Labs, Burlingame, CA). Whole eyecup preparations were cut radially and mounted en face. Labeled cells were visualized with a dual channel laser scanning confocal microscope (Sarastro/Molecular Dynamics, Sunnyvale, CA) as previously described (Hanzel et al., 1991).

Domain specific biotinylation of RPE-J and RPE in situ

The steady state distribution of RET-PE2 antigen in RPE-J cells was determined using a domain specific biotinylation assay as previously described (Hanzel et al., 1991; Zurzolo et al., 1993). To examine the RET-PE2 distribution in RPE in situ, we used a modification of a previously described quenched biotinylation assay (Lisanti et al., 1990). The assay was performed as follows: adult Long-Evans rats were euthanized by CO2 asphyxiation and the eyes enucleated. The eyes were washed in ice-cold PBS/CM, eyecups prepared as described above, washed in PBS/CM at 4°C, filled with a 2 mg/ml solution of either NHS-LC-biotin (sulfosuccinimidyl-6-(biotinamido)-hexanoate; Pierce, Rockford, IL) or the disulfide cleavable NHS-S-S-biotin sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithioproprionate; Pierce), and incubated for 20 minutes at 4°C. The labeling reaction was repeated 3 times, and the biotinylation reaction quenched in PBS containing 50 mM NH4Cl (10 minutes at 4°C). Eyecups were then incubated in PBS containing 1 mM EDTA for 3060 minutes at 4°C, the RPE was dissected from the eyecup, placed in a 1.5 ml Eppendorf tube containing ice-cold PBS, pelleted for 10 seconds in a microfuge, resuspended in 1 ml PBS containing 0.5 mg/ml NHS-LC biotin and incubated at 4°C for 20-30 minutes. The labeling reaction was repeated, and the reaction was quenched for 10 minutes with 1 ml PBS, 50 mM NH4Cl at 4°C for 10 minutes. Biotinylated RPE was lysed with 1% Triton X-100 in 50 mM TrisHCl, 150 mM NaCl, 1 mM EDTA, 0.2% BSA, pH 7.4 (TBSE), containing protease inhibitors (1 mM PMSF, 0.5 mM aprotinin, 0.5 mM leupeptin, 0.5 mM antipain) for 1 hour at 4°C. The lysate was centrifuged at 10,000 g for 10 minutes, and the supernatant prepared for SDS-PAGE.

Immunoprecipitation, SDS-PAGE and streptavidin blotting

After preclearing samples with Pansorbine (Calbiochem, San Diego, CA), RET-PE2 monoclonal antibody in the form of ascites fluid was added directly to cell lysates (1-3 µl/ml). After overnight incubation, the lysates were supplemented with 10 mg Protein-A Sepharose prebound to 10 µg rabbit anti-mouse antibodies, incubated for 1 hour at 4°C, and the beads pelleted and washed as previously described (Le Bivic et al., 1989). Immunoprecipitates were resuspended in 40 µl of SDS-PAGE sample buffer containing 0.5 M 2-mercaptoethanol, heated to 95°C for 5 minutes, and analyzed by SDS-PAGE on discontinuous 12% gels.

Gels were transferred to Immobilon-P membranes (Millipore, Bedford, MA) overnight, blocked in TBSE containing 10% dry milk for 1 hour, and incubated with either [125I]streptavidin, or streptavidin conjugated to horseradish peroxidase, in 1% milk, in TBSE for 1 hour. After 3 washes in TBSE containing 0.1% Tween-20, the blots were visualized by autoradiography or enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) on Kodak X-OMAT AR film and an enhancing screen. To quantify results, gels were scanned and the intensity of bands determined using NIH Image 1.52 software.

Detergent solubility

Freshly isolated RPE or RPE-J cells were surface biotinylated and then lysed in 1% Triton X-100 in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 0.2% BSA and protease inhibitors, as above. In some samples, 5 mM EDTA was added to eliminate potential effects of divalent cations on interactions with the cytoskeleton. Cells were lysed for 20 minutes at 4°C, and insoluble material was removed by centrifugation at 10,000 g for 10 minutes at 4°C. Insoluble pellets were heated for 5 minutes at 95°C in 1% SDS, 50 mM Tris, pH 7.4, 150 mM NaCl, 0.2% BSA. The amount of detergent in each sample was equalized by the addition of SDS or Triton X-100 to final concentrations of 0.1, and 1%, respectively. RET-PE2 antigen was immunoprecipitated and the amount of antigen determined from streptavidin blots of the immunoprecipitates as described above.

Sorting of lipids

Sorting of lipids was determined as previously described (van Meer, 1993; van Meer and van’t Hof, 1993). Differentiated monolayers of RPE-J cells were allowed to incorporate C6-NBD-ceramide in HBSS for 30 minutes at 4°C. Delivery to the cell surface of newly synthesized C6-NBD-glucosylceramide (C6-NBD-glcCer, a glycosphingolipid) and C6-NBD-sphingomyelin (C6-NBD-SM, a phospholipid) were assayed by selective depletion from either the apical or basolateral surfaces with fatty acid free bovine serum albumin in HBSS. Fluorescent lipids were separated by two-dimensional TLC, and quantified by spectrofluorimetry.

Characterization of the apical surface of RPE in situ

Given that two RPE proteins, Na+,K+-ATPase and N-CAM, have been shown to change their surface distribution upon removal of RPE from the eye and culture in vitro (Gundersen et al., 1991; Rizzolo, 1990), our initial goal in this study was to identify apical proteins in RPE in its normal eye localization. To this end, we adapted our biotinylation assay to the eye cup (Fig. 1). The RPE is conveniently fit for this type of study since it lays as a continuous monolayer with its concave apical surface in apposition to the neural retina and the interphotoreceptor matrix. The neural retina can be easily peeled off after digestion with bovine testicular hyaluronidase, with minimal damage to the RPE. To label only the apical surface of the RPE, the neural retina was removed, soluble components of the IPM washed away, and the exposed apical surface labeled with NHS-LC biotin. Examination by laser scanning confocal microscopy (LSCM) of eye cup whole mounts exposed to FITC-streptavidin in the presence of detergent (Fig. 2A) showed intense fluorescence at the apical surface and no fluorescence at the basolateral surface or the underlying choroid. For basolateral staining, apical sites were blocked with a cleavable biotin derivative, NHS-S-S-biotin (Fig. 2B), basolateral sites were exposed by treatment with 1 mM EDTA, and labeled with the uncleavable NHS-LC-biotin (Fig. 1). After removal of RPE from the underying choroid, biotin is removed from the apical proteins by reduction with the 2-mercaptoethanol in the SDS-PAGE sample buffer. After this procedure the only remaining biotinylated proteins are basolateral. Control eye cup experiments in which the apical surface was blocked with NHS-S-S-biotin, and subsequently exposed to NHS-LC-biotin, indicated that the apical surface is adequately blocked by this procedure (Fig. 2C).

Fig. 1.

In situ biotinylation of RPE. Eyes are enucleated from adult Long-Evans rats (1) and the anterior segments and neural retina dissected away. An aliquot of either NHS-LC-biotin (b) or NHS-S-S-biotin (x) is placed within the eyecup (2) to either label (b) or quench label (x) the apical membrane. After the biotinylation reaction, eyecups are incubated at 4°C (metabolic block) for 20-30 minutes in PBS containing 1 mM EDTA, and the RPE monolayer is dissected out of the eyecup (3). The apically quenched RPE monolayer is then biotinylated in suspension with NHS-LC-biotin (4). After the reaction is completed the cells are lysed in detergent (5).

Fig. 1.

In situ biotinylation of RPE. Eyes are enucleated from adult Long-Evans rats (1) and the anterior segments and neural retina dissected away. An aliquot of either NHS-LC-biotin (b) or NHS-S-S-biotin (x) is placed within the eyecup (2) to either label (b) or quench label (x) the apical membrane. After the biotinylation reaction, eyecups are incubated at 4°C (metabolic block) for 20-30 minutes in PBS containing 1 mM EDTA, and the RPE monolayer is dissected out of the eyecup (3). The apically quenched RPE monolayer is then biotinylated in suspension with NHS-LC-biotin (4). After the reaction is completed the cells are lysed in detergent (5).

Fig. 2.

Biotinylation of intact eye cups labels only the apical surface of RPE. The apical surface of RPE in eyecups was reacted at 4°C with: (A) NHS-LC-biotin (4× 20 minutes); (B) NHS-S-S-biotin (4× 20 minutes) or (C) with NHS-S-S-biotin (4× 20 minutes), followed by NHS-LC-biotin (2× 20 minutes) and reduction with 50 шM DTT. After fixation, eyecups were permeabilized and stained with FITClabeled streptavidin (green) to reveal biotinylated sites, and nuclei were stained with propidium iodide (red). Tissue was then examined by LSCM in the x-z plane. Reaction with NHS-S-S-biotin labeled the apical surface (B) sufficient to block subsequent labelling with NHS-LC-biotin (C). Bars, 5 µm.

Fig. 2.

Biotinylation of intact eye cups labels only the apical surface of RPE. The apical surface of RPE in eyecups was reacted at 4°C with: (A) NHS-LC-biotin (4× 20 minutes); (B) NHS-S-S-biotin (4× 20 minutes) or (C) with NHS-S-S-biotin (4× 20 minutes), followed by NHS-LC-biotin (2× 20 minutes) and reduction with 50 шM DTT. After fixation, eyecups were permeabilized and stained with FITClabeled streptavidin (green) to reveal biotinylated sites, and nuclei were stained with propidium iodide (red). Tissue was then examined by LSCM in the x-z plane. Reaction with NHS-S-S-biotin labeled the apical surface (B) sufficient to block subsequent labelling with NHS-LC-biotin (C). Bars, 5 µm.

Examination of [125I]streptavidin blots of RPE monolayers labeled by this procedure indicated that the apical and basolateral protein patterns were quite different (Fig. 3). Proteins with relative molecular masses of ∼36, ∼50, and ∼60, and 100120 kDa were apical, whereas proteins of ∼55, and ∼63 kDa were distinctly basolateral.

Fig. 3.

Apically and basolaterally biotinylated proteins in RPE in situ. Apical and basolateral protein profiles of 2-month-old rat RPE were generated by in situ domain specific biotinylation. After labeling RPE from either the apical (Ap) or the basolateral (Bl) side, the cells were lysed, and the cell surface proteins resolved by SDS-PAGE, transfered to PVDF, and blotted with horseradish peroxidase-conjugated streptavidin. Labeled proteins were visualized by ECL. Note the different protein patterns on the apical and basolateral surfaces (arrowheads indicate polarized proteins). At the apical surface, proteins of 36, 50, 60 and 100-120 kDa are particularly prominent. Size standards (in kDa) are shown at the left.

Fig. 3.

Apically and basolaterally biotinylated proteins in RPE in situ. Apical and basolateral protein profiles of 2-month-old rat RPE were generated by in situ domain specific biotinylation. After labeling RPE from either the apical (Ap) or the basolateral (Bl) side, the cells were lysed, and the cell surface proteins resolved by SDS-PAGE, transfered to PVDF, and blotted with horseradish peroxidase-conjugated streptavidin. Labeled proteins were visualized by ECL. Note the different protein patterns on the apical and basolateral surfaces (arrowheads indicate polarized proteins). At the apical surface, proteins of 36, 50, 60 and 100-120 kDa are particularly prominent. Size standards (in kDa) are shown at the left.

Identification of an apical protein as the RET-PE2 antigen

Few antibodies against RPE surface proteins are currently available. One of these is the antibody RET-PE2 which recognizes a 50-55 kDa antigen that is found exclusively in rat RPE (Neill and Barnstable et al., 1990). Examination of streptavidin precipitated proteins by immunoblot with the RET-PE2 antibody of RPE monolayers labeled according to the protocol described in Fig. 1 detected a predominantly apical protein band with the apparent molecular mass of RET-PE2, 50-55 kDa (Fig. 4A). Quantitation of the polarized distribution of this protein by immunoprecipitation and [125I]streptavidin blotting (Fig. 4B) indicated a 3:1 apical to basolateral ratio. Examination of 10 µm frozen sections of the retina, double stained for RET-PE2 and and laminin (a component of the Bruch’s basement membrane) (Fig. 5B,C; C was obtained by LSCM) confirmed that RET-PE2 is predominantly localized to the apical membrane.

Fig. 4.

Apical distribution of RET-PE2 antigen determined by in situ biotinylation. The polarity of RET-PE2 antigen was determined by in situ domain specific biotinylation of eyecups prepared from 2-month-old Long-Evans rats followed by immunoprecipitation and blotting with [125I]streptavidin (A). The percentage of RET-PE2 antigen present on the apical (Ap) and basolateral (Bl) surfaces is shown in B. Results are expressed as mean ± s.d. (n=3).

Fig. 4.

Apical distribution of RET-PE2 antigen determined by in situ biotinylation. The polarity of RET-PE2 antigen was determined by in situ domain specific biotinylation of eyecups prepared from 2-month-old Long-Evans rats followed by immunoprecipitation and blotting with [125I]streptavidin (A). The percentage of RET-PE2 antigen present on the apical (Ap) and basolateral (Bl) surfaces is shown in B. Results are expressed as mean ± s.d. (n=3).

Fig. 5.

Apical polarity of RET-PE2, determined by immunofluorescence and confocal microscopy. The distribution of the RET-PE2 antigen was analyzed by immunofluorescence of frozen sections from eyes fixed by intracardiac perfusion with 4% paraformaldehyde (A,B,C). The phase and fluorescence images in A and B were photographed with a fluorescence microscope. C is a confocal x-y scan. Note in B and C the separation between laminin staining at the basement membrane (red, BM) and RET-PE2 staining (green). Ap, apical; BM, basal membrane; ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigment epithelium; Ch, choroid. Bars: 20 µm (A,B), 5 µm (C).

Fig. 5.

Apical polarity of RET-PE2, determined by immunofluorescence and confocal microscopy. The distribution of the RET-PE2 antigen was analyzed by immunofluorescence of frozen sections from eyes fixed by intracardiac perfusion with 4% paraformaldehyde (A,B,C). The phase and fluorescence images in A and B were photographed with a fluorescence microscope. C is a confocal x-y scan. Note in B and C the separation between laminin staining at the basement membrane (red, BM) and RET-PE2 staining (green). Ap, apical; BM, basal membrane; ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigment epithelium; Ch, choroid. Bars: 20 µm (A,B), 5 µm (C).

RET-PE2 antigen is basolateral in primary RPE cultures and in RPE-J

To determine whether the apical polarity of RET-PE2 is maintained or changed when RPE is removed from the eye microenvironment, we examined the steady-state distribution of this antigen in primary cultures of adult RPE and in RPE-J, an immortalized rat RPE cell line, using the biotin polarity assay (Fig. 6). Strikingly, the RET-PE2 antigen was found to have redistributed to a preferentially basolateral distribution in both cases (Fig. 6A). Quantitative analysis indicated that ∼90% of RET-PE2 in RPE-J was basolateral under these conditions (Fig. 6B). LSCM and immunofluorescence analysis confirmed the basolateral distribution of RET-PE2 in RPE-J monolayers (Fig. 6C) and in primary RPE cultures (data not shown). Interestingly, the staining was confined to the lateral membrane of unpermeabilized cells, with the basal surface excluded; part of the antigen was detected in structures with a vesicular appearance. These structures are presumably blebs in the lateral membrane which are visible by TEM (unpublished observation). The RET-PE2 staining was lost if the paraformaldehyde fixed cells were permeabilized with either 0.075% saponin or 0.1% Triton X-100 (data not shown). Methanol permeabilization at-20°C did not disrupt the RET-PE2 staining; therefore, this fixative was used when nuclei were stained with propidium iodide to provide a positional marker, or to facilitate difusion of the antibody to the basolateral side in whole mount preparations of RPE.

Fig. 6.

Steady state distribution of RET-PE2 antigen in RPE-J monolayers. RPE-J monolayers cultured on Transwell filters were subjected to domain specific biotinylation. Streptavidin blots (A) were scanned and the percentage of RET-PE2 antigen present on the apical (Ap) and basolateral (Bl) surfaces were tabulated (B). Results in B are expressed as mean ± s.d. (n=8). RET-PE2 antigen was found to be predominantly basolateral. The basolateral polarity of RET-PE2 antigen in RPE-J cells was confirmed by immunofluorescence and LSCM. Cell surface staining in the x-y plane (C) had a honeycomb appearance and was confined to the lateral borders of the cells. x-z scans (D) confirm the basolateral localization. Bars, 5 µm.

Fig. 6.

Steady state distribution of RET-PE2 antigen in RPE-J monolayers. RPE-J monolayers cultured on Transwell filters were subjected to domain specific biotinylation. Streptavidin blots (A) were scanned and the percentage of RET-PE2 antigen present on the apical (Ap) and basolateral (Bl) surfaces were tabulated (B). Results in B are expressed as mean ± s.d. (n=8). RET-PE2 antigen was found to be predominantly basolateral. The basolateral polarity of RET-PE2 antigen in RPE-J cells was confirmed by immunofluorescence and LSCM. Cell surface staining in the x-y plane (C) had a honeycomb appearance and was confined to the lateral borders of the cells. x-z scans (D) confirm the basolateral localization. Bars, 5 µm.

The polarity of RET-PE2 antigen changes during post-natal development

In rats and mice, photoreceptors are without morphologically distinct inner or outer segments until after birth. Inner segments begin to form during the first week of postnatal development. While the majority of outer segment development occurs during the second postnatal week, outer segment disks can be detected as early as PN5 (Olney, 1968). Similarly apical processes are nearly absent on RPE cells until the second week of postnatal development, becoming very prominent by PN12 (Dowling and Gibbons, 1962; Micali et al., 1989). Previous work has indicated that the expression of RET-PE2 antigen is widespread in the embryonic rat eye (Neill and Barn-stable, 1990) but becomes confined to the RPE as early as PN9 (Neill et al., 1993). To determine whether the apical polarity of RET-PE2 was dependent on the maturation of the retina, we examined its distribution by LSCM in 10 µm frozen sections and in whole mounts of RPE obtained from rats at PN1, PN7, PN14, and from 2-to 3-month-old rats (Figs 7 and 8).

Fig. 7.

The polarity of RET-PE2 is similar to cultured RPE at PN1. The distribution of RET-PE2 was compared in whole mounts of adult (A) and PN1 (B) eyes stained with RET-PE2 (green, ●) and propidium iodide (red, ). LSCM was used to generate a section series (A,B). Sections were 0.1 µm in depth at 0.5 µm intervals, starting at the apical most point of staining. Mean pixel intensities for RET-PE2 and propidium iodide were determined and the percentage maxima graphed for both adult (C) and PN1 (D) eyes. The green and red lines in C and D represent mean pixel intensities for RET-PE2 and propidium iodide staining, respectively. The numbers in the lower left hand corner of A and B correspond to the sections shown. RET-PE2 staining is strongest in the first section and decays relative to the peak of propidium iodide in the adult. In contrast at PN1 RET-PE2 staining takes on a distinct honeycomb appearance similar to that observed in RPE-J cells and peaks along with the nuclear stain. Ap, apical; Ba, basal. Bars: 10 µm (A); 20 µm (B).

Fig. 7.

The polarity of RET-PE2 is similar to cultured RPE at PN1. The distribution of RET-PE2 was compared in whole mounts of adult (A) and PN1 (B) eyes stained with RET-PE2 (green, ●) and propidium iodide (red, ). LSCM was used to generate a section series (A,B). Sections were 0.1 µm in depth at 0.5 µm intervals, starting at the apical most point of staining. Mean pixel intensities for RET-PE2 and propidium iodide were determined and the percentage maxima graphed for both adult (C) and PN1 (D) eyes. The green and red lines in C and D represent mean pixel intensities for RET-PE2 and propidium iodide staining, respectively. The numbers in the lower left hand corner of A and B correspond to the sections shown. RET-PE2 staining is strongest in the first section and decays relative to the peak of propidium iodide in the adult. In contrast at PN1 RET-PE2 staining takes on a distinct honeycomb appearance similar to that observed in RPE-J cells and peaks along with the nuclear stain. Ap, apical; Ba, basal. Bars: 10 µm (A); 20 µm (B).

Fig. 8.

Change of polarity of RET-PE2 antigen during postnatal development. The distribution of RET-PE2 was studied using LSCM at various stages of post-natal (PN) development in whole mount eyecups. (A,B) PN1; (C,D) PN7; (E,F) PN14. x-y scans of the apical most staining are shown in A,C,E, x-z scans are shown in B,D,F. At PN1, RET-PE2 immunostaining was preferentially associated with the basolateral surface of the RPE. At PN7, the staining was observed both laterally and apically. At PN14, the RET-PE2 staining was predominantly apical. Pigment may have obscured some basal staining that was apparant in x-y scans of frozen crossections (not shown). Bars, 20 µm.

Fig. 8.

Change of polarity of RET-PE2 antigen during postnatal development. The distribution of RET-PE2 was studied using LSCM at various stages of post-natal (PN) development in whole mount eyecups. (A,B) PN1; (C,D) PN7; (E,F) PN14. x-y scans of the apical most staining are shown in A,C,E, x-z scans are shown in B,D,F. At PN1, RET-PE2 immunostaining was preferentially associated with the basolateral surface of the RPE. At PN7, the staining was observed both laterally and apically. At PN14, the RET-PE2 staining was predominantly apical. Pigment may have obscured some basal staining that was apparant in x-y scans of frozen crossections (not shown). Bars, 20 µm.

Whole mounts stained with RET-PE2 and propidium iodide (to label nuclei) were examined using LSCM. Section series were generated from the first (apical most) point at which any staining could be detected until both signals became weak (approx. 6 µm in depth). RET-PE2 staining in adult animals (Fig. 7A and C) was strongest in the first section and grew progressively weaker while the propidium iodide stained nuclei increased in strength before dropping off. This was evident in plots of the percentage of maximum mean pixel intensity for both channels (Fig. 7C and D) and was in contrast to whole mounts of PN1 eyes (Fig. 7B and D). At PN1 RET-PE2 staining took on a distinct honeycomb appearance similar to that observed in monolayers of RPE-J cells. The RET-PE2 staining became more intense as the plane of section moved toward the basal surface, achieving a maximum intensity at the same level as the propidium iodide (Fig. 7D). In frozen sections (data not shown) obtained from PN1 rats, RET-PE2 immunostaining was detected in both the RPE and underlying choroid, with the strongest signal along the lateral borders of the RPE and the Bruch’s membrane. A similar lateral staining was observed by examination of whole mounts (Figs 7B,D, 8A,B); however, little staining was observed along the basal plasma membrane or in the choroid (Figs 7B, 8B). It is unclear whether this is due to incomplete accessibility of the antibodies to the basal side after methanol fixation, or to interference by the abundant RPE pigment granules.

Examination of frozen sections, and x-y (Fig. 8C) and x-z (Fig. 8D) scans of whole mount eyecups at PN7 revealed a nonpolar distribution of RET-PE2 staining. By PN14, however (Fig. 8E and F), the polarity of PE2 was apical in most cells. Biochemical analysis of polarity by in situ biotinylation of eyecups was found to be technically impossible because of their small size at PN1-14. These results indicate that the polarization of RET-PE2 to the apical surface follows closely the development of rod outer segments, switching from basolateral at PN1 when outer segments are not yet formed, to fully apical at PN14 when outer segments have reached maturity.

Detergent solubility of RET-PE2 antigen

As mentioned before, RET-PE2 was basolateral and highly extractable by detergent in RPE-J cells, even after paraformaldehyde fixation. Since the basolateral localization of Na+,K+-ATPase in kidney cells (Nelson, 1991) and its apical localization in RPE cells (Gundersen et al., 1991) are thought to depend at least partially on interactions with the submembrane cytoskeleton, we wished to test whether the polarity reversal of RET-PE2 from basolateral to apical in RPE in vivo correlated with increased resistance to detergent extractability, suggestive of an interaction with an apical cytoskeleton. To test this point, we extracted RPE or RPE-J cells with 1% Triton X-100 in the presence or absence of 5 mM EDTA for 20 minutes at 4°C and sedimented insoluble proteins by centrifugation for 10 minutes at 10,000 g. RET-PE2 in both Triton X-100 soluble and insoluble fractions was solubilized in SDS and immunoprecipitated after dilution of the SDS. As shown in Fig. 9, less than 15% of RET-PE2 antigen was insoluble in detergent in the presence of EDTA, both in RPE-J cells and in RPE monolayers obtained from adult rats.

Fig. 9.

RET-PE2 is soluble in detergent in RPE in situ and in RPE-J cells. RPE monolayers from 2-month-old rats or RPE-J monolayers were surface biotinylated and then lysed at 4°C in the presence or absence of 5 mM EDTA for 20 minutes. Lysates were centrifuged at 10,000 g for 10 minutes, the supernatant (soluble, S) was decanted and the pellet (insoluble, P) resuspended by boiling for 5 minutes in 1% SDS. After equalizing the concentrations of SDS and Triton X-100 in S and P fractions, RET-PE2 antigen was immunoprecipitated and the cell surface antigen visualized by SDS-PAGE and streptavidin blotting (A). The percentage of soluble RET-PE2 antigen was determined by densitometry (B). In all cases >81% of RET-PE2 antigen was soluble.

Fig. 9.

RET-PE2 is soluble in detergent in RPE in situ and in RPE-J cells. RPE monolayers from 2-month-old rats or RPE-J monolayers were surface biotinylated and then lysed at 4°C in the presence or absence of 5 mM EDTA for 20 minutes. Lysates were centrifuged at 10,000 g for 10 minutes, the supernatant (soluble, S) was decanted and the pellet (insoluble, P) resuspended by boiling for 5 minutes in 1% SDS. After equalizing the concentrations of SDS and Triton X-100 in S and P fractions, RET-PE2 antigen was immunoprecipitated and the cell surface antigen visualized by SDS-PAGE and streptavidin blotting (A). The percentage of soluble RET-PE2 antigen was determined by densitometry (B). In all cases >81% of RET-PE2 antigen was soluble.

Sorting and delivery of lipids

It has been suggested that some proteins may be sorted to the apical surface of epithelial cells in detergent insoluble glycolipid rafts (van Meer and Simons, 1988), and that exclusion from these rafts could be a potential sorting mechanism for Na+,K+-ATPase (Mays et al., 1995). We wished to determine if, in RPE-J cells, there is preferential delivery of lipids to one plasma membrane domain which would suggest a higher rate of membrane turnover in that surface and a potential sorting mechanism in RPE cells. Lipid sorting assays were performed on RPE-J cells by first preloading with C6-NBD-ceramide, and then selectively depleting newly synthesized NBD-labelled lipids from either the apical or basal membranes with albumin. This assay provides a measure of lipid flow to the apical vs basolateral surfaces, as well as a determination of lipid polarity from the ratio of a glycosphingolipid (C6-NBD-glucosylceramide) to a phospholipid (C6-NBD-sphingomyelin) transported to either surface (van Meer, 1993, van Meer and van’t Hof, 1993). In RPE-J cells, lipids were not sorted (polarity ratio of 1.34) and both C6-NBD-glucosylceramide and C6-NBDsphingomyelin were delivered in ∼2-fold greater quantities to the apical plasma membrane of RPE-J cells (2.56±3.9 and 1.92±0.3, respectively) indicating that protein-sorting in these cells is most likely independent of lipid sorting.

In this study we set out to identify apical proteins of RPE in vivo that shift their polarity when the cells are placed in culture. To accomplish this objective, we designed a domain specific biotinylation assay that allows the quantitative determination of protein polarity in the RPE in the eye. Biotinylated RPE eyecups stained with FITC-streptavidin demonstrated that labeling is restricted to the apical surface, and that basolateral labeling can be obtained by first blocking apical sites with NHS-S-S-biotin, followed by cleavage of the S-S bonds. The effectiveness of the in situ assay is verified by our finding that RET-PE2 antigen is also seen apically polarized by LSCM in the RPE of adult rats (see Fig. 5). Due to a variable extent of contamination with the underlying choroid, this assay works best for proteins that, like the RET-PE2 antigen, are restricted to or are much more abundant in the RPE than in the choroid.

The monoclonal antibody RET-PE2 recognizes a ∼50-55 kDa protein originally described as a rat RPE-specific marker. RET-PE2 is an integral membrane protein that associates with the detergent phase upon extraction with Triton X-114 and is not anchored by glycosylphosphatidylinositol (unpublished observations). RET-PE2 displayed a predominantly apical distribution in adult RPE in situ but was basolaterally distributed in early postnatal RPE cells, primary cultures of RPE, and in the RPE-J cell line. A morphometric comparison of apical and basolateral membrane areas has shown that the apical area in adult RPE cells exceeds the basolateral one by a factor of ∼3.8. (Okami et al., 1990) Thus, the apical polarity shift that we have observed may be due to the elongation of the apical microvilli during the second postnatal week, and the ensuing change in the relative proportions of apical and basolateral membranes. In the case of Na+,K+-ATPase, the enzyme was found to be only slightly enriched in the apical membrane (0.658 vs 0.440 gold particles per linear micron of membrane; Okami et al., 1990). While this observation suggests that extension of microvilli could account for the apical polarity of Na+,K+-ATPase, and potentially RET-PE2, its static nature does not eliminate the possibility of additional mechanisms responsible for their polarity. Differential rates of membrane turnover in each surface, intracellular sorting or trapping at the cell surface by the cytoskeleton or interaction with extracellular components, probably do contribute to the observed steady state polarities of these proteins.

The redistribution of RET-PE2 from basolateral to apical during development of RPE might involve an alteration in the intracellular targeting patterns of these cells. Studies with cultured epithelial cell lines have demonstrated a remarkable flexibility of the epithelial targeting pathways (Rodriguez-Boulan and Powell, 1992). In the thyroid epithelial cell line FRT, the apical protein DPPIV is delivered to the apical surface from the basolateral by transcytosis during the first two days after plating, but is directly delivered from the Golgi to the apical surface as the monolayer matures (Zurzolo et al., 1992). Similar changes have been demonstrated for several surface proteins in MDCK cells (Wang et al., 1990a,b). Do RPE cells actively sort membrane proteins? Cultured human RPE cells possess the ability to segregate influenza hemagluttinin and VSV G proteins into apical and basolateral plasma membrane domains (Bok et al., 1992), and the same seems to be true for rat RPE in vitro and in situ (V. L. Bonilha et al., unpublished observations). Furthermore another apical marker protein, p75-NTR, which has been shown to be sorted in the TGN of MDCK cells is apically polarized both in RPE-J cells and in rat RPE in situ (A. D. Marmorstein et al., unpublished observations). The demonstration of different targeting pathways in situ versus in culture requires targeting assays to measure polarized surface delivery in the eye. We are currently attempting to develop an in situ biotin targeting assay, modelled after the quenched biotinylation assay described in Fig. 1.

RET-PE2 could be stabilized at the apical surface by association with the subapical ankyrin-fodrin cytoskeleton, as has been proposed for Na+,K+-ATPase (Gundersen et al., 1991) thus resulting in a longer half-life than on the opposite surface, from where it would be rapidly removed and degraded. However, RET-PE2 remains detergent soluble throughout normal development of the retina and in the adult animal (Fig. 9), which apparently excludes this mechanism. Furthermore, although recent studies have shown an increased detergent insolubility of Na+,K+-ATPase enzyme as it redistributes from non polar to apical during embryonic development of RPE (Gundersen et al., 1991; Rizzolo and Heiges, 1991; Huotari et al., 1995; Rizzolo and Zhou, 1995), they have also shown that fodrin appears to be excluded from microvilli. This would limit the interaction of the enzyme to ankyrin, which is present in microvilli. Lastly, Mays et al. (1995) have presented data indicating that exclusion from glycosphingolipid rafts in the Golgi apparatus might account for the basolateral sorting of Na+,K+-ATPase in certain types of MDCK cells. Na+,K+-ATPase is not polarized in RPE-J cells (Nabi et al., 1993; Marrs et al., 1995). We examined the sorting of lipids in RPE-J cells and found no evidence to suggest that glycosphingolipids are sorted during delivery to the cell surface. This may account for the nonpolar distribution of Na+,K+-ATPase in RPE-J cells, however, it is unlikely that glycolipid exclusion could account for the polarized distribution of RET-PE2 in vivo or in vitro since this protein is basolaterally polarized in RPE-J cells, even in the absence of glycolipid sorting.

Alternatively, RET-PE2 could be stabilized apically in adult RPE cells by external interaction with the photoreceptors or the interphotoreceptor matrix. In this scenario, RET-PE2 might be an adhesive molecule with affinity for the IPM or for a component of the photoreceptor plasma membrane. This mechanism is supported by our finding that RET-PE2 redis-tributes apically during the second postnatal week, which is the time when photoreceptors complete their maturation in the rat (Olney, 1968), and that RET-PE2 expression in the retina becomes RPE specific by PN9 (Neill et al., 1993), a time when photoreceptor differentiation is morphologically apparent. This is also the time when the IPM, composed of extracellular matrix, is likely to be deposited between the outer segments and RPE; it is not known to what extent this material is produced by the photoreceptors or by the RPE. Indeed, it has been shown that addition of collagen to the apical surface of MDCK cells results in apical redistribution of integrins within a few hours (Ojakian and Schwimmer, 1994) and that the localization of the anion transporter in intercalated epithelial cells is determined by the extracellular matrix (van Adelsburg et al., 1993, 1994). Molecular and biochemical characterization of the RET-PE2 antigen is necessary to determine whether involvement in adhesive interactions is plausible. It should be pointed out that, as mentioned above, such mechanism would not be exclusive of additional polarity mechanisms; in fact, interactions with the neural retina might promote the development of the extremely elongated microvilli typical of RPE in vivo.

Previous work (Neill and Barnstable, 1990; Neill et al., 1993) has shown that RET-PE2 immunoreactivity is broadly distributed in the eye during embryonic development but that, postnatally it becomes restricted to the RPE. The developmental changes in RET-PE2 polarity described in this paper suggest a functional role of this protein in the maintenance of photoreceptors, or a dependence of the distribution of this protein on some still undefined component(s) of the neural retina or the IPM. The characterization of RET-PE2 structure and function after cloning of its cDNA may yield exciting insights on mechanisms of RPE adherence to the neural retina and its role in RPE polarity.

We thank Drs Wouter van’t Hof, and Anthony Scotto for sharing their expertise in the analysis of the fluorescent lipids, Drs Silvia Finnemann and Charles Yeaman for helpful discussion, and Dr Geri Gurland for critical reading of the manuscript. Ms Leona Cohen-Gould and Ms Dena Almeida provided excellent technical assistance. Supported by NIH EY08538 to E.R.B., an NRSA award to A.D.M., a CNPq fellowship to V.L.B., and the DYSON Foundation.

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