The polarity of the membrane skeleton in retinal pigment epithelial cells of developing chicken embryos and in primary culture

Differentiation ( 1995) 58:205-2 15

Differentiation

Ontogeny. NIoplasia

and Differentiation Therapy

0 Springer-Verlag 1095

The polarity of the membrane skeleton in retinal pigment epithelial cells of developing chicken embryos and in primary culture Virva Huotari, Raija Sormunen, Veli-Pekka Lehto, Sinikka Eskelinen Biocenter Oulu and Department of Pathology, University of Oulu, Kajaanintie 52 D, FIN-90220 Oulu, Finland Accepted in revised form: 19 October 1994

Abstract. We studied the morphogenesis and the membrane skeleton in the retinal pigment epithelium during chicken embryogenesis and in culture, by using immunotluorescence and electron microscopy. During embryogenesis two distinct membrane skeletal structures were formed, an apical and a basolateral one. The former was seen in the apical surface already in the 10-day-old embryos. It was comprised of ankyrin and a-fodrin and showed a codistribution with Na+,K+-ATPaseand an as yet uncharacterized cadherin-like molecule. The basolatera1 membrane skeleton was seen in the lateral walls already in the 10-day-old embryos, and later, between the 13th and 17th embryonic days, it also appeared at the basal membrane, coincidentally with the formation of the basal infoldings. It consisted of ankyrin and a fodrin, but did not codistribute with any of the integral membrane proteins studied (Na+,K+-ATPaseand cadherins). In culture, the retinal pigment epithelial cells retained their polarized morphology. Compared with the situation in vivo, however, there was a distinct translocation of the membrane skeletal components fodrin and ankyrin from the apical surface to the lateral walls, accompanied by a similar redistribution of Na+,K+-ATPase and the cadherin-like molecule. The results suggest that ( I ) there is, in the retinal pigment epithelium, an apical Na+,K+-ATPase-membraneskeleton structure stabilized by contacts between the retinal pigment epithelium and the neural retina, possibly mediated by a cadherin-like molecule, and that (2) there is another fodridankyrinbased membrane skeleton in the basolateral walls that is important for the maintenance of the extensive folding of these surface areas.

Introduction Membrane skeleton is a lattice-like network of proteins underlying the plasma membrane in virtually all types of

cells. It was first identified in erythroid cells, in which it consists of spectrin, actin and several other peripheral membrane proteins. This network is connected via ankyrin and protein 4.1 to various integral membrane proteins, such as the anion exchanger (band 3) and glycophorin C /2]. More recently, many of the red blood cell membrane skeletal proteins or their analogues, e.g. ankyrin, protein 4.1 and fodrin (non-erythroid spectrin), have also been found in non-erythroid cells [3, 6, 13, 14, 26,441, and even in plants [30]. In non-erythroid cells, the mem%rane skeleton often shows a polarized distribution in the specialized areas of the cell surface, e.g. along the basolateral membranes of the kidney epithelium, at the nodes of Ranvier in nerves, and in the neuromuscular junctions [2]. In these specialized domains, fodrin is associated, via isoforms of ankyrin, with various integral membrane proteins, such as the erythrocyte-type anion exchanger in the intercalated cells of the collecting ducts of the kidney [lo], and Na+,K+-ATPasein the kidney and the parotid gland epithelium [19, 20, 31, 351. It has been suggested that the membrane skeleton plays an important role in the anchorage of these cell surface proteins and, thus, in the maintenance of the cell polarity [2, 321. The retinal pigment epithelium (RPE) is a monolayer of highly specialized cells separating the outer segments of the photoreceptor cells from the choroid [55]. Like in other transporting epithelia, the plasma membrane of RPE is specialized into different domains: the apical membrane facing the neural retina and the basolateral membrane facing the choroid. The apical membrane, with its long projections that ensheath the outer segments of the photoreceptor cells, is responsible for the phagocytosis of the shed photoreceptor outer segments, recycling of retinoids and the transport of ions and water between the RPE and the interphotoreceptor space [56, 631. The basal surface, which rests on a thick basement membrane, has numerous infoldings and is responsible for the exchange of ions and metabolites between the RPE and the choroid [63]. Due to their ability to retain the polarized morphology also in vitro, RPE cells have

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been used to study, for example, the role of the cytoskeleton [41] and different biochemical factors [39] in the regulation of the differentiated phenotype of epithelial cells. Their highly polarized organization and multiple interactions make them also an ideal model to study the role of the membrane skeleton in cellular polarity. Na+,K+-ATPase is considered an essential integral plasma membrane protein for all vertebrate cells. It has a role in maintaining ionic gradients, electrical potential and osmotic balance across the plasma membrane [ 171. It is a commonly used 'marker' of the cell polarity; in nonpolarized cells, Na+,K+-ATPaseshows a uniform cell surface localization, while in polarized cells, e.g. in epithelial cells, it is only present in distinct plasma membrane domains. Thus, in the transporting epithelium, e.g. in kidney and intestinal epithelium, Na+,K+-ATPaseis usually found along the basolateral membranes and shows a colocalization with the membrane skeleton [ 1, 20, 3 I , 341. In contrast to most other types of transporting epithelia, in RPE, Na+,K+-ATPaseis located in the apical surface [4,15,46,551. In this study, we set out to elucidate the structural determinants of the polarized phenotype of the RPE. Therefore, we investigated the localization of the membrane skeletal proteins ankyrin and a-fodrin, the integral membrane protein Na+,K+-ATPaseand the cell-cell adhesion proteins cadherins, in the chicken RPE during embryogenesis and in culture, by using immunofluorescence microscopy. Our results show that two different types of membrane skeletal organizations are formed in the developing chicken RPE. Furthermore, we show that the contact between the RPE and the neural retina is a prerequisite for the fully polarized phenotype.

Methods Tissrte prepmrcrtion jhr immunojluore.scence microscopy. Eyes. enucleated from the lo-, 13-, 17- and 20-day-old chicken embryos, were fixed in 8% formaldehyde in 0. I M phosphate buffer, pH 7.4, for 1-2 h, cryoprotected with 2.3 M sucrose in phosphate-buffered salt solution (PBS; 137 mM NaCI, 2.7 mM KCI, 10 mM phosphate, pH 7.4) for 30 min, and frozen in liquid nitrogen. Thereafter, 1 pm cryosections were cut with Reichert Ultracut FC4E cryo-ultramicrotome. Alternatively, the eyes were embedded in Tissue-Tek (Miles, Elkhart, USA), and frozen in liquid nitrogen. Four micrometre thick frozen sections were cut with Leitz Kryostat 1720. For immunofluorescence microscopy, 4 pm sections were used for N-cadherin stainings and I pm sections for the localization of other proteins. The sections were first washed with PBS and then incubated with 10% fetal calf serum (FCS) to saturate non-specific protein binding sites. This was followed by an incubation with the primary antibody at +4" C, and then with the fluorescein isothiocyanate- (FITC-) conjugated anti-mouse or anti-rabbit antibody (Caltag Laboratories, So. San Francisco, Calif., USA). The specimens were mounted in Immu-mount mounting liquid (Shandon, Pittsburg, Pa., USA), and viewed under Olympus BH2 microscope. Kodak TMAX 3200 ASA film was used for photography.

Antibodies. The monoclonal anti-E-cadherin antibody was from Dr. Kai Simons (EMBL, Heidelberg, Germany) and the rabbit polyclonal antibody against the cytoplasmic domain of E-cadherin, recognizing also other cadherins, e.g. B- and N-cadherins, (referred to as

pancadherin antibody 1271) from Dr. James Marrs and Dr. W. James Nelson (Department of Molecular and Cellular Physiology, University of Stanford, Stanford, Calif., USA). Rabbit polyclonal antibody K62 against chicken brain a-fodrin was prepared and characterized as described elsewhere 1541. Monoclonal anti-N-cadherin antibody, against the extracellular domain of chicken heart N-cadherin, was from Sigma Chemical Co. (St. Louis, Mo., USA), rabbit polyclonal anti-ankyrin antibody, against chicken erythroid ankyrin, from Calbiochem (San Diego, Calif., USA), and monoclonal antibody a5 against the chicken kidney Na+,K+-ATPase1241 from the Developmental Studies Hybridoma Bank maintained by the Department of Biology at the University of Iowa (Iowa City, Iowa, USA) under contract number NO1 -HD-2-3 144 from the NlCHD. Tissue preparation f o r transmission electron microscopy. For transmission electron microscopy, the eyes, enucleated from the lo-, 13-, 17- and 20-day-old chicken embryos, were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hours. They were then postfixed in I % osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, for 1 hour, dehydrated in acetone, and embedded in Epon LX 112. Thin sections were cut with Reichert Ultracut E-ultramicrotome and examined in Philips 41 0 LS transmission electron microscope using an acceleration voltage of 60 kV. Cell culture.The procedure for isolating the chicken RPE cells was modified from the method described for rat eyes by Mayerson et al. [28]. The eyes, enucleated from the 10-day-old chicken embryos, were rinsed three times with PBS and incubated in an enzyme solution confaining 0.1% collagenase A (0.327 U/mg; Boehringer Mannheim, Germany) and 50 Ulml testicular hyaluronidase (type IV-S; Sigma Chemical Co.) in PBS for 60 min, followed by 0.1% trypsin-EDTA (Gibco, Gaithersburg, Md., USA) in PBS for 15 min. The eyes were then placed into the grpwth medium consisting of Eagle's minimal essential medium with Earle's salts (E-MEM; Gibco) supplemented with 2 mM glutamine, antibiotics, antimycotics, and 20% FCS (Gibco). The eyes were opened, the vitreous and the retina were lifted off and the RPE cells were peeled off from the retina and the choroid. The cells were then washed three times with PBS, trypsinized with 0.1% trypsin-EDTA in PBS for 1-2 min, triturated gently with a Pasteur pipette, and plated in the growth medium on glass cover slips for the immunofluorescence microscopy, and on plastic petri dishes for the immunoblotting and the transmission electron microscopy. The cells were not serially passaged. MDCK cells were provided by Dr. Kai Simons. They were cultured as described elsewhere [ 1 1 ] and used for immunoblotting studies. Confocal laser scanning microscopy. For stainings with ankyrin, a-fodrin, Na+,K+-ATPase, N-cadherin and pancadherin antibodies, the cells grown on glass cover slips were fixed with cold (-20 "C) methanol for 10 min. For actin stainings, the cells were fixed in 4% formaldehyde in a cytoskeleton-stabilizing buffer (100 m M piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 5 mM EGTA, 2 mM MgCI,, pH 6.8). containing 0.1% Triton X-100 [59] for 10 min. After repeated washings with PBS, the cells were post-fixed with cold ethanol (-20 "C) for 5 min. Thereafter, the cells were prepared for immunofluorescence microscopy as described above for tissue sections, except that tetramethyl rhodamine isothiocyanate- (TRITC-) conjugated secondary antibodies (Dakopatts, Glostrup, Denmark) were used. For actin-labelling, TRITC-conjugated phalloidin (Molecular Probes, Eugene, Ore., USA) was used. The specimens were analyzed with a confocal laser scanning microscope (Leica, Leica Lasertechnik, Heidelberg, Germany), equipped with Omnichrome argon-crypton laser (75 mW; laser lines 488, 568 nm) and 525 nm and 580 nm emission filters using a 1 0 0 ~ objective. Optical sections of the specimens stained for actin, afodrin and N-cadherin were generated by scanning through the cells with the laser light beam both in x-y and x-z planes with a step size of 0.21-0.50pm. In specimens stained for ankyrin,

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Fig. 1. Transmission electron micrographs showing the structure of the retinal pigment epithelium (RPE) (A, C, E) and the organization of the junctional complex area (B, D, F) in 10- (A, B), 17- (C, D) and 20- (E, F) day-old chicken embryos. In the junctional complex areas, gap junctions (gj),tight junctions (arrowheads) and adherens junctions ( rrj) are seen. ap, apical surface; ha, basal surface; hi, basal infoldings; is, developing inner segment of the photoreceptor cell; li, lateral infoldings; olm, outer limiting membrane; 0.7, developing outer segment of the photoreceptor cell. Bars, 2 ym (A, C and E) and 0.5 pm (B, D and F)

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Fig. 2. Fluorescence micrographs showing the localization of afodrin (A, C, E) and ankyrin (B, D, F) in the RPE of 10- (A, B), 17- (C, D) and 20-day-old (E,F) chicken embryos. a-fodrin and ankyrin are seen along the apical and lateral membranes in the 10-

day-old embryos and at the base of the apical projections and along the basolateral membrane in the 17- and 20-day-old embryos. Double arrowheads show the apical and arrowheads the basal surface of the RPE. Bar, 10 pm

Na+,K+-ATPaseand pancadherin, the cells were scanned through with the laser light beam only in x-y plane with a step size of 0.08 pm ( 100- I20 sections) and the x-z sections were generated from these serial sections using a computer. The appropriate images were photographed on the screen using Agfapan APX 100 or Agfapan APX 25 film.

branes as described by Towbin et al. 1571. The nitrocellulose sheets were incubated with the primary antibodies, followed by the biotin-conjugated secondary antibodies (Dakopatts) and the avidinbiotinylated horseradish peroxidase complex (AB-HRP-complex; Dakopatts). The immunoblots were developed with diaminobenzidine tetrahydrochloride (DAB, Fluka AG, Buchs, Switzerland) in the presence of 0.0 1 % hydrogen peroxide and 0.03% NiCI,.

Cell prepciration f.r transmission electron microscopy. For transmission electron microscopy, the cells grown on plastic petri dishes were quickly rinsed with ice-cold PBS whereafter they were overlaid with 2.5% glutaraldehyde, scraped from the dish using a cell scraper, fixed in 2.5% glutaraldehyde for 1 hour, and processed as described above for the tissue specimens [53].

Results Morphology of the chicken RPE during embryogenesis

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Fig. 3. Fluorescence micrographs showing the localization of Na+,K+-ATPase (A, G), pancadherin (antibody against the cytoplasmic domain of E-cadherin; B-F, H) and N-cadherin (I) in the RPE of 10- (A, B, E), and 17-day-old ((3-1) chicken embryos, and in the neural retina (C) of 10-day-old chicken embryos. In (C-F) the RPE is detached from the neural retina and the localization of pancadherin in the neural retina (C) and the RPE (E) is shown

separately; (D) and (F) represent the corresponding bright field images. Both Na+,K+-ATPase and pancadherin fluorescence are seen in the area of the apical projections of the RPE. N-cadherin fluorescence is seen along the adherens junctions in 17-day-old embryos. Arrows show the apical and arrowheads the basal surface of the RPE. o h , outer limiting membrane. Bars, 10 pm

pigment granules (Fig. 1 A). Some infoldings were seen in the lateral but not in the basal plasma membrane. In the 13-day-old embryos, RPE appeared essentially the same as in the 10-day-old embryos (not shown). In the 17-day-old embryos, there was an extensive folding of the basal plasma membrane (Fig. IC) compared with the 10-day-old embryos. The apical projections appeared more elongated and extended between the developing inner and outer segments of the photoreceptor cells. The pigment granules were more evenly distributed throughout the cells. The gap junctions were located apical of the adherens junctions and the tight junctions were seen embedded both within the adherens and gap junctions (Fig. 1 D). On the 20th embryonic day, just before hatching, both the RPE and the photoreceptor cells appeared morphologically mature (Fig. IE). The long apical projections of the RPE interdigitated with the outer and the inner segments of the photoreceptor cells. Adherens junctions appeared large and were located at the level of the upper pole of the nucleus (Fig. lE, F). They were delineated by prominent microfilament bundles. The lateral and basal membranes appeared distinctly folded. In comparison with the earlier stages of the development, the pigment granules appeared in the apical portion of the RPE and some even in the apical projections.

apical parts of the RPE cells, a-fodrin staining was seen at the base of the apical projections. No staining was detected in the more distal area of theapical projections. In the 17- and 20-day-old embryos, an intense accumulation of a-fodrin also along the basal surface became visible (Fig. 2C, E). This coincided with the development of the basal infoldings (Fig. IC, E). Ankyrin in the RPE

The localization of ankyrin appeared precisely the same as that of a-fodrin. In the 10- (Fig. 2B) and 13-day-old (not shown) embryos, it was seen along the apical and lateral walls, and in the 17- and 20-day-old embryos (Fig. 2D, F) also along the basal surface. Na+,K+-ATPase in the RPE

At all the developmental stages studied, Na+,K+-ATPase was seen in the area of the apical projections of the RPE (Figs. 3A, G and 4A, B). In closely apposed cells it was difficult to tell whether the fluorescence was in the RPE and/or the photoreceptor cells. However, in cases in which the RPE was detached from the neural retina, a distinct staining of the apical surface of the RPE could be discerned (Fig. 3G).

Fodrin in the RPE

In the 10- and 13-day-old embryos, a-fodrin was seen along the apical and lateral, but not the basal walls of the RPE cells (Fig. 2A; 13-day-old embryos not shown). In the 17- and 20-day-old embryos, the fluorescence along the lateral walls remained the same (Fig. 2C, E). In the

Cudherins in the RPE

In the 10- and 13-day-old embryos, the pancadherin antibody showed a dot-like fluorescence that was located between the RPE and the neural retina (Fig. 3B; 13-day-

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Fig. 4. Fluorescence micrographs showing the localization of Na+,K+-ATPase (A) and pancadherin (antibody against the cytoplasmic domain of E-cadherin; C) in the RPE of 20-day-old chicken embryos. Corresponding bright field images are shown in (B) and (D). Staining for both proteins are seen in the area of the

apical projections of RPE. Arrows show the basal surface of the RPE and arrowheads show corresponding regions in fluorescence and bright field images at the apical surface of the RPE. Bor, 10 pm

Fig. 5. Transmission electron micrographs showing the structure of the monolayer ( A ) and the organization of the junctional complex area (B) in cultured chicken RPE cells. In the junctional

tight junctions (nrrowhecds) and complex area, gap junctions (a), adherens junctions ( a j ) are seen. up, apical surface; ha, basal surface; li, lateral infoldings. Bars, 2 pm (A) and 0.5 pm (B)

old embryos not shown). Especially in the younger embryos, the photoreceptor cell layer is easily detached from the RPE cell layer. In such cases, it was seen that the staining was located both along the apical membrane of the RPE (Fig. 3E, F) and the photoreceptor cell membrane facing the apical surface of the RPE cells

(Fig. 3C, D). In the 17- and 20-day-old embryos, the fluorescence was found in the area of the RPE apical projections and the photoreceptor cells (Figs. 3H and 4C, D), closely resembling the localization of Na+,K+ATPase (Figs. 3G and 4A, B). In some sections of the lo-, 13- and 17-day-old embryos, a weak horizontal tlu-

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orescence in the middle part of the cells was also detected (not shown). In the 10-day-old embryos, N-cadherin was seen along the whole length of the lateral walls of the RPE cells (not shown). In the 13- (not shown) and 17-day-old (Fig. 31) embryos, a weak horizontal fluorescence in the more apical parts of the cells was detected, possibly locating to adherens junctions (Fig. IC, D). In the 20-dayold embryos we could not detect any fluorescence in the RPE (not shown). No fluorescence in the RPE was seen using an antibody specific for E-cadherin (not shown). RPE cells in culture

In primary cultures, the RPE cells, taken from 10-day-old embryos, divided and grew in colonies, forming a single cell layer and maintaining their polarized morphology. In an apical view, the cells in the central parts of the colonies appeared tightly packed and hexagonal in shape. In electron microscopy, the cells appeared cuboidal (Fig. 5A) with intermixed cell-cell adhesions (tight, adherens and gap junctions; Fig. 5B), closely resembling those seen in the 10-day-old embryos (Fig. 1B). The cells possessed distinct apical projections (Fig. 5A). Numerous pigment granules were seen in the cells. The lateral infoldings, unlike the basal infoldings, appeared well-developed. In confocal microscopy, the antibodies against afodrin and ankyrin showed a circumferential staining along the lateral walls of the cells (Fig. 6A, C). In the vertical (x-z) sections (Fig. 6B, D), they were bdth seen colocalized along the whole length of the lateral walls. Unlike in the RPE in vivo, there was no fluorescence in the apical surface. Also Na+,K+-ATPasewas found along the whole length of the lateral walls and no apical or basal fluorescence was detected (Fig. 6E, F). Pancadherin fluorescence was, on the other hand, seen in the most apical parts of the lateral walls and no apical fluorescence could be seen (Fig. 6G, H). Also the staining with the anti-N-cadherin antibody showed also a fluorescence restricted to the apical part of the lateral membrane (Fig. 61, J), probably corresponding to the adherens junctions. To localize the adherens junctions, actin was stained using TRITC-conjugated phalloidin. The fluorescence was seen as a peripheral, circumferential belt in the apical parts of the cells (Fig. 6K, L), resembling the fluorescence seen with pancadherin (Fig. 6G, H) and N-cadherin (Fig. 61, J) antibodies. lmmunoblotting of chicken RPE with antibodies to cadherins

The expression of the different cadherins in the RPE in vivo and in vitro was also studied by immunoblotting (Fig. 7). Equal amounts of proteins from the 10-day-old RPE tissue and the cultured RPE cells were loaded on SDS-PAGE (8%). Pancadherin antibody recognized two major and two minor protein bands with the molecular weights of 130 and 125 kDa and 135 and 120 kDa, respectively, both in the tissue and in the cultured cells (Fig. 7, lanes I and 2). The 130 kDa band, showing an enhanced expression in cultured cells, was also recog-

Fig. 6.Confocal laser scanning images showing the localization of a-fodrin (A, B), ankyrin (C, D), Na+,K+-ATPase(E. F), pancadherin (G, H),N-cadherin (I, J) and actin (K,L) in cultured chicken RPE cells. Panels A, C, E, G, I and K represent horizontal (x-y) confocal sections through the middle (A, C, E) or apical (G, I, K)parts of the cells. Panels B, D, F, H,J and L represent vertical (x-z) confocal sections. Vertical (x-z) sections were obtained either by scanning through the cells with the laser light beam in x-z direction (B, J, L) or by constructing the x-z section from the data collected by serial x-y sectioning (D, F, H).In C, E and G the white line denotes the site of the corresponding x-z sections (D, F, H),whereas in B, J and L the x-z sections are not from the area shown in A, I and K,respectively. a-fodrin, ankyrin and Na+,K+-ATPaseare seen along the whole length of the lateral walls, whereas pancadherin, N-cadherin and actin are seen at the most apical parts of the lateral walls. In actin staining (L) some fluorescence is also seen along the basal wall due to another cell that is growing underneath the cell layer. Arrows show the apical and arrowhecrds the basal cell surface. Bars, 5 pm

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2 3 4

5

6 7

8 9

Fig.7. lmmunoblotting with cadherin antibodies of the RPE of 10-day-old chicken embryos and the cultured RPE cells. Lanes I , 3, 5 and 8 are RPE cell lysates of 10-day-old chicken embryos, kmes 2, 4 , 6 and 9 are lysates of cultured RPE cells and lane 7 is lysate of cultured MDCK cells. Lanes 1 and 2 are labelled with anti-pancadherin antibody (against the cytoplasmic domain of Ecadherin), lanes 3 and 4 with anti-N-cadherin antibody and lanes 5-7 with specific anti-E-cadherin antibody. Lanes 8 and 9 are controls and were labelled without a primary antibody. Arrowhead shows the molecular weight standard P-galactosidase ( 1 16 kDa)

nized by the antibody to N-cadherin (Fig. 7, lanes 3 and 4). None of the bands were recognized by the antibody to E -cadherin (Fig. 7, lanes 5 and 6). The I20 kDa polypeptide in the pancadherin immunoblots had the same mobility as E-cadherin detected in MDCK cells by antiE-cadherin antibodies (Fig. 7, lane 7). Thus, the antipancadherin reactive 120, 125 and 1 35 kDa polypeptides do not seem to belong to any of the major cadherin subfamilies tested. Henceforth, these uncharacterized antipancadherin reactive polypeptides are referred to as cadherin-like polypeptides.

Discussion During embryogenesis, a precisely timed occurrence of proteins is a prerequisite for the formation of the correct polarity of the membrane domains. A coincidental expression in the same location, on the other hand, is highly suggestive of functionally important interactions. The formation of the membrane skeleton has been mostly studied in nonvertebrates, such as sea urchin [50,611 and Drosophila [25, 421, or in vertebrates during early embryogenesis [7, 12, 50, 521. On the other hand, there are only a few studies concerning the formation of the membrane skeletal structures in differentiating cells at the later stages of embryogenesis. During the development of certain neuronal cells of brain, two distinct membrane skeletal' structures, consisting of different isoforms of P-fodrin and ankyrin and locating to partly separate membrane domains, are formed at different stages of both avian and mammalian embryogenesis [21, 22, 23, 33, 45, 511. In epithelial cells, the formation of the membrane skeleton has only been studied in rat embryonic intestine [I]. In this study, we explored the formation of the membrane skeleton in chicken RPE during embryogenesis and in culture. Memhrune skeleton und the polarization of the chicken RPE in vivo

A membrane skeleton composed of fodrin and ankyrin was seen at the base of the apical projections in the RPE.

It colocalized with the integral membrane protein Na+,K+-ATPase and as yet uncharacterized anti-pancadherin-immunoreactive polypeptides. Na+,K+-ATPase and the cadherin-like molecules, but not fodrin or ankyrin, were also seen in the contact area involving the apical projections of the RPE and the overlying photoreceptor cells. Fodrin and ankyrin were, on the other hand, seen also along the basolateral membranes of RPE with no accompanying Na+,K+-ATPaseor cadherins. In accordance with our results, Philp and Nachmias [43] found fodrin along the lateral walls in isolated chicken embryo RPE sheets and in the apical projections of the isolated chicken embryo RPE cell aggregates. In frozen sections of the whole eyes of an adult rat, fodrin appears both along the apical and the basal surfaces but not along the lateral walls or the apical projections [37]. In isolated adult rat RPE, on the other hand, fodrin and ankyrin only occur along the apical surface in colocalization with Na+-K+-ATPase[ 151. The molecular basis of the differential organization of the apical and basolateral membrane skeleton/cell surface assemblies in the chicken RPE is not known. It may be due to the presence of specific forms of, for example, P-fodrin and/or ankyrin in the membrane skeleton with varying affinities for cadherins, Na+,K+-ATPaseand other integral membrane proteins. In a subset of neuronal cells of brain [21, 22, 331 and in the epithelium of the distal tubuli of kidney [2, 81, for example, two isoforms of ankyrin have been found in the-same cell localizing to different membrane domains. In MDCK cells, a widely used cell type to study the role of the membrane skeleton in cell polarity, fodrin and ankyrin colocalize with E-cadherin and Na+,K+-ATPase along the lateral walls of the cells and form complexes with either E-cadherin or Na+,K+-ATPase[31, 32, 34, 361. In fibroblasts, the expression of exogenous E-cadherin brings about a redistribution of fodrin and Na+,K+ATPase from an unpolarized distribution to a restricted localization at the cell-cell contact sites [29]. Thus, it has been suggested that the accumulation of E-cadherin to cell-cell contacts directs the assembly of the membrane skeleton, which, in turn, restricts the localization of Na+,K+-ATPase.In embryonic rat intestine, there is a sequential expression of the membrane skeletal and the cell surface components such that first fodrin and E-cadherin, and only later Na+,K+-ATPaseappear in the cellcell contact sites [I]. This also supports the view that cadherins and/or membrane skeletal components direct and impose restraints on the Na+,K+-ATPase location during embryogenesis. On the other hand, not all epithelial cells possess similar membrane skeleton-integral membrane protein assemblies. In the choroid plexus epithelium, for example, complexes between fodrin/ankyrin with either Na+,K+-ATPaseor cadherins are located in different membrane domains in the cell: the Na+,K+ATPase-containing complexes are found at the apical plasma membrane and the cadherin-containing complexes along the lateral walls. Moreover, the cadherins in these complexes are different from E-cadherin [27]. The apical membrane of the RPE cells is a highly specialized membrane structure. Unlike in many other

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epithelial cells, it is not facing a lumen but is in contact with the neural retina. The apical projections ensheath the outer segments of the photoreceptor cells and are responsible for the phagocytosis of the shed outer segments [63]. We found the apical projections already in the 10-day-old embryos and, from the day 17 onwards, they extended between the developing photoreceptor cells. The linkage molecules between the RPE and the neural retina have not been clearly identified, but both calcium-dependent and calcium-independent cell-cell adhesion molecules seem to be responsible for the adhesion [9, 16, 621. In the area of the apical projections of the RPE, there was a similar staining pattern with anti-pancadherin and anti-Na+,K+-ATPaseantibodies at all the developmental stages studied. It was not possible, by using immunofluorescence microscopy, to distinguish whether the staining was associated with the apical projections of the RPE and/or with the photoreceptor cells. However, in cases in which the neural retina was detached from the RPE, a distinct staining of the apical surface of the RPE was clearly seen, In such cases, also the staining of the neural retina with the pancadherin antibody could be seen (Fig. 3C-F). These two proteins showed a colocalization with the membrane skeletal proteins fodrin and ankyrin at the base of the projections. Analogously to MDCK cells, this could be a reflection of either direct or indirect interactions between these proteins. Notably, the membrane skeleton did not extend to the more distal area of the apical projections. Membrane skeleton could, however, sequester the cadherin-like molecules of Na+,K+-ATPaseto the apical projections also by forming a ‘fence’ at the base of the projections. In erythrocytes, for example, the membrane skeleton restricts the lateral mobility of all the anion exchanger molecules even though only a portion of them is in direct association with the membrane skeleton [2, 581. A common mechanism to segregate the cadherin-like molecules, membrane skeleton and Na+,K+-ATPaseinto the apical area in vivo is also supported by a coincidental redistribution of these proteins in RPE cells in culture (see below). Also, apically localized protein complexes consisting of Na+,K+-ATPase,ankyrin and fodrin in colocalization with calcium-independent cell-cell adhesion molecule N-CAM have been described in the rat [ 15, 161. The basolateral surface of the RPE displays an extensive folding. Such infolding is also seen in kidney tubule epithelial cells [ 19, 311, and it is important in increasing the surface area of the transporting epithelia. There is, however, very little data on the structural organization and the determinants of the infoldings. In the present study we saw a stepwise development of the basolateral folding; folding of the lateral walls was apparent already in the day 10 embryos while the basal folding was only seen in the day 17 embryos. In the basolateral membrane, the formation of the membrane skeleton assembly consisting of ankyrin and fodrin, coincided with the occurrence of folds. In these assemblies, there was no Na+,K+-ATPase,in contrast to the infoldings of kidney epithelium, where membrane skeleton and Na+,K+-ATPasecolocalize [ 19, 3 I]. Inter-

estingly, in the basal membrane infoldings of the kidney epithelium, the membrane skeleton and Na+,K+-ATPase are only found along the actual loops of the membrane foldings and are excluded from the regions where the basal plasma membrane is in contact with the basement membrane [19, 311. Moreover, in the rat intestinal epithelium, in which the entire basal plasma membrane is in contact with the basement membrane and does not show such infoldings, Na+,K+-ATPaseand fodrin are excluded from the basal cell membrane and are found only along the lateral walls [I]. This seems to be true also in rat and chicken choroid plexus epithelium [27]. We could not discern whether also in the chicken RPE the membrane skeleton is excluded from the sites which are in contact with the basement membrane. That this is the case, however, is strongly suggested by a simultaneous appearance of the folding and the basal membrane skeleton components. The coincident occurrence of the membrane skeletal proteins and the basal membrane folding suggests that the membrane skeleton has a role in the maintenance of this complex membrane organization. From the biophysical point of view, it could be expected that the folding of the lipid bilayer, without any constraints, would lead to a vesiculation of the membrane. Thus, by inference, we suggest‘that in the folded parts of the membrane, the fodrin-based membrane skeleton serves the purpose of ‘freezing’ the membrane in the folded state. Our previous results with MDCK cells give further evidence for such a supporting function of the-membrane skeleton; disassembly of the fodrin-based membrane skeleton in MDCK cells by lowering intracellular pH [ I I ] or PMA treatment [ 18, 601 led to the destabilization of the lateral walls. Concomitantly, the cells acquired an unpolarized morphology. Reversal of the poluritv in culture

In RPE cells, taken to culture from the 10-day-old chicken embryos, there was a dramatic reversal of the membrane skeleton organization. The cells retained their polarized morphology. However, the membrane skeletal proteins, fodrin and ankyrin, were only seen along the lateral walls and not in the apical membrane. The same was true for Na+,K+-ATPaseand the cadherin-like molecules. A similar type of distribution of a-fodrin along the lateral walls [38, 401 and an unpolarized distribution of the P-subunit of Na+,K+-ATPaseboth along the apical and lateral membranes [46,48] have been seen in earlier studies on cultured chicken embryonic RPE. In cultured rat RPE, on the other hand, fodrin and ankyrin appear only along the apical membrane and, analogously, Na+,K+-ATPaseremains at the apical surface [ 151. More recently, it has been suggested that Na+,K+-ATPasebecomes located to the basolateral membranes also in the rat RPE maintained in culture for a longer period of time [ 161. The distribution of P-Na+,K+-ATPasein the cultured chicken RPE does not seem to be affected by the growth substratum [47]. Interestingly, however, the dif-

214

fusible factors from the neural retina are reported to increase the barrier properties of the tight junctions (the transepithelial electrical resistance) in cultured embryonic chicken RPE cells and to induce a change in the localization of Na+,K+-ATPasefrom an unpolarized distribution to a polarized localization along the basolateral membranes [49]. The reversal of the Na+,K+-ATPase and the membrane skeleton polarity in vitro was accompanied by an extensive redistribution of the cadherin-like molecules. This further strengthens the notion formulated on the basis of the in vivo results that there are direct or indirect contacts between these proteins. Furthermore, it suggests that the contact between the RPE and the overlying photoreceptor cells is important for the apical localization of these proteins. A similar redistribution of the calcium-independent adhesion molecule N-CAM is seen in rat RPE in culture [ 161. Ac.k,iori.lr~l~emerir.s. This work was supported by grants from the

Academy of Finland, the Sigrid Juselius Foundation and the Finnish Cancer Research Fund for Professor Veli-Pekka Lehto, and by postgraduate grants from the Medical Research Fund of Oulu, the University of Oulu, Oulu University Foundation, the Finnish Cultural Foundation, the Research and Science Foundation of Farmos, the Ida Montin Foundation, and the Cancer Research Fund of Northern Finland for Virva Huotari. Dr. James Marrs and Dr. W. James Nelson are kindly acknowledged for providing the antibody against the cytoplasmic domain of E-cadherin and Dr. Kai Simons for providing the MDCK cells and the E-cadherin antibody. We want to thank Ms. Anna-Liisa Oikarainen and Ms. Tarja Piispanen for their skillful technical assistance, and Mr. Tapio Leinonen for preparing the micrographs.

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