Steps in the morphogenesis of a polarized

II. Disassembly and assembly of plasma membrane domains during reversal of epithelial polarity in multicellular epithelial (MDCK) cysts

ALLAN Z. WANG1, GEORGE K. OJAKIAN2 and W. JAMES NELSON1-* xInstitute for Cancer Research, 7701 Burholme Avenue, Philadelphia, PA 19111, USA 2Department of Anatomy and Cell Biology, State University ofNeiv York Health Sciences Center, Brooklyn, New York, USA

* Author for correspondence

Summary

A fundamental aspect in the morphogenesis of a that, when fully developed cysts formed in suspen- polarized epithelium is the formation of structur- sion culture are placed in a collagen gel, polarity is ally and functionally distinct apical and basal- rapidly reversed without cell dissociation. We show lateral domains of the plasma membrane. The that during the process of polarity reversal, plasma formation of these membrane domains involves the membrane domains are disassembled by uptake of accumulation of domain-specific proteins and re- proteins into cytoplasmic vesicles, followed by pro- moval of incorrectly localized proteins. The mech- tein degradation that probably occurs in lysosomes. anisms involved in these processes are not well The disassembly and assembly of the apical and the understood. We have approached this problem by basal-lateral membrane domains occur in a se- detailed analysis of the distribution and fate of quential order with different kinetics. Our results proteins specific for different membrane domains provide further insights into the establishment of during reversal of epithelial polarity. In the preced- protein specificity of plasma membrane domains in ing paper we showed that MDCK cells form multi- polarized cells. cellular cysts comprising a closed monolayer of polarized cells. The orientation of depends upon whether cysts are formed in suspen- sion culture or in a collagen gel. Here, we show Key words: morphogenesis, polarized epithelium, adhesion.

Introduction teins, the cells must first generate specific membrane domains as the targets for these proteins. Studies on the The plasma membrane of polarized epithelial cells is morphogenesis of different polarized epithelia, including characterized by apical and basal-lateral domains of kidney tubule epithelia (Saxen et al. 1968; Ekblom et al. specific structure and function (Simons and Fuller, 1985; 1981; Saxen, 1987), the trophectoderm in preimplan- Rodriguez-Boulan and Nelson, 1989). Each domain com- tation mammalian embryos (Ekblom et al. 1986; Fleming prises a set of specific proteins. This polarized distri- and Johnson, 1988), and MDCK cells in vitro (Balcar- bution of proteins is functionally important, since it ova-Stander et al. 1984; Vega-Salas et al. 1987; Wang provides the basis for the vectorial uptake, transcytosis, et al. 1990), indicate that cell—cell and cell—substratum and secretion of ions and solutes between biological contact play important roles in initiating the formation of compartments separated by the epithelium (Berridge and structurally and functionally distinct domains of the Oschman, 1972). A fundamental problem in the morpho- epithelial plasma membrane. genesis of polarized epithelia is to understand the mech- The process of generating a plasma membrane domain anisms involved in generating and maintaining these is complex. First, it requires the accumulation of specific plasma membrane domains. proteins in distinct areas of the plasma membrane. Recent studies have focused on the problem of how Second, since the presence of an ion channel or transport newly synthesized membrane proteins are sorted to protein within the incorrect domain may be deleterious to different membrane domains (reviewed by Rodriguez- the functioning of the cell and organism, formation of a Boulan, 1983; Matlin, 1986; Bartlesand Hubbard, 1988). membrane domain requires the removal of proteins that However, for correct sorting of newly synthesized pro- happen to be trapped in the incorrect membrane domain Journal of Cell Science 95, 153-165 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 153 upon cell-cell or cell-substratum contact. It has been a round-bottomed Falcon polypropylene tube that had been coated at the bottom with Iodogen (20jUg; Pierce Chemical suggested that initial accumulation of specific proteins to 12S different regions of the plasma membrane is the result of Co.). A 1 mCi sample of I(ICN) was added to the medium limiting the diffusion of existing proteins, perhaps as a and the cysts were agitated gently for 30 min at room tempera- consequence of the assembly of the tight junction (Pisam ture. The cysts were removed from the tube, diluted with 5 ml DMEM/FBS and pelleted by centrifugation (1500#for 5 min). and Ripoche, 1976; Hertzlinger and Ojakian, 1984; The cysts were washed three times in 10 ml DMEM/FBS. The Ziomek et al. 1980; van Meer and Simons, 1986) and cysts were then either processed directly for sectioning and membrane-cytoskeleton (Nelson and Veshnock, 1986, autoradiography (zero time point) or embedded in collagen gel 1987; Salas et al. 1988). However, the fate of proteins as described above. A fraction of the iodinated cysts were kept trapped in the incorrect domain upon cell-cell or cell- in suspension culture for a further 24 h. At various times, cysts -substratum contact is poorly understood. One approach embedded in collagen gel were fixed with 2% formaldehyde, to this problem has been to implant a foreign protein, that dehydrated and embedded in paraffin. Sections (5 ,um) were cut is normally targeted to the basal-lateral membrane (G with a Leitz microtome, and mounted on glass slides coated protein of vesicular stomatitis virus), into the apical with Histostick (Accurate Chemical Co.) and 10% gelatin. The membrane of polarized MDCK cells and determine its sections were deparaffinized in 100% xylene (20min), rehy- drated through an ethanol series from 100% ethanol to distilled fate (Matlin et al. 1983; Pesonen and Simons, 1983). water, and air dried. The slides were dipped in 50% Kodak These studies showed that G protein was endocytosed atomic emulsion. Excess emulsion was allowed to drain off the from the apical membrane, and a fraction was redirected slides for 1 h. The slides were stored at 4°C in the dark for 3-7 to the basal- lateral membrane by transcytosis. The days, and then developed with Kodak D76 developer. Follow- remainder was apparently degraded. ing fixation and further washing, the sections were counter- In this study, we have taken a different approach to stained with hematoxylin and eosin, and mounted in Perma- these problems by following the fate of endogenous mount. proteins in multicellular MDCK cysts that were induced to reverse cell polarity. Previously, we showed that Antibodies MDCK cysts formed in suspension culture comprise The antibodies used in this study have been extensively characterized: (1) rabbit polyclonal antibody against canine closed monolayers of polarized cells with their apical + + membranes facing the outside and basal-lateral mem- kidney cv-subunit Na ,K -ATPase (for details, see Nelson and Veshnock, 1986; Nelson and Hammerton, 1989). (2) Rat branes facing the central lumen (Wang et al. 1990). In monoclonal antibody against ZO-1, a peripheral membrane this report, we show that transfer of these cysts into a protein specific for the tight junction (for details, see Stevenson collagen gel induces a rapid reversal of polarity, which et al. 1986). (3) Mouse monoclonal antibody to MDCK cell gives rise to cysts in which the apical membranes of cells apical membrane glycoprotein, gpl35 (for details, see Ojakian face the central lumen and basal-lateral membranes face and Schwimmer, 1988). (4) Rabbit polyclonal antibody against the exogenous collagen gel (see also, Chambard et al. canine kidney uvomorulin (Nelson et al. 1990). (5) Rabbit 1981; Nitsch and Wollman, 1980; Wohlwend et al. 1985; polyclonal antibody against type IV collagen (Chemicon). All Garbi et al. 1987). Detailed analysis of the steps involved antibodies were used at the same dilutions as described in the in the disassembly and assembly of membrane domains preceding paper (Wang et al. 1990). indicates that reversal of polarity is accomplished by uptake and intracellular degradation of proteins from the Microscopy old membrane domain, and subsequent accumulation of Transmission electron microscopy and indirect immunofluor- proteins at the new membrane domain. escence microscopy were performed as described in the preced- ing paper (Wang et al. 1990).

Materials and methods Results

Cell culture Morphology of MDCK cysts transferred to collagen gels The growth conditions for forming multicellular MDCK cysts Electron microscopy was used to analyze the ultrastruc- in suspension culture and three-dimensional collagen gels has ture of MDCK cysts transferred from suspension culture been described in detail in the preceding paper (Wang et al. into collagen gel (Fig. 1). MDCK cysts formed in 1990). For transfer to a collagen gel, fully developed cysts suspension culture comprise a closed monolayer of polar- formed in suspension culture for 5-7 days were concentrated by ized cells that surround a central lumen. The nucleus is s low-speed centrifugation (ISOOg'), and approximately 10 cysts located at the pole of the cell facing the lumen and the were resuspended in chilled growth medium containing type I Golgi complex is localized above the nucleus and facing collagen (Noda, 1960) as described in the preceding paper (Wang et al. 1990). The suspension of cysts in collagen was the apical surface (see also, Wang et al. 1990). A few pipetted into plastic Petri dishes and placed in a 37 °C incubator microvilli are present on the outside cell surface facing in a humidified atmosphere of 5 % CO2 in air. The collagen the growth medium. Upon transfer of the cysts into gelled within 5 min and was overlayered with DMEM/FBS collagen gel, little or no morphological changes are (Dulbecco's modified Eagle's medium/foetal bovine serum). detected within ~4h. Approximately 4-6 h after trans- fer, there is a change in the distribution of the nucleus Iodination of MDCK cyst surface proteins and cytoplasmic organelles. The nucleus becomes local- A total of 104 to 10s cysts, that had been formed in suspension ized to the pole of the cell facing the new collagen gel culture, were resuspended in 0.5 ml DMEM/FBS and placed in matrix at the outside surface. The Golgi complex is

154 A. Z. Wang et al. Fig. 1. Electron micrographs of MDCK cysts following transfer from suspension culture to collagen gel. MDCK cysts formed in suspension culture for 5 days (A) comprise closed monolayers of cells surrounding a lumen (1); mierovilli (arrows) are located on the outside surface membrane. Approximately 8 h after transfer to collagen (B) mierovilli are still located on the outside surface membrane. However, in some cells, the Golgi complex (G) is oriented towards the luminal (1) surface membrane. Approximately 16 h after transfer to collagen gel (C) mierovilli (arrows) are detected on both the outside (top) and luminal (1) surface membranes; higher magnification (D) shows two electron-dense structures (tight junctions, tj) at the apices of the contact zone between cells at the boundary of the outside (top) and luminal (1) surface membranes (arrows in D; boxed area in C). Approximately 36h after transfer to collagen gel (E), cells exhibit morphologically complete reversal of polarity; mierovilli (arrows) are exclusively detected on the luminal (1) surface membrane, and the Golgi (G) complex is located between the nucleus and the luminal surface membrane. Bar, 1 jxm. located perinuclearly opposite the new collagen gel incubation. Note also that external cell surface labelling matrix (i.e. facing the luminal surface). These changes in with 125I did not result in labelling of the lateral or the distribution of the nucleus and Golgi complex are the luminal membranes, suggesting that the tight junction first indications of reversal of cell polarity upon transfer remained impermeable to ions and proteins (see Fig. 4, of cysts to collagen gel. below). This was also confirmed by fixing cells in the Subsequent changes in cell morphology are detected presence of Ruthenium Red (data not shown). on the cell surfaces. Approximately 12 h after transfer into collagen gel, we detected the appearance of mierovilli Disassembly and assembly of the apical membrane on the inside-facing, luminal cell surfaces. At this time, domain mierovilli are detected on both inside- and outside-facing Reversal of polarity of an apical membrane protein, cell surfaces (Fig. 1C). This distribution of mierovilli is gpl35. Staining of cryostat sections of MDCK cysts transient and, approximately 16-24 h after transfer, all formed in suspension culture shows that gpl35 is located mierovilli disappeared from the outside-facing cell sur- exclusively at the external plasma membrane (Fig. 2A). faces and are present only on the inside cell surfaces The staining appears continuous along the plasma mem- facing the lumen (Fig. IE). brane. Upon transfer of cysts to collagen gel little or no An important observation of cyst morphology during change is detected in this distribution of gpl35 up to 4 h. these changes in cell organization is that the continuity of However, between 4 and 6 h after transfer, gpl35 staining the epithelial monolayer is always maintained. We did not on the cell surface appeared punctate rather than continu- detect any cell-cell dissociation upon transfer of suspen- ous (Fig. 2B), and staining in the cytoplasm became sion-formed cysts into collagen gel or during subsequent prominent (Fig. 2B,C). The cytoplasmic staining is

Reversal of epithelial polarity 155 gpi35

Fig. 2. Reversal of polarity of the apical membrane protein gpl35 following transfer of MDCK cysts to collagen gel. Cryosections of MDCK cysts stained with a monoclonal antibody against gpl3S. MDCK cysts formed in suspension culture for ~5 days (A) exhibit intense gpl35 staining at the outside surface membrane (arrows); there is little or no staining of the membrane at the contact zone between cells, or at the luminal (1) surface membrane. Approximately 4-6 h after transfer of these cysts to collagen gel (B), the outside surface membrane staining becomes distinctly punctate (arrows) and gpl3S staining appears in the cytoplasm (open arrow). At successive times (C and D, ~8-12h; E, •—12—16h) gpl35 staining of the outside surface membrane decreases in intensity (arrows in E). There is extensive gpl35 staining in the cytoplasm (open arrow in C,D,E), but no luminal surface staining. Approximately 16-24h after transfer to collagen gel (F), cysts begin to exhibit intense gpl35 staining of the luminal surface membrane. In these cysts there is little or no staining of the membrane at the contact zone between cells, the outside surface membrane (open arrows), or cytoplasmic vesicles. Bar, 16jUm. irregular and may represent staining of cytoplasmic ing of the luminal (inside-facing) cell surfaces of cysts vesicles. Between 8 and 12 h after transfer, the amount of (Fig. 2F; Fig. 3A). In the majority of cases, luminal plasma membrane staining relative to that present in- surface staining was detected in only those cysts that itially in the cyst in suspension culture appeared to exhibited little or no outside-facing cell surface staining decrease (compare Fig. 2A, D and E). In some cysts, (Fig. 3A). Thus, the polarity of a marker protein of little or no surface staining was detected (Fig. 2E). The apical membrane domain is completely reversed upon appearance of gpl35 staining in the cytoplasm coincides transfer of a polarized cyst formed in suspension culture with the loss of cell surface staining, suggesting that the to collagen gel. cytoplasmic gpl35 is derived from the plasma membrane Fate of apical membrane proteins labelled with I25I (Figs 2 and 3A). The loss of gpl35 staining at the outside upon transfer of suspension-formed cysts to collagen gel. surface membrane was relatively uniform throughout the To analyze the fate of other apical membrane proteins cells of given cysts (Fig. 3A), indicating that the cell upon transfer of cysts to collagen gel, we surface-labelled surface reorganization was synchronous within individual cysts with 1ZSI. One hour after iodination of cysts in cysts. suspension culture, cell surface labelling is confined to Sixteen to twenty four hours after transfer to collagen the external plasma membrane (Fig. 4A). We did not gel, we began to detect intense, continuous gpl35 stain- detect labelling of the lateral or luminal cell surfaces,

156 A. Z. Wang et al. indicating the presence of a functional tight junction in outside cell surface upon transfer into a collagen gel, and these cysts (Fig. 4A). Six hours after transfer of cysts into are not transferred to the newly forming membrane collagen gel, we detected extensive labelling of the domain on the luminal surface. external surface and subjacent cortical cytoplasm (data not shown). However, 18-24h after transfer little or no labelling of the cysts was observed (Fig. 4C, D). Analysis Assembly and disassembly of the tight junction protein of 125I-labelled cysts kept in suspension culture during ZO-1 upon transfer of cysts to collagen gel this time retained extensive external surface labelling Cysts formed in suspension culture exhibit intense ZO-1 after 16h (Fig. 4B). Taken together these results provide staining of cells at the apex of lateral membranes at the strong evidence that proteins are internalized from the boundary with the external cell surface (Fig. 5A). Upon

gpl35 0 outside cell surface ZO-1 0 apex of lateral membrane at outside surface • cytoplasmic vesicles H apex of lateral membrane • luminal cell surface 100 at lumen surface both

80 1 80 L 60 60

40 1 40

20 20

1 4 8 12 16 24 48 0 8 16 24 48 (32) (13) (15) (16) (16) (21) (11) (15) (14) (13) (17) (17) Hours (AT) Hours (AO

+ + c Na ,K -ATPase 0 lateral and outside surfaces lateral and luminal surfaces both 120

100

80

60 Fig. 3. Histograms of gpl3S, ZO-1 and Na+,K+-ATPase distributions in MDCK cysts during reversal of epithelial polarity. Cryosections of MDCK cysts transferred to 40 collagen gel for different times (1-48 h) were stained with gpl35 (A), ZO-1 (B), or Na+,K+-ATPase (C) antibodies. At different times, populations of cysts (A7) were scored for surface membrane (outside or luminal), and cytoplasmic 20 vesicle staining; note that cysts exhibited either outside or luminal surface membrane staining (=100%); in the case of 0 8 16 24 48 gpl3S, a high proportion of cysts exhibiting outside surface (16) (15) (14) (23) (11) staining also exhibited cytoplasmic vesicle staining. The Hours (AO standard error of the mean is indicated.

Reversal of epithelial polarity 157 transfer into collagen gel this distribution appeared cysts is apparently transient, since 16-24 h after transfer unchanged for approximately 12h (Figs 5B, and 3B). to collagen gel ZO-1 staining at the original external Between 12 and 16 h after transfer, additional spot-like location begins to disappear (Fig. 5B and D). Thus, distributions of ZO-1 are detected at the apex of the upon transfer of cysts to collagen gel the polarity of the lateral membrane bordering the luminal cell surface tight junction is reversed completely. (Figs 5C and 3B). Significantly, we still detected the original ZO-1 distribution in many of these cysts (Figs 5C and 3B). In grazing sections, we did not detect Distribution of endogenous type W collagen during linear ZO-1 staining along the lateral membrane, indi- reversal of cell polarity cating that the external and luminal tight junctions are Loss of type W collagen from the lumen of MDCK cysts distinct entities and are not linked along the contact zone upon transfer into a type I collagen gel. Staining of between adjacent cells. Independent evidence of the suspension-formed cysts with antibodies to type IV presence of two tight junctions at this stage is provided by collagen shows that the luminal cell surface is lined with electron micrographs of cells showing electron-dense type IV collagen (see preceding paper, Wange£ al. 1990). patches at both ends of the lateral membrane (Fig. ID). Upon transfer of these cysts into a type I collagen gel, The presence of tight junctions on both sides of these there is little or no change in the presence of type IV

Fig. 4. Autoradiograms of MDCK cysts formed in suspension culture and labelled at the outside surface membrane with 125I, and then transferred to collagen gel. MDCK cysts formed in suspension culture for 5 days were labelled at the external surface membrane with 125I catalyzed with Iodogen. Labelled cysts were embedded in collagen gel. At Oh (A), 18h (C) or 24h (D) after transfer to collagen gel, cysts were fixed, embedded in paraffin, sectioned and processed for autoradiography. After emulsion development, the sections were counterstained with hematoxylin/eosin and photographed. At Oh (A), the outside surface membrane of cysts is heavily labelled; there is no labelling of the membrane at the contact zone between cells, or the luminal surface membrane. At 18-24h after transfer (C,D), there is little or no labelling of any of the surface membranes (arrows). Note that in populations of 125I-labelled cysts kept in suspension culture during this period, extensive labelling of the outside surface remained after 16 h (B). Bar, 10 /Am.

158 A. Z. Wang et al. collagen in the central lumen after 4h (Fig. 6A, C). ZO-1 However, between 12 and 16 h after transfer, the inten- sity of type IV collagen staining decreases in the lumen (not shown). Sixteen to twenty-four hours after transfer, type IV collagen staining was not observed at the luminal cell surface for the majority of cysts (Fig. 6B, D). Loss of type TV collagen coincides with the appearance of gpl35 and ZO-1 at the luminal surface. Double immunofluorescence was used to compare the distri- butions of type IV collagen with either gpl35 or ZO-1 during reversal of cell polarity (Fig. 6). Four hours after transfer to collagen gel, gpl35 is present on the external cell surface (Fig. 6A') as described in detail above (see Fig. 2). Type IV collagen is detected at the luminal membrane of the same cysts (Fig. 6A). Sixteen hours after transfer we detect cysts that stained intensely at the luminal cell surface with gpl35 antibodies (Fig. 6B'), but that did not exhibit type IV collagen staining (Fig. 6B). A similar result was obtained when the distributions of type IV collagen and ZO-1 were compared. Staining of ZO-1 is confined to the apex of the lateral membrane at the external cell surface (Fig. 6C) when type IV collagen was detected in the lumen of the cyst (Fig. 6C). How- ever, 16-24 h after transfer to collagen gel, we detected cysts with little or no type IV collagen staining at the lumen (Fig. 6D) and ZO-1 staining at the luminal side of the lateral membrane (Fig. 6D').

Redistribution of a basal-lateral plasma membrane protein, Na+,K+-ATPase, upon transfer of cysts into collagen gel The distribution of Na+,K+-ATPase, a marker protein of the basal-lateral plasma membrane, is polarized in sus- pension-formed MDCK cysts. It is located at the lateral and luminal membranes but not at the outside surface membrane (Figs 7A,B and 3C). Upon transfer of cysts to a collagen gel, little or no change in this distribution was detected up to 16-20h (Figs 7C and 3C). Twenty-four hours after transfer, Na ,K+-ATPase staining also became localized to the external membrane surface. At Fig. 5. Reversal of polarity of the tight junction protein ZO- this time we also detected staining on the lateral and 1 in MDCK cysts formed in suspension culture and then luminal membranes (Figs 7D and 3C). Further analysis transferred to collagen gel. Cryosections of MDCK cysts of cysts 48 h after transfer to collagen gel shows distinct were stained with a monoclonal antibody against ZO-1. Na+,K+-ATPase staining on the lateral membrane sur- MDCK cysts formed in suspension culture (A) exhibit a dot- faces and also less-distinct staining on the external like staining pattern of ZO-1 at the apex of the cell contact zone at the boundary with the outside surface membrane. surface. There is little or no staining detected on the luminal surface (Figs 7E and 3C). Thus, the distribution Arrowheads demarcate the apices of the contact zone at the + + external and luminal (1) surface membranes (see also below). of Na ,K -ATPase is reversed upon transfer of cysts to At —12h after transfer to collagen gel (B), the ZO-1 staining collagen gel. of MDCK cysts appears similar to that in cysts formed in suspension culture (compare with A). However, ~16h after Distribution of the cell adhesion protein, uvomorulin, transfer (C), ZO-1 staining begins to be detected at the apex during reversal of epithelial cell polarity of the contact zone between cells at both the boundary with Indirect immunofluorescence of uvomorulin in suspen- the external surface membrane and the luminal surface sion-formed cysts shows the protein is localized predomi- membrane (arrowheads demarcate the ends of the contact nantly to the membrane at the contact zone between zone between cells). After ~24-36h in collagen gel (D), ZO- 1 staining is now detected only at the apex of the contact zone adjacent cells (Fig. 8A). Little or no staining is located on at the boundary with the luminal surface membrane. Bar, the outside cell surface, and the staining of the luminal 16 jum. surface is much less intense compared with that on the lateral membranes. Upon transfer of cysts into a collagen gel, staining of the lateral membranes was maintained at all times examined (Fig. 8B-D). This is consistent with

Reversal of epithelial polarity 159 the earlier observation that cell-cell contact is not lost Discussion upon transfer of cysts to collagen gel and reversal of cell surface polarity (Fig. 1). The patchy, less-intense stain- In the preceding paper, we showed that MDCK cells ing of the luminal membrane disappears approximately have retained the ability to form cystic structures in vitro 24h after transfer (Fig. 8C). After 24-30h a patchy-like that comprise a closed monolayer of polarized cells that staining is also detected on the external cell surface in surround a fluid-filled lumen (Wang et al. 1990). These addition to the prominent staining at the contact zone structures appear to develop in stages that are similar to between cells (Fig. 8D). those in the formation of polarized epithelia in complex

Collagen gp135

Fig. 6. Distributions of endogenous type IV collagen, the apical membrane 16h protein gpl35 and the tight junction protein ZO-1 upon transfer of MDCK cysts to collagen gel. Cryosections of MDCK cells were processed for double immunofiuorescence with antibodies to type IV collagen (A,B,C,D; rabbit polyclonal, TRITC channel), and either gpl3S (A',B'; mouse monoclonal, FITC channel) or ZO-1 (C',D'; rat monoclonal, FITC channel). Four hours after transfer of MDCK cysts formed in suspension culture (5 days) to collagen gel, type IV collagen staining is detected at the luminal (1) surface membrane (A,C). Double immunofluorescence of these cysts shows gpl35 staining exclusively to the outside surface membrane (A', open arrow) and ZO-1 staining exclusively to the apex of the contact zone between cells at the outside surface membrane (C, open arrow); neither protein is located at the luminal surface membrane (A',C, arrowhead). Approximately 16—24h 16h after transfer of cysts to collagen gel, type IV collagen staining is no longer detected at the luminal surface membrane (B,D, arrowhead). gpl3S and ZO-1 staining are now detected at the luminal membrane surface Collagen ZO-1 (B',D'). Bar, 16,um. 160 .4. Z. Wangetal. N a+ K+- ATPase

Fig. 7. Reversal of polarity of the basal-lateral membrane protein Na+,K+-ATPase following transfer of MDCK cysts to collagen gel. Cryosections of MDCK cysts were stained with antibodies against ce-subunit Na+,K+-ATPase. MDCK cysts formed in suspension culture (~S days) exhibit intense staining of the membrane at the contact zone between adjacent cells, and the luminal (1) surface membrane (A,B; arrows). The external surface membrane exhibits little or no Na+,K+-ATPase staining. Approximately 12h after transfer to collagen gel (C) there is little change in Na+,K+-ATPase staining. Twenty-four hours after transfer (D) some cysts exhibit staining at the external surface membrane. After ~48h (E), MDCK cysts show intense Na+,K+-ATPase staining on the membrane at the contact zone between cells and on the external surface membrane (arrows); there is little or no staining of the luminal (1) surface membrane. Note that throughout the time in collagen gel, Na+,K+- ATPase staining at the contact zone between cells appeared invariable. Bar, 16 fim.

three-dimensional environments in vivo. The polarity of Maintenance of cell-cell contact during reversal of the cells in these cysts depended upon the presence of epithelial cell polarity cell-cell contacts and the location of cell-substratum Electron and light microscopy showed that upon transfer contact. We have extended this analysis in the present of a cyst to collagen gel, cell-cell contact within the study to show that upon transfer of MDCK cysts formed closed monolayer of polarized MDCK cells is not dis- in suspension culture to a type I collagen gel matrix the rupted. Thus, the structural continuum of the epithelium cells reverse cytoplasmic and plasma membrane polarity. was maintained throughout the time of reversal. Import- Although the phenomenon of reversal of epithelial cell antly, indirect immunofluorescence microscopy revealed polarity in culture has been reported previously (Cham- the presence of the cell adhesion protein, uvomorulin bard et al. 1981; Nitsch and Wollman, 1980; Wohlwend (Fig. 8), and the tight junction protein, ZO-1 (Fig. 5), at et al. 1985; Garbi et al. 1987), this study is the first to the contact zone between cells throughout the period of analyze the reversal of polarity of specific membrane and incubation of the suspension-formed MDCK cysts in membrane-associated proteins. This approach provides collagen gel. In addition, following external surface insights into how plasma membrane domains are disas- labelling with 125I, we did not detect labelling of the sembled and assembled, and the role of extracellular cues lateral surfaces of cells, indicating that the tight junction in triggering the development of membrane domains and remained impermeable (Fig. 4). These observations are cell polarity. significant, since they rule out the possibility that reversal

Reversal of epithelial polarity 161 of plasma membrane polarity is simply a consequence of 125I-labelled proteins from the external surface reflected loss of cell-cell contact and diffusion of membrane normal turnover of the labelled proteins rather than proteins between different domains. Reversal of mem- specific internalization and degradation due to transfer of brane polarity must, therefore, be an active process the cyst to collagen gel. However, analysis of cysts requiring co-ordinate disassembly and assembly of differ- labelled and subsequently maintained in suspension cul- ent membrane domains. ture demonstrated extensive outside cell surface labelling remaining at 16 h. In addition, the metabolic half-life of gpl35 on the apical membrane is normally >24h (D. Disassembly of membrane domains: mechanism(s) for Wollner and W. J. Nelson, unpublished results). This removing membrane proteins half-life is too long to explain the loss of protein from the Upon transfer of MDCK cysts into collagen gel we cell surface of cysts transferred to collagen gel as due to detected a dramatic change in the distribution of the normal protein turnover. Thus, we conclude that gpl35 apical membrane protein gpl35 (Fig. 2). In cysts formed and other apical proteins are rapidly internalized from the in suspension culture, gpl35 is located as a continuous cell surface and degraded intracellularly. line of staining at the outside cell surface. Approximately Our observations on the tight junction protein ZO-1 6h after transfer, the cell surface staining appeared during reversal of polarity provide indirect evidence that distinctly punctate in the cytoplasm subjacent to the components of the original tight junction are also not re- plasma membrane. This punctate gpl35 staining may used in the formation of a new junction (Fig. 5). Twelve represent cytoplasmic vesicles. Sixteen to twenty four to sixteen hours after transfer of cysts to collagen gel, we hours after transfer all of the gpl35 staining of the detected by immunofluorescence and electron mi- external cell surface had disappeared. croscopy the presence of two tight junctions at either end Two scenarios can be envisaged for the fate of gpl35 of the contact zone between adjacent cells. There did not taken up from the external cell surface. First, gpl35 may appear to be any staining of ZO-1 along the contact zone be targeted directly to the luminal side of the cell and between these two points, indicating the presence of contribute to the formation of the new apical membrane. separate ZO-l/tight junction complexes. Subsequently, Alternatively, gpl35 may be targeted to lysosomes and the original ZO-1 staining at the external apex of the degraded. Detailed analysis shows that gpl35 together lateral membrane disappeared, but after the appearance with other proteins of the outside surface domain are not of the new ZO-1 staining at the luminal end of the relocated to the luminal cell surface domain (Figs 2 and membrane. Hence, there is no evidence at present of a 4). Proteins labelled with 12SI on the external cell surface precursor-product relationship between the two junc- of suspension-grown cysts were not detected on the tions. These results strongly suggest that components of luminal cell surface. The degree of cell surface labelling the original tight junction are not re-utilized during decreased rapidly with kinetics similar to those for the reversal of tight junction polarity. That other membrane loss of gpl35. It is formally a possibility that the loss of and membrane-associated proteins are not re-utilized Uvomorulin

Fig. 8. Reversal of polarity of the cell adhesion protein uvomorulin in MDCK cysts transferred to collagen gel. Cryosections of MDCK cysts were stained with antibodies against uvomorulin. MDCK cysts formed in suspension culture (5 days) exhibit intense uvomorulin staining on the membrane at the contact zone between cells (A, arrow). Twelve hours after transfer to collagen gel, there is little change in uvomorulin localization; note that there is some uvomorulin staining on the luminal (1) surface membrane (arrow). Approximately 24h after transfer (C), uvomorulin staining remains intense on the membrane at the contact zone between cells, and staining at the outside surface membrane can be detected (arrows). Little or no staining is detected on the luminal surface membrane. After 48 h (D), uvomorulin staining appears similar to that in cysts at 24 h. Note that throughout the time that the cysts were in collagen gel, uvomorulin staining appeared constant on the membrane at the contact zone between cells. Bar,

162 A. Z. Wang et al. during reversal of cell polarity has been more difficult to biosynthetic pathways of apical and basal-lateral mem- determine. The change in polarity of Na+,K+-ATPase brane proteins changes upon transfer of cysts to collagen occurs considerably more slowly than does that of gpl35 gel. This problem is currently under investigation. How- (Fig. 3). A problem in this analysis was that the lateral ever, ultrastructural analysis of cysts indicates that this membrane staining of Na+,K+-ATPase and uvomorulin may be an early event in reversal of cell polarity. Electron appeared constant during reversal of polarity. Although micrographs of MDCK cysts at different times following we detected loss of staining of these proteins from the transfer to collagen gel revealed a change in the orien- luminal membrane, the intensity of staining was usually tation of the Golgi complex relative to the location of the low relative to that of gpl35. Hence, it has been difficult apical membrane domain and the nucleus approximately to determine whether loss of Na+,K+-ATPase and uvo- 4-6 h after transfer. At present, we do not know how the morulin from the luminal membrane involved uptake spatial polarity of the Golgi complex is reversed. It is into cytoplasmic vesicles and then degradation (see possible that microtubules may play a role, since, in below). polarized epithelial cells, microtubules appear to interdi- Although these results indicate that several classes of gitate near the Golgi complex and subjacent to the apical proteins are not transferred between membrane domains membrane (Drenckhan and Dermietzel, 1988). Further- and re-utilized during reversal of epithelial polarity, more, recent studies have reported that drugs that recent studies show that cellular mechanisms exist for disrupt microtubule structure also cause a loss of polar- transporting of proteins from one membrane domain to ized delivery of proteins to the apical membrane (Rindler another as part of the vectorial function of polarized et al. 1987; Eilers et al. 1989). Current studies are epithelial cells (Bartles et al. 1987). In intestinal epi- involved in analyzing the distribution of microtubules thelia, specific proteins internalized from the basal-la- during polarity reversal to address their role in this teral surface are targeted to the apical membrane (e.g. process. poly Ig receptor), while other specific proteins internal- Although the biosynthetic machinery of the cells may ized from the apical membrane are redirected to the reorient rapidly (3-6 h) following transfer of cysts to basal-lateral membrane (e.g. Fc receptor) (reviewed by collagen gel, the accumulation of proteins at the new Mostov and Simister, 1985). Significantly, transfection membrane domains is much slower. In the case of the studies with the poly Ig receptor show that regulated apical membrane protein gpl35, the appearance of pro- transcytosis exists also in MDCK cells (Mostov and tein on the luminal cell surface begins —16 h after Deitcher, 1986). However, there is a distinction between transfer. This apparent delay may be caused by the transcytosis of the poly Ig receptor and the uptake and presence of an endogenous containing type apparent degradation of membrane proteins observed IV collagen (Fig. 6) and laminin (data not shown) on the during reversal of cell polarity. Uptake of the poly Ig luminal cell surface, which did not disappear until receptor from the basal-lateral surface is induced by approximately 16-20 h after transfer of cysts to collagen binding poly Ig, resulting in the formation of a ligand-re- gel. We have no direct evidence that the loss of type IV ceptor complex that then enters the endocystic pathway collagen from the luminal cell surface is the result of and is transported to the apical membrane (Sztul et al. specific degradation by a collagenase (a metalloprotei- 1983; Solari and Kraehenbuhl, 1984; Hopped al. 1985). nase). However, it is interesting to note that incubation of At present, we do not know how uptake of gpl35 from the cysts in the presence of the metalloproteinase inhibitor, membrane is induced in MDCK cysts transferred to 1,10-phenanthroline, inhibited the loss of type IV col- collagen gel. However, it seems unlikely that it is a result lagen from the lumen of the cysts (data not shown). of binding a ligand. In this context, analysis of Na+,K+- Concomitant with the loss of type IV collagen staining ATPase in MDCK cells also indicates that this basal-la- from the luminal surface, we detected the appearance teral membrane protein is not normally internalized into there of gpl35 and ZO-1. These results indicate that endosomes (Gottlieb et al. 1986). although the orientation of the protein biosynthetic machinery may have reversed polarity soon after transfer Assembly of membrane domains of cysts to collagen gel, the presence of type IV collagen Given that proteins disassembled from old membrane at the luminal cell surface blocks the appearance of newly domains are not reassembled into new membrane synthesized protein at this membrane domain. The basis domains, the accumulation of protein in the latter must for this apparent inhibitory effect is not known at present. be a consequence of the targeting of newly synthesized It is possible that gpl35 and other apical membrane proteins. Studies on the biosynthesis of viral glyco- proteins are targeted to the luminal membrane but either proteins in polarized MDCK cells have concluded that are not inserted into the membrane, or are inserted and the sorting of proteins destined for either the apical or then rapidly internalized (see above). basal—lateral membranes occurs intracellularly, possibly The appearance of Na+,K+-ATPase on the outside cell in the late Golgi complex (Rodriguez-Boulan and Saba- surface may be a consequence of the gradual accumu- tini, 1978; Matlin and Simons, 1984; Rindlere* al. 1985; lation of newly synthesized protein targeted from the Misek et al. 1984). In polarized MDCK cells, the Golgi Golgi complex to this membrane domain (Caplan et al. complex is located in the perinuclear area towards the 1987). It is noteworthy that accumulation of Na+,K+- apical pole of the cell indicating that the biosynthetic ATPase on the external membrane domain was not machinery of these cells may be spatially polarized (see detected by immunofluorescence until ~16 to 24 h after Fig. 1). At present, we do not know when the polarity of transfer. This coincided with the time when gpl35 and

Reversal of epithelial polarity 163 other components of this membrane domain had been 06927, RR 05539). W. J. Nelson is a recipient of an Established internalized and degraded. Investigator Award from the American Heart Association.

Conclusions References In this and the preceding paper (Wang et al. 1990) we have sought new insights into the morphogenesis of a BALCAROVA-STANDER, J., PFEIFFER, S. E., FULLER, S. D. AND polarized epithelium. We have shown that MDCK cells SIMONS, K. (1984). Development of cell surface polarity in have retained the morphogenetic potential to form three- epithelial Madin-Darby canine kidney (MDCK) cell line. EMBOJ. dimensional cysts comprising a closed monolayer of 3, 2687-2694. polarized cells surrounding a central lumen. The orien- BARTLES, J. R., FERACCI, H. M., STEIGER, B. AND HUBBARD, A. L. (1987). Biogenesis of the rat hepatocyte plasma membrane in vivo: tation of cell polarity depends upon the location of the comparison of the pathways taken by apical and basolateral basal lamina. The steps involved in the morphogenesis of proteins using subcellular fractionation. J. Cell Biol. 105, a polarized three-dimensional MDCK epithelium in- 1241-1252. volve: (1) cell aggregation; (2) formation of extensive BARTLES, J. R. AND HUBBARD, A. L. (1988). Plasma membrane cell-cell contacts; (3) development of cell surface and protein sorting in epithelial cells. Do secretory pathways hold the key? Trends biochem. Sci. 13, 181-184. cytoplasmic polarity; (4) accumulation of a basal lamina; BERRIDGE, M. J. AND OSCHMAN, J. L. (1972). Transporting and (5) establishment of the epithelial axis and the Epithelia, pp. 91-108. Academic Press, London, New York. development of a central lumen. These steps appear CAPLAN, M. J., ANDERSON, C. H., PALADE, G. E. AND JAMIESON, J. similar to those described for the formation of the D. (1987). Intracellular sorting and polarized cell surface delivery + + trophectoderm in preimplantation mammalian embryos, of (Na ,K )ATPase, an endogenous component of MDCK cell basolateral plasma membranes. Cell 46, 623-631. and for the development of kidney tubule epithelium, CHAMBARD, M., GABRION, J. AND MAUCHAMP, J. (1981). Influence suggesting that the mechanisms involved may be com- of collagen gel in the orientation of epithelial cell polarity: follicle mon to different epithelia. formation from isolated thyroid cells and from preformed monolayers. J. Cell Biol. 91, 157-167. Significantly, we have demonstrated directly several DRENCKHAN, D. AND DERMIETZEL, R. (1988). Organization of the important contributions of cell-cell and cell-substratum filament cytoskeleton in the intestinal brush border: a contact in the morphogenesis of a polarized epithelium. quantitative and qualitative immunoelectron microscope study. Our results show that in the absence of cell-substratum J. Cell Biol. 107, 1037-1048. contact, cell—cell contact alone is sufficient to induce EILERS, U., KLUMPERMAN, J. AND HAURI, H.-P. (1989). Nocodozole, a microtubule-active drug interferes with apical protein delivery in formation of two biochemically distinct membrane cultured intestinal cells (Caco-2). J. Cell Biol. 108, 13-22. domains containing either an apical membrane protein, EKBLOM, P., MlETLlNIN, A., VlRTANEN, I., WAHLSTROM, T., gpl35, or a basal-lateral membrane protein, Na+,K+- DAWNAY, A. AND SAXEN, L. (1981). In vitro segregation of the ATPase. In the presence of a defined substratum, the metanephric . Devi Biol. 84, 88-95. apical membrane protein gpl35 accumulates on plasma EKBLOM, P., VESTWEBER, D. AND KEMLER, R. (1986). Cell-matrix interactions and cell adhesion during development. A. Rev. not in contact with cells or substratum. Biol. 2, 27-47. Cell-cell contact is necessary for the development of the FLEMING, T. P. AND JOHNSON, M. H. (1988). From egg to epithelial axis, and the formation of a central lumen, but epithelium. A. Rev. Cell Biol. 4, 459-485. it is not sufficient. Inhibition of type IV collagen accumu- GARBI, C, HASCIA, A. AND NITSCH, L. (1987). Cell polarity and lation inhibits tight junction and lumen formation. morphogenetic properties of Fischer rat thyroid cells (FRT) cultured in suspension or embedded in different gels. Cell molec. Hence, formation of a basal lamina and cell—substratum Biol. 33, 293-305. contact are required for the development of the epithelial GOTTLIEB, T. A., GONZALEZ, A., RIZZOLO, L., RINDLER, M. J., axis. Significantly, the formation of membrane domains ADESNIK, M. AND SABATINI, D. D. (1986). Sorting and involves the internalization and degradation of incor- endocytosis of viral glycoproteins in transfected polarized cells. J. Cell Biol. 102, 1242-1255. rectly localized proteins, and the gradual accumulation of HERTZLINGER, D. A. AND OJAKIAN, G. K. (1984). Studies on the domain-specific proteins either by recruitment of pro- development and maintenance of epithelial cell surface polarity teins from existing pools on the membrane, or by with monoclonal antibodies. J. Cell Biol. 98, 1777-1787. targeting newly synthesized proteins to the appropriate HOPPE, C. A., CONNOLLY, T. P. AND HUBBARD, A. L. (1985). Transcellular transport of polymeric IgA in the rat hepatocyte: membrane domain. biochemical and morphological characterization of the transport pathway. J. Cell Biol. 101, 2113-2123. HUBBARD, A. L., STEIGER, B. AND BARTLES, J. R. (1989). Biogenesis We thank Pam Veshnock and Dr Steven Warren for their of endogenous plasma membrane proteins in epithelial cell. A Rev. early contributions to defining conditions for the growth of Physiol. 51, 755-770. MDCK cysts, Jane Wang for excellent technical assistance in MATLIN, K. S. (1986). The sorting of proteins to the plasma preparing samples for immunofluorescence and electron mi- membrane in epithelial cells. J. Cell Biol. 103, 2565-2568. croscopy, Betty Tallardy (Experimental Histopathology Ser- MATLIN, K., BAINTON, D. F., PESONEN, M., LOUVARD, D., vice) for the preparation of cryo-sections, and to Secretarial GENTYN, N. AND SIMONS, K. (1983). Transepithelial transport of a Services for typing the manuscript. In addition, we thank Dr viral membrane glycoprotein implanted into the apical plasma Bruce Stevenson, Yale University, for the generous gift of ZO-1 membrane of Madin-Darby canine kidney cells. I. Morphological antibodies, and members of the Nelson laboratory for anti- evidence. J. Cell Biol. 97, 627-637. MATLIN, K. S. AND SIMONS, K. (1984). Sorting of an apical plasma bodies and discussions. This work was supported in part by membrane glycoprotein occurs before it reaches the surface in grants from the NSF to W. J. Nelson (DSB 8609091 and DIR cultured epithelial cells. J. Cell Biol. 99, 2131-2139. 8811434) from the NIH to W. J. Nelson (GM 35527), G. K. MISEK, D. E., BARD, E. AND RODRIGUEZ-BOULAN, E. (1984). Ojakian (DK-30537) and the Institute for Cancer Research (CA Biogenesis of epithelial cell polarity: intracellular sorting and

164 A. Z. Wang et al. vectorial exocytosis of an apical plasma membrane glycoprotein. infected with temperature sensitive viruses. J. Cell Biol. 104, Cell 39, 537-546. 231-252. MOSTOV, K. E. AND DEITCHER, D. L. (1986). Polymeric RlNDLER, M. J., IVANOV, I. E. AND SABATINI, D. D. (1987). immunoglobulin receptor expressed in MDCK cells transcytoses Microtubule-acting drugs lead to the non-polarized delivery of the lgA. Cell 46, 613-621. influenza hemagglutinin to the cell surface of polarized Madin- MOSTOV, K. E. AND SIMISTER, N. E. (1985). Transcytosis. Cell 43, Darby canine kidney cells. J. Cell Biol. 104, 231-252. 389-390. SALAS, R. J. I., VEGA-SALAS, D. E., HOCHMAN, J., RODRIGUEZ- NELSON, W. J. AND HAMMERTON, R. W. (1989). A BOULAN, E. AND EDIDIN, M. (1988). Selective anchoring in the membrane-cytoskeletal complex of Na+,K+-ATPase, ankyrin and specific plasma membrane domain: a role in epithelial cell polarity. fodrin in Madin-Darby canine kidney (MDCK) cells: implications J. Cell Biol. 107, 2363-2376. for the biogenesis of epithelial cell polarity. J. Cell Biol. 108, SAX£N, L. (1987). Organiogenesis of the Kidney. Cambridge 893-902. University Press, Cambridge. NELSON, W. J., SHORE, E. M., WANG, A. Z. AND HAMMERTON, R. SAXEN, L., KOSKIMIES, O., LAHTI, R., MIETINEN, M., RAPOLA, J. W. (1990). Identification of a membrane-cytoskeletal complex AND WASHIOVAA, J. (1968). Differentiation of kidney mesenchyme containing the cell adhesion protein uvomorulin, ankyrin and in an experimental model system. Adv. Morph. 7, 251-293. fodrin in Madin-Darby canine kidney epithelial cells. J. Cell Biol. SIMONS, K. AND FULLER, S. D. (1985). Cell surface polarity in (in press). epithelia. A. Rev. Cell Biol. 1, 243-288. NELSON, W. J. AND VESHNOCK, P. J. (1986). Dynamics of SOLARI, R. AND KRAEHENBUHL, J.-P. (1984). Biosynthesis of the IgA membrane-skeleton (fodrin) organization during development of antibody receptor: a model for transepithelial sorting of a polarity in Madin-Darby canine kidney epithelial cells, jf. Cell Biol. membrane glycoprotein. Cell 36, 61-71. 103, 1751-1765. STEVENSON, B. R., SILICIANO, J. D., MOOSEKER, M. S. AND NELSON, W. J. AND VESHNOCK, P. J. (1987). Ankyrin binding to GOODENOUGH, D. A. (1986). Identification of ZO-1: a high Na+,K+-ATPase and implications for the organization of molecular weight polypeptide associated with the tight junction membrane domains in polarized cells. Nature, Land. 328, 533-536. (zonula occludens) in a variety of epithelia. jf. Cell Biol. 103, NITSCH, L. AND WOLLMAN, S. H. (1980). Ultrastructure of 755-766. intermediate stages in polarity reversal of thyroid epithelium in SZTUL, E. S., HOWELL, K. E. AND PALADE, G. E. (1983). follicles in suspension culture. J. Cell Biol. 86, 875-880. Intracellular and transcellular transport of secretory component NODA, H. (1960). Physico-chemical studies on the soluble collagen of and albumin in rat hepatocytes. J. Cell Biol. 97, 1582-1591. rat-tail tendon. Biochim. biophys. Ada 17, 92-98. VAN MEER, G. AND SIMONS, K. (1986). The function of tight OJAKIAN, G. K. AND SCHWIMMER, R. (1988). The polarized junctions in maintaining differences in lipid composition between distribution of an apical cell surface glycoprotein is maintained by the apical and the basolateral cell surface domains of MDCK cells. interactions with the cytoskeleton of Madin-Darby canine kidney EMBOJ. 5, 1455-1464. cells. J. Cell Biol. 107, 2377-2388. VEGA-SALAS, D. E., SALAS, P. J. I., GUNDERSEN, D. AND PESONEN, M. AND SIMONS, K. (1983). Transepithelial transport of a RODRIGUEZ-BOULAN, E. (1987). Formation of the apical pole of viral membrane glycoprotein implanted into the apical plasma epithelial (Madin-Darby canine kidney) cells: polarity of an apical membrane of Madin-Darby canine kidney cells. II. Immunological protein is independent of tight junctions while segregation of a quantitation.J. Cell Biol. 97, 638-643. basolateral marker requies cell-cell interactions. J. Cell Biol. 104, PlSAM, M. AND RlPOCHE, P. (1976). Redistribution of surface 905-916. macromolecules in dissociated epithelial cells. .7. Cell Biol. 71, WANG, A. Z., OJAKIAN, G. K. AND NELSON, W. J. (1990). Steps in 907-920. the morphogenesis of a polarized epithelium. I. Uncoupling the RODRIGUEZ-BOULAN, E. (1983). Membrane biogenesis, enveloped roles of cell-cell and cell-substratum contact in establishing plasma RNA viruses and epithelial polarity. In Modern Cell Biology (Satir, membrane polarity in multicellular epithelial (MDCK) cysts. B. M., ed.), vol. 1, pp. 119-170. New York: Alan R. Liss. J. Cell Sci. 95, 137-151. RODRIGUEZ-BOULAN, E. AND NELSON, W. J. (1989). Morphogenesis WOHLWEND, A., MOTESANO, R., VASSALLI, J.-D. AND ORCI, L. of the polarized epithelial cell phenotype. Science 245, 718-725. (1985). LLC-PK! cysts: a model for the study of epithelial RODRIGUEZ-BOULAN, E. AND SABATINI, D. D. (1978). Asymmetric polarity. J. cell. Physiol. 125, 533-539. budding of viruses in epithelial monolayers: a model system for ZlOMEK, C. A., SCHULMAN, S. AND EDIDIN, M. (1980). study of epithelial polarity. Proc. natn. Acad. Sci. U.S.A. 75, Redistribution of membrane proteins in isolated mouse intestinal 5071-5075. epithelial cells. J. Cell Biol. 86, 849-857. RlNDLER, M. J., IVANOV, I. E., PLESKEN, H. AND SABATINI, D. D. (1985). Polarized delivery of viral glycoproteins to the apical and basolateral membranes of Madin-Darby canine kidney cells (Received 7 September 1989 - Accepted 20 October 1989)

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