Steps in the morphogenesis of a polarized epithelium II. Disassembly and assembly of plasma membrane domains during reversal of epithelial cell 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 cell polarity 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.