Abstract. The time course of development of polarity of an apical (184-kD) and a basolateral (63-kD) plasma membrane protein of Madin-Darby canine kidney cells was followed using semiquantitative immunofluorescence on semithin (,,o0.5-gm) frozen sections and monoclonal antibody probes. The 184-kD protein became highly polarized to the apical pole within the initial 24 h both in normal medium and in 1-5 IxM Ca 2÷, which results in well-spread, dome-shaped cells, lacking tight junctions and other lateral membrane interactions. In contrast, the basolateral 63-kD membrane protein developed full polarity only after incubation in normal Ca 2+ concentrations for >72 h, a time much longer than that required for the formation of tight junctions (,,o18 h) and failed to polarize in 1-5 IxM Ca 2+. These results demonstrate that intradomain restriction mechanisms independent of tight junctions, such as self-aggregation or specific interactions with the submembrane cytoskeleton, participate in the regionalization of at least some epithelial plasma membrane proteins. The full operation of these mechanisms depends on the presence of normal cell-cell interactions in the case of the basolateral 63-kD antigen but not in the case of the apical 184-kD protein. SECRETORY and transporting epithelia carry out a variety of vectorial functions that depend on the polarization of the plasma membrane into apical and basolateral domains (52, 59). Tight or occluding junctions, located at the boundary between basolateral and apical regions (16,20), confer on the cell layer the property of a selective barrier to the diffusion of ions and macromolecules (42) and appear to play a role in keeping molecules from both domains segregated (10). Recent work has shown that tight junctions constitute a fence for the movement of lipids in the outer leaflet in the bilayer but not for those in the inner leaflet (11,68).A passive fence, however, can maintain but not generate polarity. Other mechanisms have to be postulated to perform this task. In confluent monolayers, with well-developed apical and basolateral poles separated by fight junctions, intracellular sorting mechanisms operating at the level of the Golgi apparatus, have been shown to participate in a vectorial delivery of apical and basolateral proteins to the respective surface domain (4, 37, 39, 46, 51). However, epithelial cells can also generate their surface polarity (and therefore their apical and basal poles) de novo. A typical example in vitro is the passage from a round cell suspension to an asymmetric monolayer after trypsinization and replating (6). There are also examples in vivo; perhaps the most striking is the generation of tubule cells from mesenchymal cells during kidney development (15). It is clear that specific cell-substrate and cell-cell interactions must play a crucial role in these cases. In fact, a previous study has shown that junction-free subconfluent epithelial cells, attached to a collagen substrate, can sustain asymmetric virus budding, a polarity property of confluen...

Abstract. Experimental conditions that abolish or reduce to a minimum intercellular contacts between Madin-Darby canine kidney epithelial cells result in the appearance of an intracellular storage compartment for apical membrane proteins. Subconfluent culture, incubation in 1-5 ~tM Ca ++, or inclusion of dissociated cells within agarose or collagen gels all caused the intracellular accumulation of a 184-kD apical membrane protein within large (0.5-5 ~tm) vacuoles, rich in microvilli. Influenza virus hemagglutinin, an apicaUy targeted viral glycoprotein, is concentrated within these structures but the basolateral glycoprotein G of vesicular stomatitis virus and a cellular basolateral 63-kD membrane protein of Madin-Darby canine kidney cells were excluded. This novel epithelial organelle (VAC), which we designate the vacuolar apical compartment, may play an as yet unrecognized role in the biogenesis of the apical plasma membrane during the differentiation of normal epithelia. DJRING growth and differentiation, epithelial ceils establish contact with other cells and with various extracellular matrix (ECM) ~ components which, in many instances, they synthesize themselves. There is considerable evidence that cell-substratum interactions promote or stabilize the expression of genes carrying out differentiated cellular functions. For example, ECM components promote growth and differentiation of mammary epithelial cells (17, 67), promote casein gene expression and secretion (32,33), and regulate the nature and distribution of glycosaminoglycans (47). Likewise, ECM regulates hepatocyte proliferation and differentiation (18,21). Hay and co-workers have shown that collagen and other ECM components stimulate differentiation and ECM synthesis by corneal epithelium (27,(37)(38)(39)59). In fibroblasts, both messenger translatability and the synthesis of cytoskeletal components require cell-substratum interactions (4,19).The role of cell-ceU contacts in the process of cell differentiation is not well defined. There is evidence that cell-cell interactions control cytoskeletal protein synthesis in epithelial cells (3), mammary epithelial cell differentiation (34), and endothelial cell growth (28). In Dictyostelium, synthesis and stability of mRNA are regulated by cell-cell contact (12). In epithelial cells in culture, the levels of expression of apical markers of differentiation, such as Na+-dependent hexose 1. Abbreviations used in this paper: ECM, extracellular matrix; ILIA, radioimmunoassay; VAC, vacuolar apical compartment. transport and various hydrolase activities, increase markedly when the cells become confluent (1,43,49,68). Even cilia and microvilli, typical morphological markers of the apical surface, are infrequent in subconfluent cells but are readily expressed on the apical surface as soon as the cells become confluent (42,43).This study explores regulatory aspects of the biogenesis of the apical membrane components in polarized Madin-Darby canine kidney (MDCK) epithelial cells, derived from dog kidney (9,51,58). It...

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+– K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.

Abstract. The vacuolar apical compartment (VAC) is an organelle found in Madin-Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 ~tM Ca ++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (VegaSalas, D. E., P. J. I. Salas, and E. . J. Cell Biol. 104:1249-1259. The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. RodriguezBoulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca ++ to monolayers developed in 5 ~tM Ca ++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo.