Monoclonai Polarization Antibodies As Probes of Epithelial Membrane

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen. The plasma membrane of epithelial cells shows a striking polarization into two morphologically and functionally distinct domains: the apical and basolateral membranes which face the external and internal milieu, respectively (1). The functional polarity of an epithelium results from the specific localization of various membrane components (enzymes, receptors, transport systems, etc.) to one or the other of these cell surfaces. The apical and basolateral membranes are separated from one another by tight junctions where neighboring cells attach to each other (2). The tight junction is thought to contribute to the maintenance of epithelial polarity by preventing the lateral diffusion of at least some proteins and lipids between these two membranes (3-5). Little is known about the basic question of how this polarity in membrane structure is generated and, in particular, at which intracellular stems) membrane components destined for the apical or basolateral surface are sorted. Recent observations have also shown that many plasma membrane components in a variety of cell types, including epithelia, undergo or can be induced to undergo some form of internalization (endocytosis) and that at least some of these components are subsequently recycled back to the cell surface (4, 6). Clearly, in epithelia THE JOURNAL OF CELL BIOLOGY , VOLUME 101 DECEMBER 1985 2173-2180 © The Rockefeller University Press • 0021-9525/86/01/2173/08 $1.00 such recycling events must be integrated into the sorting mechanism for apical and basolateral membrane components. Continuous cultured epithelial cell lines provide particulady convenient and powerful tools for the study of epithelial biogenesis and function (1, 7). A number of these lines have now been extensively characterized and shown to retain many of the differentiated properties associated with naturally occurring epithelia, including morphological and functional polarity (1, 7). The A6 cell line is derived from the kidney of the aquatic toad Xenopus laevis (8). In culture these cells form confluent polarized monolayers oriented with their basal surface against the supporting substrate. Morphologically they exhibit apical microviUi, occasional apical cilia, and tight junctions (9). When grown on a solid support they form domes or hemicysts, structures thought to be due to transepithelial transport of solute and water. When grown on a permeable support confluent A6 monolayers exhibit a high electrical resistance (5,000 ft.cm2), a mean transepithelial potential difference of 10 mV (apical side negative), and short circuit currents of 2 ~A/cm 2, which are equivalent to their net sodium transport (9). This net transepithelial sodium flux 2173 on Jauary 2, 2018 jcb.rress.org D ow nladed fom is competitively inhibited by apical amiloride and reversibly stimulated by cAMP and adrenal steroid hormones (9, 10). These properties of the A6 cells, together with their ease of handling in culture, have made them a particularly valuable system for studying salt transport in high resistance epithelia (7, 9-11). The present study focuses on the behavior of epithelial cell surface components using the A6 cells as a model epithelium. We have employed monoclonal antibodies (MAb's) ~ directed against specific membrane components of these cells as probes of the phenomena that underlie the genesis and maintenance of epithelial polarity. A number of MAb's directed against the A6 cell surface were generated using the hybridoma technique developed by Kohler and Milstein (12). Two MAb's have been selected for detailed study here. We show that one of these is directed against a 23-kD peptide found on both surfaces of the A6 cells and the other recognizes a lipid localized to the apical membrane only. This is, to our knowledge, the first demonstration of the existence of a lipid that is localized to one side of an epithelium. We report the results of a series of experiments that characterize the internalization and recycling properties of the antibody-antigen complexes formed by these MAb's. Our data show that although both MAb-antigen complexes are internalized by the A6 cells, only the MAb-lipid complex is recycled. Surprisingly, however, this complex is recycled to both the apical and basolateral surface, which indicates that MAb binding interferes in some way with the normal sorting process for this apical lipid antigen. The mechanism of this interference remains to be determined. MATERIALS AND METHODS