Analysis of epithelial cell surface polarity with monoclonal antibodies

The hybridoma technique of K?hler and Milstein was utilized to isolate hybrid cell lines secreting monoclonal antibodies against cell surface proteins on the Madin-Darby canine kidney (MDCK) epithelial cell line. These antibodies were employed as high-affinity ligands to study the development and maintenance of epithelial cell polarity in MDCK cells and for the identification of nephron segment-specific proteins. Using standard procedures, we were able to immunoprecipitate glycoproteins with molecular weights of 25,000 ( 25K ), 35,000 ( 35K ), and 50,000 (50K). Immunofluorescence and immunoelectron microscopy of MDCK demonstrated that the 35K and 50K proteins could be localized on both the apical and basolateral membranes of subconfluent cells but primarily on the basolateral membranes of confluent cells. By determining the cell surface distribution of the 35K and 50K proteins on MDCK cells during growth into a confluent monolayer, and after the experimental disruption of tight junctions, evidence was obtained that the polarized distribution of these cell surface glycoproteins required the presence of tight junctions. We propose that confluent MDCK cells have a mechanism that is responsible for the establishment and maintenance of epithelial apical and basolateral membranes as distinct cell surface domains. These monoclonal antibodies were also used to localize the 25K and 35K glycoproteins in the kidney. The distribution of these proteins was mapped by immunofluorescence and immunoelectron microscopy and was determined to be on the basolateral membranes of epithelial cells in only certain tubular segments of the nephron. The possible functional implications of these distributions are discussed.     

Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.     

Involvement of the membrane-cytoskeleton in development of epithelial cell polarity

The generation of cell surface polarity in transporting epithelial cells occurs in three distinct stages that involve cell-cell recognition and adhesion, cell surface remodelling to form biochemically and functionally distinct cell surface domains, and development of vectorial function. A widely used model system to study mechanisms involved in these stages is the Madin-Darby canine kidney (MDCK) cell line. Under appropriate growth conditions, MDCK cells develop in similar stages into polarized, multicellular epithelial structures. Analysis of membrane-cytoskeletal proteins ankyrin and fodrin during development of MDCK cell surface polarity shows that they gradually assemble into an insoluble protein complex on the basal-lateral membrane domain upon cell-cell adhesion, concomitantly with the redistribution of Na+,K(+)-ATPase, a marker protein of the basal-lateral membrane. Biochemical analysis shows that ankyrin, fodrin occur in a complex with Na+,K(+)-ATPase and the cell adhesion molecule uvomorulin in MDCK cells. A model is presented in which assembly of membrane-cytoskeletal complexes at sites of uvomorulin-induced cell-cell contact causes a remodelling of the cell surface distribution of specific membrane proteins which, in turn, contributes to the generation of epithelial cell surface polarity.     

Molecular networks controlling epithelial cell polarity in development

During embryonic development, polarized epithelial cells are either formed during cleavage or formed from mesenchymal cells. Because the formation of epithelia during embryogenesis has to occur with high fidelity to ensure proper development, embryos allow a functional approach to study epithelial cell polarization in vivo. In particular, genetic model organisms have greatly advanced our understanding of the generation and maintenance of epithelial cell polarity. Many novel and important polarity genes have been identified and characterized in invertebrate systems, like Drosophila melanogaster and Caenorhabditis elegans. With the rapid identification of mammalian homologues of these invertebrate polarity genes, it has become clear that many important protein domains, single proteins and even entire protein complexes are evolutionarily conserved. It is to be expected that the field of epithelial cell polarity is just experiencing the 'top of the iceberg' of a large protein network that is fundamental for the specific adhesive, cell signalling and transport functions of epithelial cells.     

Changes in cell surface glycoproteins during Dictyostelium development analysed using monoclonal antibodies

We have produced a series of monoclonal antibodies that recognize carbohydrate epitopes on cell surface glycoproteins of developing amoebae of Dictyostelium discoideum. The antibodies were found to have differential specificity for amoebae at different stages of development and were classified into types A to E on the basis of their temporal pattern of reactivity with the developing amoebal cell surface. Evidence from Western Blots and digestion of the glycoproteins with alkaline phosphatase were consistent with previous reports that the cell surface glycoproteins are extensively processed during development, leading at 16 h of development to the exposure of a highly antigenic core recognized by antibodies in group E. The nature of this core structure is indicated by the finding that antibodies in group E were found also to bind with high avidity to the plant glycoprotein horse radish peroxidase.     

Role of laminin A chain in the development of epithelial cell polarity

Kidney organ culture was used to study the conversion of embryonic mesenchymal cells into a polarized, differentiated kidney epithelium. We examined the expression of laminin, a basement membrane glycoprotein, during this conversion. The B chains of laminin were constitutively expressed, whereas the appearance of the A chain of laminin was dependent on embryonic induction and coincided with the onset of cell polarization. Antisera against the carboxy-terminal end of laminin inhibited polarization but did not affect the developmental events that precede polarization. Antisera against N-terminal parts of laminin failed to inhibit morphogenesis. Since the fragments at the carboxy-terminal end contain parts of the A chain, we suggest that the appearance of this chain is fundamental for initiation of cell polarity.     

Ocular surface epithelial and stem cell development

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Control of epithelial cell shape and polarity

The polarised character of a cell is often obvious from its shape and is largely dependent on the actin cytoskeleton and the membrane-associated cell cortex---a dense network comprising spectrin and other related proteins. Spatially and functionally distinct protein scaffolds, assembled from transmembrane and cytoplasmic proteins, provide the cues for polarisation. Recent data have provided new insights into the molecular nature of these cues and the mechanisms by which they may be translated into a polarised phenotype.     

Integrins in epithelial cell polarity: using antibodies to analyze adhesive function and morphogenesis

Epithelial cells polarize in response to cell-substratum and cell-cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin. Investigation of the roles of integrins in epithelial cell polarization has depended on the use of function-blocking antibodies both to determine ligand specificity of individual integrins and to disrupt and redirect normal morphogenesis. In this article we describe techniques for employing function-blocking anti-integrin antibodies in adhesion assays of the polarized Madin-Darby canine kidney (MDCK) cell line and to demonstrate the involvement of beta1 integrins in collagen-induced tubulocyst formation. These techniques can be easily expanded to other antibodies and epithelial cell lines to characterize specific functions of individual integrins in epithelial morphogenesis.     

Cell surface antigens during submerged development of Myxococcus xanthus examined with monoclonal antibodies

Eighteen monoclonal antibodies directed against cell surface antigens of Myxococcus xanthus were followed by enzyme-linked immunosorbent assay. Three of the monoclonal antibodies were specifically directed against antigens present only on cells undergoing fruiting body development. These cell surface antigens became detectable by the early preaggregation stage (2 to 4 h) of development and increased until early aggregation (9 to 10 h), after which the concentrations of two of the cell surface antigens remained constant and the concentration of the third decreased. The remaining 15 monoclonal antibodies recognized cell surface antigens that were shared by vegetative and developing cells. Based on their relative concentrations during development, these shared antigens can be grouped into three classes. In the first class antigen concentration remained constant, in the second it decreased, and in the third it increased. Western blots of cell surface antigens were probed with monoclonal antibodies. Five monoclonal antibodies reacted with material in distinct bands, five monoclonal antibodies reacted with multiple, diffuse bands, and eight monoclonal antibodies were not reactive in Western blots.     

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

A novel monoclonal antibody against the second extracellular loop of occludin disrupts epithelial cell polarity.

The tight junction (TJ) regulates epithelial cell polarity and paracellular permeability. In the present study, to investigate whether the second extracellular loop of occludin affects the localization of carcinoembryonic antigen (CEA) and CD26 expressed on apical membranes, and the fence function of the TJ, the human intestinal epithelial cell line T84 was treated with the monoclonal anti-occludin antibody (MAb) 1H8, corresponding to the second extracellular loop of occludin. In T84 cells treated with MAb 1H8, occludin disappeared, and CEA and CD26 were observed to diffuse from the apical membrane to the basolateral membrane. Furthermore, a decrease in the fence function of TJ was observed without changes in the TJ strands and barrier function. When T84 cells precultured in low calcium (Ca) medium were recultured in normal Ca medium in the presence of MAb 1H8, recruitment of occludin to the apical-most membranes and recovery in distribution of CEA and CD26 were markedly retarded compared with the control. These results suggested that MAb 1H8 against the second extracellular loop of occludin selectively affected formation of the apical/basolateral intramembrane diffusion barrier and that the second extracellular loop of occludin plays a crucial role in the maintenance of epithelial cell polarity by the TJ.     

Analysis of the murine sperm surface with monoclonal antibodies

Hybridomas secreting monoclonal antibodies reactive with murine spermatozoa were produced by fusion of myeloma cells with spleen cells from C57BL/6J mice immunized with spermatozoa from mice of the same strain. All antisperm antibodies were of the mu (mu) immunoglobulin heavy chain class; only one (MS-1) bound S. aureus protein A. Antibody MS-1 recognized an antigen present on the sperm acrosomal cap, on the surface of cells from liver and kidney and from some cultured cell lines. The subunit molecular weight (69000) of the polypeptide reactive with MS-1 was determined by SDS-PAGE analysis of sperm membrane proteins followed by their electrophoretic transfer to nitrocellulose.     

Studies of tracheal secretion using serous cell cultures and monoclonal antibodies

The glycoconjugate composition of tracheal secretions varies with physiological and pathophysiological parameters. Believing that these differences might be explained by metabolic or regulatory modifications of particular cell types, we have developed strategies for biochemical analysis at the cellular level. We have produced monoclonal antibodies whose determinants are restricted to a single secretory cell type (serous, mucous, or goblet cell granules, or ciliated cell glycocalyx). By enzyme immunoassay (ELISA), we have characterized four of the antibodies biochemically, and have also used the antibodies as quantitative molecular probes to detect release of antigen from mixed cell explants. Four of the antigens are carried by carbohydrate moieties of high molecular weight glycoproteins. Western blot analysis shows their molecular weight in reducing gels (SDS-PAGE) to exceed 200 kD. When used in parallel with pulse-chase labeling studies, the antibodies are both more sensitive and specific (than bound radioactivity) in detecting gland or goblet cell secretion in response to autonomic drugs or proteases. We have also isolated and cultured serous gland cells for physiological and biochemical studies. These cells express serous cell phenotype as reflected by ultrastructure, histochemistry, and lysozyme activity. Biochemical analysis of their secretory products reveals glycoconjugate components which are heterogeneous with respect to both molecular weight and charge. Radiolabeled secretory products eluting in the void volume of Sepharose C1 4B were completely degraded by chondroitinase ABC. This indicates that the major glycoconjugate produced by serous cell is a proteoglycan resembling chondroitin sulfate.