Determinants of apical membrane formation and distribution in multicellular epithelial MDCK cysts.

Madin-Darby canine kidney epithelial cells form three-dimensional cysts in spinner culture with a defined cell surface polarity. Transfer of cysts from spinner culture to a collagen gel matrix results in rapid loss of apical membrane proteins from the outside surface of the cyst, degradation of extracellular matrix (ECM) from the cyst lumen, and de novo formation of the apical membrane at the luminal surface. Degradation of endogenous ECM was inhibited with 1,10-phenanthroline, an inhibitor of metalloproteinases, resulting in cysts in which cells are surrounded by either cell-cell or cell-substratum contacts. The consequence of the lack of a free cell surface on the formation of a new apical membrane domain in these cysts was analyzed. Changes in cell surface polarity were followed with antibodies to marker proteins of the apical or basolateral membranes. In the absence of a free cell surface, the apical membrane formed de novo by accumulation and fusion of presorted vesicles containing apical membrane proteins; the coalescence of these vesicles results in the formation of a central lumen. These results provide novel insights into the generation of membrane domains and formation of a lumen in complex, three-dimensional epithelial structures in development.

Improved in vitro models for assay of rheumatoid synoviocyte chemotaxis.

OBJECTIVES: (1) To define the optimal conditions for transwell assay of synoviocyte chemotaxis. (2) To develop a live cell imaging chemotaxis assay for synoviocytes. (3) To characterize the chemotaxis of rheumatoid synoviocytes (RAS). RESULTS: Optimal conditions for transwell assay of synoviocyte chemotaxis were 8 microns pore size filters coated with collagen I (C1), assayed for 24 hours. Without the C1 coating 2.9 x 10(3) RAS migrated to the lower chamber. This increased to 4.7 x 10(3) when 20 micrograms/ml fibronectin (Fn) was added. With the C1 coating 4.3 x 10(3) cells migrated through the filter without chemotactic stimulation compared to 12.8 x 10(3) with interleukin 1 beta (IL-1 beta) 5 ng/ml, 12.2 x 10(3) with granulocyte-macrophage colony stimulating factor (GM-CSF) 25 ng/ml, 11.7 x 10(3) with Fn 20 micrograms/ml, and 9.0 x 10(3) with transforming growth factor-beta 1 (TGF-beta 1) 20 ng/ml (all p < 0.01). In the imaging assay, 50.7% of the RAS migrated toward the C1 coating without bound chemoattract. The percentage of cells migrating toward each chemoattractant at its optimal concentration was 64.3% for IL-1 beta, 60.8% for IL-8, 64.7% for GM-CSF, 61.0% for Fn, 58.9% for IL-6, and 69.1% for TGF-beta 1 (all p < 0.01). All of these chemoattractants increased directed migration without changing the random migration. Indomethacin (100 ng/ml) and Dexamethasone (10 ng/ml) inhibited Fn-induced chemotaxis. CONCLUSION: We report two in vitro assays for synoviocyte chemotaxis adapted and optimized for the study of synoviocytes. The live cell imaging assay had the advantage that it could separate directed and random migration.

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.

Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin-Darby canine kidney epithelial cells.

Cell-cell contact is an important determinant in the formation of functionally distinct plasma membrane domains during the development of epithelial cell polarity. In cultures of Madin-Darby canine kidney (MDCK) epithelial cells, cell-cell contact induces the assembly and accumulation of the Na+,K+-ATPase and elements of the membrane-cytoskeleton (ankyrin and fodrin) at the regions of cell-cell contact. Epithelial cell-cell contact appears to be regulated by the cell adhesion molecule uvomorulin (E-cadherin) which also becomes localized at the lateral plasma membrane of polarized cells. We have sought to determine whether the colocalization of these proteins reflects direct molecular interactions which may play roles in coordinating cell-cell contact and the assembly of the basal-lateral domain of the plasma membrane. Recently, we identified a complex of proteins containing the Na+,K+-ATPase, ankyrin, and fodrin in extracts of whole MDCK cells (Nelson, W.J., and R. W. Hammerton. 1989. J. Cell Biol. 108:893-902). We have now examined cell extracts for protein complexes containing the cell adhesion molecule uvomorulin. Proteins were solubilized from whole MDCK cells and fractionated in sucrose gradients. The sedimentation profile of solubilized uvomorulin is well separated from the majority of cell surface proteins, suggesting that uvomorulin occurs in a protein complex. A distinct portion of uvomorulin (30%) cosediments with ankyrin and fodrin (approximately 10.5S). Further fractionation of cosedimenting proteins in nondenaturing polyacrylamide gels reveals a discrete band of proteins that binds antibodies specific for uvomorulin, Na+,K+-ATPase, ankyrin, and fodrin. Significantly, ankyrin and fodrin, but not Na+K+-ATPase, coimmunoprecipitate in a complex with uvomorulin using uvomorulin antibodies. This result indicates that separate complexes exist containing ankyrin and fodrin with either uvomorulin or Na+,K+-ATPase. These results are discussed in the context of the possible roles of uvomorulin-induced cell-cell contact in the assembly of the membrane-cytoskeleton and associated membrane proteins (e.g., Na+,K+-ATPase) at the contact zone and in the development of cell polarity.

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.

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

Steps in the morphogenesis of a polarized epithelium. I. Uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts.

The development of cell polarity in Madin-Darby canine kidney (MDCK) cells has been analyzed under conditions in which cells are induced to form multicellular epithelial cysts in stages that mimic the ontogeny of epithelial tissues and organs in vivo. The morphogenesis of MDCK cysts in suspension culture or in a collagen gel proceeds in distinct stages involving the initial aggregation of cells followed by development of a closed monolayer of polarized epithelial cells that surrounds a central lumen. The polarity of cells was determined at each stage by analyzing the distributions of marker proteins of the apical (gp135) and basal-lateral (Na+,K(+)-ATPase) domains of the plasma membrane, the tight junction (ZO-1) and proteins involved in cell-cell (uvomorulin) and cell-substratum contact (type IV collagen). We show that cells have a distinctive and opposite polarity in cysts formed in suspension culture compared to those formed in collagen gels. In suspension culture, the basal-lateral membrane faces the central lumen and the apical membrane faces the outside, whereas in collagen gel, the basal-lateral membrane faces the outside collagen and the apical membrane faces the central lumen. Detailed analysis of the distributions of marker proteins during the morphogenesis of these three-dimensional structures indicates that: (1) cell-cell contact is sufficient to trigger the segregation of marker proteins of the apical and basal-lateral membrane domains to distinct regions of the membrane; (2) cell-cell contact induces association of the tight junction protein ZO-1 with the contact zone between cells; (3) localization of the tight junction protein ZO-1 to the apex of the lateral membrane and the establishment of the epithelial axis, however, requires the formation of a basal lamina and cell-substratum contact; (4) in suspension culture, MDCK cysts secrete and establish a basal lamina in the central lumen. These results show that cell-cell and cell-substratum contact have distinct roles in the morphogenesis of polarized epithelia. We suggest that the mechanisms involved in triggering cell polarity may be common to different polarized epithelia in vivo.