Two epithelial cell lines, MDCK and NMuMG, growing as monolayers on type I collagen gels, respond to an overlay of additional collagen by altering their polarity and reorganizing to form lumina. This provides a model system for studying the mechanisms of a morphogenetic event, specifically the role of extracellular matrix (ECM) material in the determination of cellular polarity and organization of epithelia. Lumen formation is a biological assay which may be correlated with detectable biochemical and cytological activities. The inducibility, availability, accessibility and relative simplicity gives this model many advantages over other epithelial morphogenetic systems. This proposal questions 1) the specificity of the ECM material that induces cell polarity and lumen formation, that is, whether the effect is specific to collagen, 2) what macromolecular synthesis, particularly that of ECM material, accompanies and is required for lumen formation, and 3) if the mechanism of polarity determination and lumen formation entails transmembrane associations of the cytoskeleton with the ECM and restructuring of the cytoskeleton. ECM components such as glycosaminoglycans (GAGs), fibronectin, laminin as well as other compounds will be added either in solution, coupled to dextrans, or incorporated into gels of hydroxyethylmethylmethacrylate and tested for their ability to induce lumen formation. Synthesis of ECM components will be followed by metabolic incorporation of radioisotopes. GAGs will be characterized by two dimensional cellulose acetate electrophoresis and column chromatography and proteins by two dimensional gel electrophoresis. Immunological techniques will be utilized for the identification of specific ECM and cytoskeletal proteins. Differential solubilization will be used to fractionate the cells to explore possible polarized transmembrane associations of the cytoskeleton with the ECM. The upper and lower collagen gels will be analyzed separately to follow changes in the associations that may occur during the course of lumen formation. Light and electron microscopic immunolocalization methods will be used to visually follow the polarization of the ECM and cytoskeletal proteins and their possible redistribution during lumen formation. To determine if there is a functional role for the cytoskeleton in lumen formation, the effects of colchicine, taxol and cytochalasin B will be tested.
| Hall, H G; Bissell, M J (1986) Characterization of the intermediate filament proteins of murine mammary gland epithelial cells. Response to collagen substratum. Exp Cell Res 162:379-89 |
