The role of extracellular matrix in the regulation of epithelial cell surface polarity development was studied using MDCK cells. Previous work has demonstrated that MDCK cells cultured in suspension form epithelial cysts having polarized cell surface distributions of several membrane proteins. When MDCK suspension cysts are incubated within collagen gel, a dynamic epithelial membrane remodeling occurs that is accompanied by the reversal of cell surface polarity (Wang et al., 1990b, J. Cell Sci. 95, 153–165), suggesting that extracellular matrix is important in the modulation of epithelial polarity development. To determine if members of the integrin receptor family were involved, MDCK cyst binding studies were done utilizing antifunctional monoclonal antibodies (AIIB2 and AJ2) against the beta 1 integrin subunit. These antibodies inhibited cyst binding to type I collagen, type IV collagen and laminin, providing evidence that functional beta 1 integrin heterodimers were present on the cyst outer membrane. Integrin localization on suspension cysts demonstrated that the alpha 2, alpha 3 and alpha 6 integrin subunits had a non-polarized cell surface distribution and were localized to both the apical and basolateral membranes. Interestingly, immunofluorescence microscopy determined that the beta 1 subunit had a polarized, basolateral membrane distribution although cyst binding studies using inhibitory monoclonal antibodies suggested that functional beta 1 subunits were present on the cyst outer membrane. After incubation of suspension cysts in collagen gel for 8 hours, the beta 1 integrin subunit was detected on the outer membrane, suggesting that the formation of additional integrin alpha/beta heterodimers could be involved in epithelial remodeling. To establish the role of beta 1 integrins in polarity reversal, experiments were done on cysts incubated in collagen gel. After 6 hours in collagen gel, considerable membrane remodeling had occurred as determined by a reduction in outer membrane microvilli. However, the presence of monoclonal antibody AIIB2 inhibited membrane remodeling by preventing both microvillar loss and the endocytosis of the apical membrane glycoprotein gp135. These results provide strong evidence that members of the beta 1 integrin family are involved in the regulation of epithelial polarity reversal, and demonstrate that MDCK cysts constitute an excellent model system for studying the role of cell-extracellular matrix interactions in the regulation of epithelial plasticity and cell surface polarity development.
Metal-Assisted Chemical Etching for Anisotropic Deep Trenching of GaN Array
