Analysis of epithelial cell surface polarity with monoclonal antibodies.
The hybridoma technique of Köhler 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.
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D004285:Dogs; D004848:Epithelium / Q000502:physiology / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis; D007668:Kidney / Q000502:physiology / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008970:Molecular Weight; D009399:Nephrons / Q000502:physiology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D006023:Glycoproteins