| Abstract: | Substrate-attached material (SAM) prepared from murine BALB/c 3T3 cells and various derivatives contains adhesion sites which pinch off from the cell surface during EGTA-mediated detachment but which remain bound to the serum-coated tissue culture substratum. SAM contains the related adhesive glycoproteins cold-insoluble globulin (CIG) (from serum in the medium) and fibronectin (synthesized by the cells) as detected by immune staining of electrophoretically separated proteins, using antibodies of defined specificity. Serum and SAM contain cross-linked multimers of serum-derived CIG (not disulfide-mediated) but not of cell-derived fibronectin; therefore, thiol-resistant cross-linking between CIG and fibronectin is not involved in adhesion of these cells. Immunofluorescence microscopy of SAM from sparse cultures reveals fibrillar pools containing cellular fibronectin, although most retraction fibers seen on EGTA-treated cells do not stain, even after treatment with non-ionic detergent. Very little specific staining can be detected in SAM prepared from dense cultures, although gel electrophoretic analysis reveals proportionately as much murine fibronectin as is found in SAM from sparse cultures. Hyaluronidase digestion of SAM has no effect on the immunofluorescent staining, while gentle trypsin digestion completely abolishes staining without removing all biochemically detectable fibronectin. We conclude that some of the fibronectin and CIG in adhesion sites is masked and unavailable for antibody binding and that multiple pools of fibronectin exist in this adhesive material. |
