Initial studies of the molecular organization of the cell-substrate adhesion site.
Using selective extraction reagents and non-penetrating probes, studies have been initiated on the molecular organization of substrate-attached material, adhesion sites which pinch off from the cell surface of normal Balb/c 3T3 or SV40-transformed Balb/c 3T3 (SVT2) cells and which remain bound to the serum-coated substrate during EGTA-mediated detachment of cells. Extraction of SVT2 adhesion sites with non-ionic detergents resulted in (a) only small amounts of leucine-radiolabeled protein and glucosamine-radiolabeled polysaccharide being solubilized; (b) selective solubilization of 80% of the adhesion site actin, and (c) solubilization of 95% of the phospholipid from these membranous pools. ATP in combination with potassium chloride extracted 60% of the actin. The 3T3 and SVT2 adhesion site proteins which are accessible to lactoperoxidase-catalyzed iodination were also determined. Many of the serum-derived proteins, bound to the substrate, were iodinated during iodination treatment of serum-coated or substrate-attached material-coated substrates, whereas the cellular proteins in the adhesion sites were not iodinated even though they were present in larger quantity as revealed by Coomassie blue staining. Iodination of cells, followed by their EGTA-mediated detachment and reattachment to fresh serum-coated substrates, indicated that the principal iodinated cell surface component deposited in new adhesion sites is the large external transformation-sensitive glycoprotein (even though large external transformation-sensitive glycoprotein is not the only principal iodinated cell surface component of these cells). These studies further establish the selective enrichment in this adhesive material of specific cell surface components and indicate that they are tenaciously bound at the interface between the serum coating and the undersurface of the adhesion site membranous pools.
D000199:Actins / Q000032:analysis; D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002999:Clone Cells; D006023:Glycoproteins / Q000032:analysis; D007202:Indicators and Reagents; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D013499:Surface Properties
D000199:Actins; D006023:Glycoproteins; D007202:Indicators and Reagents; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D000255:Adenosine Triphosphate