Molecular composition and origin of substrate-attached material from normal and virus-transformed cells

The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein C_o (the LETS glycoprotein); protein C_a (a myosin-like protein); protein C_b (MW 85,000); protein C₁ (MW 56,000, which is apparently not tubulin); protein C₂ (actin); proteins C₃–C₅ (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins C_c, C_d, C_e, and C_f. The LETS glycoprotein (C_o) and C_d appear in newly-synthesized SAM (which is probably enriched in “footpad” material – “footpads” being focal areas of subsurface membranous contact with the substrate) in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in “footprint” material – remnants of footpads left behind as cells move across the substrate). C_o and C_d turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell “surface” complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of “footpad” vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.