Experiments are described which show that while the presence of calcium in the medium is required for the cells to maintain their adhesion, it is not necessary for the initial attachment of 3T3 cells to solid substrates. Cells are detached by treatment with urea at 4 °C suggesting that adhesion may involve hydrogen bonding between the cell surface and the substratum. Although most of the cell-bound calcium is removed by trypsin, the detaching effect of trypsinisation can be inhibited at low temperature suggesting that ionic calcium bridges are probably not directly involved in retaining the cells on the surface. Cells are made totally insensitive to removal by trypsin by prior washing with lanthanum. Our findings suggest that the external role of calcium in cell adhesion is exerted indirectly. We conclude that the cell presents to the exterior at least two physiochemical classes of molecule. One class composed of hydrogen bond-forming adhesive material (possibly proteins) and another class of anti-adhesive molecules (possibly glycoproteins). These two components are somehow separated in the formation of adhesive ‘plaques’ and this process is apparently uninfluenced by the calcium concentration in the medium. However, the maintenance of the localised zones of adhesion is aided by factors which prevent their disruption by the intrusion into them of anti-adhesive molecules diffusing from adjacent regions of the cell membrane. These factors include cooling below the transition temperature of the membrane lipids and lateral cross-linking of non-adhesive elements by calcium. By contrast, conditions which reduce the stability of the separation of adhesive and non-adhesive surface components would be expected to diminish the overall adhesiveness of cells to the substratum.
In a temporal analysis of the mitogenic response to serum, a critical period has been demonstrated just prior to the onset of replicative DNA synthesis during which transient calcium depletion blocks the subsequent entry of the cells into the S phase of the mitotic cycle. Transient washing of monolayer cultures of 3T3 cells with 2.5 mM EGTA between 6 and 8 h after serum-stimulated initiation of DNA synthesis was found to reduce cell-associated calcium levels and to inhibit thymidine incorporation, whereas similar treatment before (1–5 h) and after (8–9 h) had no detectable effect on either of these parameters when estimated after 21 h incubation. The effects during the chelation-sensitive period were reversed by the subsequent addition of fresh serum.
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