On the Role of Calcium in Adhesion of Cells to Solid Substrates

Abstract

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.

Time Dependency of a Critical Effect of EGTA on DNA Synthesis in Cultures of Swiss 3T3 Cells

“…Approxima tely 96% of the residual cell-associated calcium was removed by 3 subsequent 0.1 M lanthanum chloride washes, 89% by washing the cultures with EGTA (2.5 mM at pH 7.2) and 78% by trypsinising the cells. These results imply that a significant proportion of the cell-associated calcium is external as has been previously demonstrated (3,4,6). The washing schedule that was finally employed in the present study consisted of two saline washes in which the wells were rinsed in a stream of 0.9% saline, allowed to stand in 1 ml/well of saline for 10 min, rinsed again and given a second wash (1 ml/well) for 3 -5 min.…”

“…While demonstrating a correlation between DNA synthesis and total cell-associated calcium our results do not distinguish between different localisations of cellular calcium. It is likely, from the examination of the washing schedules, particularly in the light of previous work (3,4,6,7), that a significant proportion of the CACa is bound to the cell externally. In view of the findings of Rubin and Sanoi (8) and Barnes and Colowick (9) it is also probable that some of the calcium binding is 'non-specific'.…”

“…Experiments were carried out on monolayer cultures of Swiss 3T3 cells, mycoplasma free, grown in MEM with Earle's salts and 20 mAf Hepes buffer with 10% FBS (4). Cells were seeded at 10s cells/well as initial seeding density and grown to confluent monolayers in tissue culture plates (Linbro EB-4 multidishes with 4 wells each of 28.28 cm2 surface area).…”

Cellular Adhesion to Growth Surfaces: a Possible Role for Low Molecular Weight Surface Components

“…One could also envisage that the detachment of cells would expose proteins occurring on the undersurface of the cells. It is also known that the high molecular weight adhesive proteins, such as fibronectin, are loosely bound to the cell membrane and that the process of adhesion by means of fibronectins requires divalent ions such as Ca2+ for stabilising these macromolecules by lateral bridging [3]. A depletion of these ions may cause breakage of the loose linkage of the macromolecule and release the cell from the substratum leaving the fibronectin mole cules adhering to the substratum.…”

Du Temps Perdu a la Recherche

“…The problem of motility in relation to migration attracted my attention and with Rayya Damluji we examined the effects of calcium on adhesion (99, 100). Later, with Bryan Groves, I devised a chamber to measure cell‐to‐substratum adhesion by shear resistance (101).…”