Divalent cations and extracellular matrix receptor function during newt epidermal cell migration

Abstract

Skin explants were placed in plastic dishes coated with fibronectin (FN), fibrinogen (FGN) or collagen. Explants were cultured for 16 h in serum-free medium containing calcium (Ca) and magnesium (Mg), or in medium containing either Ca-only, Mg-only or manganese (Mn)-only. In Ca/Mg, migrating keratinocytes on all test substrata produced a sheet of contiguous cells that formed a robust halo around each explant. When Ca was the only divalent cation added, the halos in FN- and FGN-coated dishes were approximately 70-80% as large as in Ca/Mg. On collagen, however, the halos were significantly smaller than on the other two substrata. This substratum-specific response in Ca-only suggests that migration on collagen is fundamentally different than migration on FN and FGN. Halos as large or larger than those in Ca/Mg formed on all three substrata in Mg-only. In this case, the halos were not in the form of a sheet of contiguous cells, but were composed of dissociated cells that had migrated from the explant. Individual cells likewise migrated from explants cultured in medium containing Mn-only; however, these halos were never as large as in Mg-only. Thus, while exogenous Ca appears to be an absolute requirement for maintenance of cell-cell connections, the cell-substratum interactions that lead to migration can utilize either Ca, Mg or, to a lesser extent, Mn. Additionally we found that migration on the generally nonpermissive protein, BSA, was not improved by the presence of Mn. Hence, even though Mn can mediate to some extent migration on FN, FGN and collagen, presumably by binding to integrin-like receptors on the cell surface, Mn does not alter receptor specificity in a way that permits migration on BSA. To analyze the possibility that individual newt keratinocytes possess receptors for more than one ECM molecule, we cultured skin explants in Mg-only on a narrow zone of FN bordered on one side by collagen and on the other by uncoated plastic. Individual keratinocytes were able to migrate across the FN strip and continue on collagen but not on uncoated plastic, indicating that at least some keratinocytes have receptors for at least two matrix molecules.