A major difference between serum and fibronectin in the divalent cation requirement for adhesion and spreading of BHK21 cells

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Abstract
Adhesion and spreading of BHK21 cells on adsorbed bovine and foetal bovine serum require addition to the medium of a divalent cation. Divalent cations are effective in the order Mn2+>Co2+>Mg2+>Ca2+, with Ca2+ ineffective below 10−4M. On purified fibronectin, however, no added divalent cation is required, since the requirement is largely met by adventitious Ca2+ (circa 10−5M) in nominally divalent cation-free saline. In such background Ca2+, adhesion and spreading on fibronectin are only slightly slower than in optimal Mg2+, and appear identical, morphologically, and in sensitivity to cytochalasin D. Cells also spread on fibronectin in response to Mg2+, when Ca2+ is buffered below 10−6M, showing that external Ca2+ is not needed as a source for an increase in internal Ca2+. Cells can be induced to spread on serum in low Ca2+ by substantially increasing the fibronectin concentration; this supports other evidence that at its unsupplemented concentration, fibronectin contributes little to the spreading of these cells on serum. The Ca2+ requirement for spreading on preparations of vitronectin (serum spreading factor), partially purified from bovine serum, is similar to that on whole serum. Thus the difference in divalent cation requirement between serum and fibronectin may arise because, on serum, the dominant protein responsible for induction of spreading is vitronectin rather than fibronectin. We argue that such a difference in ion requirement between these surfaces points to a site of action of the ions at the cell surface, perhaps directly on binding of the adsorbed proteins by their receptors, or in receptor-specific transduction events, rather than via ion fluxes, on cyto-skeletal responses common to different surfaces. Models are discussed to account for why the divalent cation requirement should differ for interaction of cells with different substrate-adsorbed proteins.