Complementary adhesive responses of human skin fibroblasts to the cell-binding domain of fibronectin and the heparan sulfate-binding protein, platelet factor-4

Plasma fibronectin (pFN) contains binding domains for an unidentified receptor on the surface of fibroblasts and for heparan sulfate chains of proteoglycans on these same cells. A series of experiments were designed to assess the relative importance of these activities in mediating substratum adhesion of human skin fibroblasts (strain 4449) grown in the absence of ascorbate (asc-) or in its presence (asc+) to minimize or maximize collagen production-maturation, respectively. The cell-binding fragment (CBF) of pFN was purified from chymotryptic digests free of any heparan sulfate-binding activity. The responses of cells to CBF were then compared with those mediated by the heparan sulfate-binding protein, platelet factor-4 (PF4). At early time points when cells had spread effectively on pFN, both asc- or asc+ cells extended spiky projections on PF4 and long projections on CBF with actively ruffling membranes at their tips. By 4 h, asc+ cells had spread much more effectively on CBF than asc+ cells on PF4 or asc- cells on either binding activity. Mixtures (w/w) of CBF:PF4 between 1:1 and 9:1 generated a more physiologically normal response than to either of the binding proteins alone, particularly for asc+ cells. Examination of cytoskeletal reorganization by fluorescence analysis with an antibody to 7S tubulin (for microtubules) and NBD-phallacidin (for F-actin) revealed condensations of microfilaments at the ruffling edges of asc- cells on CBF or on PF4 and for asc+ cells on PF4; in contrast, asc+ cells on CBF generated long bundles of microfilaments in their spreading lamellae within 4 h. Microtubule networks reorganized very well on CBF but only partially on PF4 with either cell type. Microfilament reorganization was comparable to that on intact pFN with CBF:PF4 mixtures of 1:1 and 9:1 for asc+ cells, whereas asc- cells generated condensations of microfilaments but little bundling. These studies reveal that the adhesive responses to mixtures of these two binding activities are significantly greater than to the individual activities and that the responses of asc+ cells approach the properties of cells on intact pFN, whereas asc- cells remain incapable of forming stress fiber-like bundles of microfilaments under all conditions.