Adhesion sites of murine fibroblasts on cold insoluble globulin‐adsorbed substrata

Abstract

The attachment and detachment behavior of three mouse fibroblast cell lines adhering to plastic tissue culture substrata coated with the serum protein cold-insoluble globulin (CIg) resembles that seen on the usual serum-coated substrata. The transformed cell line SVT2 spreads more extensively on the CIg-coated than on the serum-coated substratum, while the nontransformed Balb/c 3T3 line and concanavalin A-selected "revertant" of SVT2 are equally well spread on both substrata. In all three cases, immunofluorescence microscopy using antibodies to CIg suggests that the cells are more tightly apposed to the CIg-coated substratum than to the serum-coated substratum. Substrate-attached material (SAM), which contains cell-substratum adhesion sites and which is left after EGTA-mediated detachment of cells, is enriched for cell surface fibronectin and glycosaminoglycans (GAG). When cells are seeded onto CIg-coated substrata rather than serum-coated substrata, there is an increased deposition of GAG but a comparable deposition of cellular proteins. The protein distribution of the two types of SAM are identical as analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, including fibronectin content. This indicates that substratum-bound CIg cannot functionally substitute for cell surface fibronectin in these adhesion sites. Analysis of the GAG deposited on CIg-coated substrata reveals that hyaluronate and the chondroitins are increased to a much greater extent than heparan sulfate; however, the ratio of hyaluronate to the various chondroitin species is invariant. These data provide further evidence that hyaluronate and the chondroitins are deposited in adhesion sites in well-defined stoichiometric proportions, possibly as supramolecular complexes, and that CIg may mediate adhesion of cells in the serum layer by binding to GAG-containing proteoglycans.

Adhesion site composition of murine fibroblasts cultured on gelatin‐coated substrata

“…This result is consistent with the observation that cells on gelatin spread more extensively (Fig. 1) and so will leave more SAM behind when they are detached; a similar increase in the amount of SAM occurs when cells are grown on pFn-coated plastic (Murray et al, 1980). Although SAM by its mode of formation is considerably enriched in adhesion site proteins, it is still very complex biochemically (gels are shown below).…”

“…These changes are probably due to plasma fibronectin affinity concentrated from the medium onto the gelatin, since similar effects have been observed in cells grown on plastic coated with cell-t. surface fibronectin (Yamada et al, 1976. At confluence cells on gelatin displayed a swirling aligned growth pattern also characteristically due to pFn (Murray et al, 1980;Haas and Culp, 1982); this did not occur on plastic even when the medium contained dissolved gelatin (not shown).…”

“…In this study we present a combined morphological and biochemical analysis of the adhesion of fibroblasts to denatured collagen (gelatin). (We have used gelatin, as is regularly used to culture myoblasts [Richler and Yaffe, 19701, because first, it provides a simple, uniform, reproducible, and well-defined model collagenous substratum, and second, it binds more fibronectin than does undenatured fibrillar collagen [Kleinman et al, 1981;Yamada, 19831, permitting us to extend our earlier studies of the influence of pFn on cell adhesion [Murray et al, 1980;Haas and Culp, 19821 to the case of pFn specifically bound in native form to a protein matrix, rather than nonspecifically adsorbed and denatured on plastic.) We find that the gelatin substratum causes some changes in 1) the growth pattern and morphology of the cells, as determined by scanning electron microscopy, 2) the nature of binding of pFn adsorbed from the medium, as monitored quantitatively with 1251-radiolabeled pFn, and 3) the protein composition of the adhesion sites of adherent cells, as determined by gel analysis of "substratum-attached material" (SAM), a selectively enriched preparation of adhesion sites left behind after adherent cells have been removed.…”

Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin.

“…Since PF4 and FN both bind heparan sulfate independent of the presence of Ca ++ (24,27), it is likely that these EGTA-resistant adhesions represent areas of highly stabilized interactions between focally high concentrations of heparan sulfate and the substrata. Consistent with this hypothesis is that the cellular material remaining on FN-containing substrata after EGTAmediated cell release is significantly enriched relative to whole cells in cell surface heparan sulfate (33). EGTA may thus effect cell retraction from these substrata by destabilizing Ca++-dependent cell surface or cytoplasmic interactions that are distinct from those which occur directly between cell surface heparan sulfate and the substratum.…”

Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: Modulation of affinity binding to fibronectin

“…Fractions (0.5 ml) were assayed as described above. Goat antibody to human plasma fibronectin, which is highly crossreactive with neuroblastoma cellular fibronectin , was purified by affinity methods as described by Murray et al (1980) and linked to Sepharose 4B columns by the cyanogen bromide reaction as described here.…”