Fibronectin and proteoglycans as determinants of cell‐substratum adhesion

Abstract

When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.

Transcriptional repression of type I procollagen genes during adipocyte differentiation

“…ECM is not merely a structural or an architectural entity, but plays an important role in transducing signals into cells for various cell behaviors. Collagen is the most abundant component of ECM and serves as substratum for cell adhesion, migration, spreading, and anchorage-dependent growth (Culp et al, 1979;Yamada, 1982;Spiegelman and Ginty, 1983). Prior to differentiation, 3T3-L1 preadipocytes resemble typical fibrobalsts.…”

The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface.

“…Cell surface proteoglycans have been implicated in a variety of cellular events, such as proliferation (29,35,48), migration (10,14), and adhesion (3,5,10,31), and it has been proposed that they may influence cellular behavior by acting as receptors for components of the extracellular matrix and for various circulating molecules (23). Proteoglycans are known to bind to several components of the extracellular matrix, including fibronectin (50, 53,67), laminin (52,65), and collagen (32,53,54), and also other proteoglycans (15).…”

Separation of human colostral macrophages and neutrophils on gelatin and collagen-serum substrata.

“…Balkwell et al (2) enriched human colostral macrophages by adherence to microexudates prepared from baby hamster kidney (BHK) cell line. Microexudates are composed in part of collagen, fibronectin, and glycosaminoglycans (5,9). Since fibronectin binds to collagen and gelatin with high affinity (6), acid soluble collagen or gelatin substrata preincubated with serum were expected to substitute for microexudates as a defined attachment surface.…”