Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response.

Abstract

Abstract. Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4°C were on the order of 10 -5 dynes/cell and did not involve the cytoskeleton. Adhesions to fibronectin after 15 min at 37°C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37°C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37°C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37°C, glioma cells increased their surface area within close contact to the substrate by ",,125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex. C ELL-SUBSTRATUM adhesion to fibronectin (FN) ~ orto glass often has been associated with several morphological and cytological changes including cell spreading and the formation of focal contacts, close contacts, and stress fibers (1, 9, 12, 19, 32-34, 40, 42, 45, 49, 51). While a spread morphology and focal contacts provide physical evidence that an adhesion has occurred, the actual adhesion and its relationship to the cell's behavioral changes that follow must be defined sequentially and on functional terms. As in cell-cell adhesion (39, 48), cell-substrate adhesion has been suggested to involve at least two measurable steps: the first involves initial contact between cell and substrate and the second step strengthens the adhesion through processes requiring metabolic energy (10,27,35,44,48).A number of extracellular matrix proteins including FN and laminin have been found to serve as adhesive substrates for cells. A more recently discovered extracellular matrix Dr. Lotz's present address is New England

Quantitative switch in integrin expression accompanies differentiation of F9 cells treated with retinoic acid

“…There was also little difference in the rate of strengthening to fibronectin and laminin for the two cell types (strengthening of adhesion over several orders of magnitude occurs when the cells, in contact with the substrate, are incubated for several minutes at 37°C (Lotz et al, 1989;McClay et al, 1981) (Fig. 3).…”

“…Cells were resuspended in DME-H a t a concentration of 4.5 x lo5 cells/ml or less and used in adhesion assays. The adhesion assay used is described in (Lotz et al, 1989). At the concentration of cells used for the assay, cell-cell adhesion is not a factor since the surface area covered by the cells as they attach to the substrate is less than 10% of the available surface.…”

“…Interference reflection microscopic analysis was performed as previously described (Lotz et al, 1989). For cell spreading assays, coverslips were coated with either fibronectin or laminin (same concentrations as in adhesion assays).…”

“…Though differentiation of F9 cells to RA cells is accompanied by a number of molecular changes it is not known which one or more of those changes might be responsible for the phenotypic differences. The known change in secretion of fibronectin to laminin could, by itself, account for the phenotypic change, although experimentally this is very unlikely since RA cells quickly assume the same flattened morphology on substrates of either fibronectin or laminin (Lotz et al, 1989). Alternatively, changes in integrins, cytoskeleton, or a combination of all of these could account for the observed differences in cellular morphology.…”

“…We determined the subclasses of integrins that are expressed during differentiation using antibodies in quantitative immunoprecipitation studies. We measured the mRNA levels of these subtypes by Northern analysis, and we examined adhesion properties using a quantitative adhesion assay (Lotz et al, 1989). The adhesion assay distinguishes initial cell attachment, due simply to receptor-ligand interactions, from strengthened adhesion achieved after short incubations at temperatures higher than 4°C (McClay et al, 1981;Lotz et al, 1989).…”