The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4°C or 37° C, but phagocytosis was observed only at 37°C. In addition, degradation of 3H-pFN from ingested beads occurred at 37°C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4°C and ingestion at 37°C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was ~250 ng pFN/cm2. When various sized beads (0.085-1.091 μm), coated with similar densities of pFN, were incubated with cells at 4°C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from <100 molecules on the 0.085-μm beads to >15,000 molecules on the 1.091-μm beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is ~18nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 μM pFN-coated beads to the cells were analyzed. There appeared to be ~62,000 binding sites and the K(D) was 4.03 x 10-9 M. Assuming a bivalent interaction, it was calculated that BHK cells have ~120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is ~6 x 10-5 M.
ASJC Scopus subject areas
- Cell Biology