Figure 1.

Involvement of gelsolin in the initial binding step of collagen phagocytosis. (A) Fibroblasts from WT mice were plated on collagen-coated beads for 20 min, fixed, and immunostained with polyclonal antibody to gelsolin. Cells show enrichment of gelsolin around collagen beads. DIC, differential interference contrast microscopy of same cells immunostained for gelsolin. (B) Gsn^- and WT cells were plated at indicated collagen bead:cell ratios, incubated for 30 min, and collagen bead binding was analyzed by flow cytometry. There was significantly lower percentage of cells binding beads in Gsn^- cells compared with WT cells. Data are mean ± SEs of mean percentage of cells binding collagen beads for B and C (n = 4 independent samples/data point). (C) Gsn^- cells transfected with cDNA gelsolin expression vector for 48 h show gelsolin expression at ∼50% of WT cells. When analyzed with collagen bead binding assay, there was 50% enhancement of collagen bead binding in transfected cells. (D and E) α2β1-mediated collagen bead binding and phagocytosis in Gsn^- and WT cells. Cells were plated on collagen-coated beads at 8:1 (bead:cell ratios) at 37°C for the indicated times, put on ice, and collagen bead attachment (D) and phagocytosis (E) were determined. Internalized beads were discriminated by quenching extracellular bound beads with trypan blue. Fluorescent and quenched beads were counted in 60 cells/sample at each time point. The numbers of beads binding per cell were significantly lower in Gsn^- cells (p < 0.05), which also showed delayed bead internalization.