Figure 2.

Involvement of actin filaments and actin binding proteins in collagen bead binding. (A) Incubation of cells with cytochalasin D reduces the percentage of cells binding collagen beads in both WT and Gsn^- cells by >10-fold. Data are mean ± SEs of mean percentage of cells binding collagen beads (n = 4 independent samples/data point). (B) De novo actin assembly around beads. Cells were allowed to bind to collagen beads for 2, 10, and 20 min and subsequently treated with 0.2% OG-PHEM buffer for 2 s, incubated with rhodamine-actin monomers, fixed, and stained with FITC-phalloidin. There was reduced and much slower incorporation of actin monomers into actin filaments in Gsn^- cells. Inset, WT cells incubated with collagen beads for 2 min show staining of actin filaments with FITC-phalloidin (left), rhodamine actin monomer incorporation into nascent filaments around beads (middle), and phase contrast showing collagen beads. (C) Accumulation of actin and actin binding proteins during early events of bead binding. Collagen-bead associated proteins were isolated at indicated time points and immunoblotted. From immunoblots normalized for bead counts and protein abundance (by Bio-Rad assay), there was marked reduction in amounts of actin, cortactin, and Arp3 associated with collagen beads in Gsn^- cells. Data are mean ± SEs of blot density adjusted for number of beads in sample (n = 3/sample). Immunoblotting of whole cell lysates from unstimulated Gsn^- and WT cells is shown for corresponding proteins at right side of panel.