Fig. 6. Inhibition of resealing by biologically mediated actin polymerization is reversible with cytochalasin or DNase1. (a) RGM1 cells lining a scratch site 30 minutes after its creation were heavily stained in their cortices with TRITC-phalloidin. (b) When this scratch maneuver was carried out in FDx, so that those cells along the scratch site incurring plasma membrane disruptions could be identified, those cells most heavily labeled with TRITC-phalloidin were co-labeled with the FDx marker of a plasma membrane disruption event (arrowheads). (c) A monolayer was scratched first in PBS containing cytochalasin B (3 µg/ml) and FDx (10 mg/ml), and then rinsed with fresh medium minus cytochalasin. Two hours later it was scratched a second time (scratch lines approximately at right angle to first set) in the presence of PBS containing fixable Texas Red-labeled dextran (TRDx; Mr 70,000; 10 mg/ml). Double-labeled (yellow) cells (arrows) are clearly present in the population and represent those that survived two PMD events. (d) A monolayer was treated identically to that above, except that it was scratched first in PBS minus cytochalasin. No double-labeled (yellow) cells are observed. (e) The percentage of yellow (double-labeled) relative to green cells (single label from first scratch only) observed as a function of scratching the first time plus (CyB+) or minus (CyB-) cytochalasin B (3 µg/ml). The number of yellow cells relative to green were scored along a total of 200 mm of scratch site length in five separate experiments. The mean and s.d. of these measurements are plotted. Comparison of the two data sets yielded P=0.00882 (Kruskal-Wallis test). (f) Analysis of cells surviving two disruptions made by glass beads instead of scratching the monolayer. During the first plasma membrane disruption event induced by beads, the PBS medium contained no additives (CyB-), or cytochalasin B at 3 µg/ml (CyB+) or DNase1 at 100 µg/ml (DN1). The data represents the mean and s.d. of five experiments. The CyB- (*) sample differs significantly from the other two samples (P< 0.01, Kruskal-Wallis test). Bars, 20 µm.
