Fig. 7. F-actin binding is required for efficient and stable leading edge localization of coronin 1B. (A) Rat2 fibroblasts expressing GFP-tagged R30D mutant and lacking endogenous coronin 1B were EGF-stimulated (2 minutes) and stained for the Arp2/3 complex (p34Arc subunit), cortactin, F-actin (phalloidin) and VASP. (B) Rat2 fibroblasts lacking endogenous coronin 1B and expressing GFP-tagged wild-type protein (WT) or R30D mutant protein were stained for the Arp2/3 complex (p34Arc subunit) and cortactin following recovery from ATP depletion. (C) The lamellipodial colocalization analysis was performed on the protruding areas of the Rat2 fibroblasts prepared as in B. Fluorescence intensity values were normalized, combined from multiple cells. N, number of cells analyzed per treatment; data are given as the mean ± s.e.m. (Top) Endogenous coronin 1B, soluble GFP and Cortactin. (Middle and bottom) GFP-tagged coronin 1B (WT or R30D), p34Arc (Arp2/3) and cortactin. The indexes of relative leading edge enrichment of GFP signals from various cells were calculated and presented as the percentile mean ± s.d. in the center of the graphs. (D) Representative Rat2 fibroblast, whose endogenous coronin 1B was replaced with GFP-tagged wild-type protein, was subjected to fluorescence recovery after photobleaching (FRAP) analysis. A differential interference contrast (DIC) image is presented on the left and individual frames from time-lapse imaging of GFP are presented on the right. Bleached regions are circled; 1, lamellipodia; 2, cytoplasm). (E,F) Representative fluorescent intensity profiles from the two-spot FRAP analysis of Rat2 fibroblasts whose endogenous coronin 1B was replaced with GFP-tagged wild-type protein or GFP-tagged R30D mutant. (G) Statistical analysis of the FRAP immobile fraction across multiple cells. Data are presented as the mean, error bars indicate 95% confidence intervals. Dunnett multiple comparison test was performed as the post-test for one-way ANOVA using the first column as control. N, number of cells analyzed per treatment; ^**P<0.01, ^***P<0.001.