Figure 4.

ASAP1^BARPH stabilizes F-actin bundles and decreases the spontaneous polymerization of G-actin to F-actin. A, increasing concentrations of ASAP1^BARPH protect F-actin bundles against depolymerization. ASAP1^PH (B) and V^Tail (C) were used as negative and positive controls, respectively. A premixing concentration of 4 μm F-actin and the indicated concentrations of ASAP1^BARPH, ASAP1^PH, or V^Tail was used. The representative plotted fluorescence intensities are normalized to 1 for the initial fluorescence at t = 0 of each sample shown in panels A–C. D, ASAP1^BARPH decreases the spontaneous polymerization of 3.5 μm G-actin (10% pyrene labeled) to F-actin. ASAP1^PH does not affect actin polymerization (E), whereas V^Tail slightly decreases the actin polymerization rate under the same experimental conditions (F). Representative data are shown. G, montage of images showing the elongation of individual actin filaments alone (top) or in the presence of 500 nm ASAP1^BARPH (bottom). Time interval, 30 s; scale bar, 1 μm. (H) Actin elongation rates as a function of ASAP1^BARPH concentration (n = 28–55). ****, p < 0.0001, and **, p < 0.01, using one-way ANOVA with Dunnett's multiple-comparison test. I, F-actin does not influence the enzymatic GAP activity of ASAP1^BARPZA in vitro (n = 2).