Formin-mediated actin assembly is inhibited by the introduction of Cap1/2 or CapZ. (A–C) Assembly of 1.5 μM rabbit muscle actin, 10% pyrene-labeled, was initiated in the presence of 7.5 μM yeast profilin, Pfy1 (A and B), or vertebrate profilin (C) by the addition of 50 nM Bni1 (A), 5 nM Bnr1 (B), or 2 nM mDia1 (C) and monitored by pyrene fluorescence enhancement. At 140 (A) or 127 s (B and C), Cap1/2 or vertebrate CapZ at the indicated concentration was added to the reaction. Each curve is the average of two technical replicates. (D) Actin assembly rate during a 230-s window 130 s after the addition of Cap1/2 or CapZ. Rates are shown relative to the condition with formin only (Bni1, Bnr1, or mDia1). Bars represent the mean and error bars indicate the SD. Statistical significance is calculated by two-way ANOVA (n.s., no significance; P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). Letters a, b, and c indicate conditions that are statistically significantly different from the buffer alone control for Bni1, Bnr1, and mDia1, respectively. There is a significant difference between the three formins (**, P = 0.0039). (E and F) mf-TIRF reactions where at t = 0 formin-elongated filaments were exposed to different concentrations of vertebrate CapZ. The percent of capped filaments over time was fit with a one-phase exponential to calculate the observed on rate of Cap1/2 for Bni1- (E) and Bnr1-bound (F) barbed ends. From 10 to 100 nM Cap1/2, n = 23, 107, and 40 Bni1-bound filaments and n = 50, 25, and 26 Bnr1-bound filaments. (G) Data from E and F plotted as a function of CapZ concentration and fit with a linear regression equation to calculate the on-rate (k+) of CapZ to Bni1- and Bnr1-bound barbed ends shown in the figure ± SD. Error bars indicate the 95% CI from the curve fits in F and G. The SD of the residuals (Sy.x) for the curve fit is 5.8 × 10−3 and 1.2 × 10−3 for Bni1 and Bnr1, respectively.
