Figure 2.
Cap1/2 joins yeast formins at filament barbed ends. The behavior of formin-elongated actin filaments with their pointed ends anchored to the coverslip was monitored in mf-TIRF experiments with 1 μM yeast G-actin and 2 μM Pfy1 (profilin). (A) Schematic showing the orientation of actin filaments and the possible conditions at the barbed end: formin-bound, free, or capped. (B) Two kymographs, the top example showing a formin dissociation event where the filament transitions from growing at the formin-enhanced rate (cyan dashed line) to the free barbed end rate (yellow dashed line), and the bottom example showing an event where Cap1/2 halts formin elongation and the filament immediately transitions from growing at the formin rate (cyan dashed line) to paused (red dashed line). (C) The elongation rates of yeast actin filaments with free barbed ends and Bni1-bound barbed ends (left) or Bnr1-bound barbed ends (right) growing side-by-side in the same TIRF reaction in the presence of 1 μM yeast actin, 2 μM yeast profilin (Pfy1), and 100 nM SiR-actin (n = 130 free barbed ends and 115 Bni1-bound barbed ends, and n = 122 free barbed ends and 94 Bnr1-bound barbed ends). (D) At time 0, 100 nM Cap1/2 was introduced to yeast actin filaments growing in the presence of 1 μM yeast actin, 2 μM yeast profilin (Pfy1), and 100 nM SiR-actin, and the percent of capped filaments was monitored over time (n = 223 filaments, error bars indicate 95% confidence interval [CI]). This data was fit with a one-phase exponential curve (R2 0.99) and used to calculate the observed on-rate of Cap1/2 to the free barbed end, 1.6 ± 0.3 (±95% CI) × 106 M−1s−1. (E) A table comparing the barbed end binding kinetics of capping protein from different species in different studies (Kim et al., 2004; Kuhn and Pollard, 2007). (F) Percentage of filaments elongating at the formin rate (red, Bni1, n = 160 filaments; blue, Bnr1, n = 225 filaments) in reactions without Cap1/2. Data were fit with a one-phase exponential decay (R2 0.90 and 0.93 for Bni1 and Bnr1, respectively) to calculate the dissociation rate constants (k−) for Bni1 and Bnr1, shown on the graph ±95% CI. (G and H) TIRF reactions where at t = 0 formin-elongated filaments were exposed to different concentrations of Cap1/2. 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- (G) and Bnr1-bound (H) barbed ends. From 100 to 2,000 nM Cap1/2, n = 266, 225, 160, and 133 for Bni1-bound ends and n = 68, 178, 312, and 195 for Bnr1-bound barbed ends. (I) Data from G and H plotted as a function of Cap1/2 concentration and fit with a linear regression equation to calculate the on-rate (k+) of Cap1/2 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 3.2 × 10−4 and 1.1 × 10−3 for Bni1 and Bnr1, respectively.

Cap1/2 joins yeast formins at filament barbed ends. The behavior of formin-elongated actin filaments with their pointed ends anchored to the coverslip was monitored in mf-TIRF experiments with 1 μM yeast G-actin and 2 μM Pfy1 (profilin). (A) Schematic showing the orientation of actin filaments and the possible conditions at the barbed end: formin-bound, free, or capped. (B) Two kymographs, the top example showing a formin dissociation event where the filament transitions from growing at the formin-enhanced rate (cyan dashed line) to the free barbed end rate (yellow dashed line), and the bottom example showing an event where Cap1/2 halts formin elongation and the filament immediately transitions from growing at the formin rate (cyan dashed line) to paused (red dashed line). (C) The elongation rates of yeast actin filaments with free barbed ends and Bni1-bound barbed ends (left) or Bnr1-bound barbed ends (right) growing side-by-side in the same TIRF reaction in the presence of 1 μM yeast actin, 2 μM yeast profilin (Pfy1), and 100 nM SiR-actin (n = 130 free barbed ends and 115 Bni1-bound barbed ends, and n = 122 free barbed ends and 94 Bnr1-bound barbed ends). (D) At time 0, 100 nM Cap1/2 was introduced to yeast actin filaments growing in the presence of 1 μM yeast actin, 2 μM yeast profilin (Pfy1), and 100 nM SiR-actin, and the percent of capped filaments was monitored over time (n = 223 filaments, error bars indicate 95% confidence interval [CI]). This data was fit with a one-phase exponential curve (R2 0.99) and used to calculate the observed on-rate of Cap1/2 to the free barbed end, 1.6 ± 0.3 (±95% CI) × 106 M−1s−1. (E) A table comparing the barbed end binding kinetics of capping protein from different species in different studies (Kim et al., 2004; Kuhn and Pollard, 2007). (F) Percentage of filaments elongating at the formin rate (red, Bni1, n = 160 filaments; blue, Bnr1, n = 225 filaments) in reactions without Cap1/2. Data were fit with a one-phase exponential decay (R2 0.90 and 0.93 for Bni1 and Bnr1, respectively) to calculate the dissociation rate constants (k−) for Bni1 and Bnr1, shown on the graph ±95% CI. (G and H) TIRF reactions where at t = 0 formin-elongated filaments were exposed to different concentrations of Cap1/2. 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- (G) and Bnr1-bound (H) barbed ends. From 100 to 2,000 nM Cap1/2, n = 266, 225, 160, and 133 for Bni1-bound ends and n = 68, 178, 312, and 195 for Bnr1-bound barbed ends. (I) Data from G and H plotted as a function of Cap1/2 concentration and fit with a linear regression equation to calculate the on-rate (k+) of Cap1/2 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 3.2 × 10−4 and 1.1 × 10−3 for Bni1 and Bnr1, respectively.

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