Panel A shows a series of line graphs depicting the fraction of surviving branches over time under different constant forces, ranging from 0.54 pN to 6.03 picoNewtons. The x-axis represents time in seconds, and the y-axis represents the fraction of surviving branches. Each line graph is fitted with a single exponential curve, and shaded areas indicate 95 percent confidence intervals. Panel B presents a scatter plot with error bars showing debranching rates as a function of constant pulling forces for branches pre-aged for 4 minutes (light blue) and 30 minutes (blue). The x-axis represents force in picoNewtons, and the y-axis represents debranching rate in inverse seconds. A dashed line represents a single exponential fit of the 30-minute pre-aging data. Panel C is a microscopy image showing branches (cyan) on mother filaments (magenta) under specific experimental conditions. The scale bar is 10 micrometers. Panel D shows line graphs depicting the fraction of surviving branches over time for branches in ADP and ADP-Pi states. The x-axis represents time in seconds, and the y-axis represents the fraction of surviving branches. Shaded areas indicate 95 percent confidence intervals, and black curves are single exponential fits.
Impact of Arp2/3 complex aging and force on the debranching rate. (A) Surviving fractions of branches monitored over time using different flow rates to apply different constant forces, while flowing in 0.15 µM Alexa Fluor 568 (10%)–G-ATP-actin. Black curves are single-exponential fits. The average force for each experiment is indicated. Each curve is from a single experiment with at least 50 analyzed branches. Shaded areas are 95% confidence intervals. Vertical ticks on the surviving curves are times where censoring events occurred. (B) Debranching rates as a function of constant pulling forces, for ADP-Arp2/3 complex branch junctions, pre-aged for 4 (light blue) or 30 (blue) minutes. Error bars are standard deviations for the force, and confidence intervals on the single-exponential fits, obtained by fitting the upper and lower bounds of 95% confidence intervals of the survival fractions of debranching experiments. Data are obtained from experiments with at least 50 and 18 analyzed branches for 4- and 30-min pre-aging duration, respectively. The dashed line is a single-exponential fit of the 30-min pre-aging data. (C) Branches (cyan) on mother filaments (magenta) are exposed to a solution of 0.04 µM 10% Alexa Fluor 568–labeled G-actin in F-buffer, 50 mM KCl, and 0.5% methylcellulose, in the presence or absence of 50 mM phosphate. Images were acquired at a frame rate of 1 frame every 30 s in TIRF for at least 60 min. The scale bar is 10 µm. (D) Surviving fractions of branches monitored over time. Shaded areas are 95% confidence intervals. Vertical ticks on the surviving curves are times where censoring events occurred. Black curves are single-exponential fits with characteristic rates of 1.49 (±0.9) × 10−4 (n = 60 branches) and 1.7 (±0.7) × 10−4 (n = 90) s−1 for ADP-Arp2/3 complex branches; 3.2 (±1.6) × 10−5 (n = 60) and 1.48 (±1) × 10−5 (n = 90) s−1 for Arp2/3 complex branches in the presence of 50 mM phosphate.