Panel A shows a schematic diagram illustrating measurement of debranching rates through branch loss under actin monomer conditions. Panel B shows a scatter plot with x-axis Force (piconewtons) and y-axis Debranching rate (per second). Panel C shows a schematic diagram and fluorescence microscopy images illustrating branch renucleation on mother filaments over time. Panel D shows a scatter plot with x-axis Force (piconewtons) and y-axis Branch renucleation ratio.
Stability of the Arp2/3 complex interfaces is nucleotide-dependent. (A) Schematics of the experiment where the debranching rate is probed at different pulling forces in the presence of 0.15 µM actin. (B) Debranching rate as a function of the pulling force for ADP (blue)- and ADP-BeFx (light green)-Arp2/3 complex branch junctions, and for Arp2/3 complex branch junctions exposed to 50 mM phosphate buffer (dark green, dashed). Each point is from a single experiment with at least 40 analyzed branches. The error bar for each individual data point is the standard deviation of the force. All data points are from Figs. 1 and 2. (C) (Left) Schematics of the experiments where the branch renucleation ratio is probed at different pulling forces, in the presence of 1.5 µM Alexa Fluor 568 (10%)–G-actin. (Right) Time-lapse images showing the branch renucleation from ADP-BeFx Arp2/3 complexes, with the actin mother filament in magenta, and actin branches in cyan. The scale bar is 5 µm. (D) Branch renucleation ratio as a function of the pulling force for ADP (blue)- or ADP-BeFx (light green)-Arp2/3 complexes, and for Arp2/3 complexes exposed to 50 mM phosphate (dark green, dashed). Each point is from a single experiment with at least 30 analyzed branches. The error bar for each individual data point is the standard deviation of the force, and the 95% confidence interval for the branch renucleation ratio. In B and D, continuous lines are best fits of the data, obtained by the simultaneous least-square error minimization of the debranching rate and of the branch renucleation ratio, for each Arp2/3 complex nucleotide state (see the main text). For the experiments performed in the presence of 50 mM phosphate, the dashed lines are directly obtained by considering branch junctions that detach 68% of the time with their Arp2/3 complex in the ADP state, and 32% in the ADP-Pi state that do not interconvert, and using the parameter obtained from the fits of the ADP- and ADP-BeFx-Arp2/3 complex branch junction data (see Materials and methods).