Arp2/3 complex nucleotide state tunes its stability and its ability to renucleate branches. (A) Schematic of the debranching experiment with fluorescently labeled Arp2/3 complexes. Previously formed branches are exposed to a buffer containing 200 µM ATP, without actin in order to observe the dissociation of fluorescent Arp2/3. Images are acquired every second for ADP-Arp2/3 branches in a regular buffer or exposed to a 50 mM Pi buffer, and every 2 s for ADP-BeFx-Arp2/3 complex branches. (B) Time-lapse images of an experiment using Alexa Fluor 488–labeled Arp2/3 complex in 50 mM Pi buffer. (C) Fraction of surviving fluorescently labeled Arp2/3 complex still bound to the mother filament as a function of time, observed as described in A, pooled from two experiments for each condition, in the ADP (n = 70 branches, blue) or ADP-BeFx (n = 74 branches, light green) nucleotide state, or in the presence of 50 mM phosphate (n = 80 branches, dark green). For all experiments, branches are pulled on with an average force of 5.9 pN, and the ionic strength of the buffer is adjusted to match the one of the 50 mM phosphate buffer. Fits (black lines) by a monoexponential decay function yield k_off^ADP = 0.56 (±0.08) s⁻¹ and k_off^ADP-BeFx = 4.6 (±0.7) × 10⁻³ s⁻¹ for ADP and ADP-BeFx Arp2/3 complex experiments, respectively. For the 50 mM phosphate buffer condition, the fit using a model explained in the main text and the Method section yields k_{Pi rel.} = 0.057 (±0.03) s⁻¹. Error bars indicate 95% confidence intervals. (D) Branch renucleation ratio measured for Arp2/3 complex branch junctions in ADP, ADP-BeFx states, or in the presence of 50 mM Pi, in the presence of 0.5 μM G-actin in solution and an average pulling force of 4.9 pN. Each small data point indicates a single experiment with at least 30 analyzed branches. Large data points are averages for each condition.