Simultaneous fit of debranching rate and branch renucleation ratio. (A) Same data and fit as in Fig. 4, C and D, displayed from 0 to 20 pN. The dashed lines indicate the contribution to the overall debranching rate of the Arp2/3 complex interface with the daughter filament, while the dash-dot lines the one with the mother filament. (B) Branch renucleation ratio does not depend on the flow rate on surviving Arp2/3 complexes. Branch renucleation ratio from ADP-Arp2/3 (average pulling force on branch junctions is 3.8 (±0.9) pN) force as a function of applied flow rate in the microfluidics chamber (n = 26, 53, 20, 23, 22, 26, 21, 99, 60, 60, 91, 35, 81, 36, 88, 35, 81, 29, and 89 branches in flow rate ascending order), in the presence of 0.5 µM actin in F-buffer. Black data points are average values from individual data points binned by flow rates, with standard deviation error bars. Pairwise Welch’s tests result in P values >0.13. (C) Tension exerted on mother filaments at the branch location has no impact on the debranching rate, and on the branch renucleation ratio for ADP-Arp2/3 complex branches. Surviving fractions of branches monitored over time for branches grouped according to the tension experienced by the mother filament at the branch junction (n = 23, 23, and 22 branches for an average tension on the mother filament at the branching point of 15.9, 23.3, and 30.8 pN, respectively). All daughter filaments are exposed to the same flow rate, are pulled with an average force of 4.1 pN, and are exposed to 1.5 µM Alexa Fluor 568 (10%)–G-ATP-actin. Shaded areas are 95% confidence intervals. Black curves are single-exponential fits. (D) Debranching rates as a function of the tension experienced by the mother filament at the branching point, obtained from single-exponential fits of the data in C. Pairwise logrank tests performed between the survival fractions of the three populations show no statistically significant difference (all P values >0.05). (E) Branch renucleation ratio as a function of the tension experienced by the mother filament at the branch point, from the three groups of branches used in C. (F) Branch renucleation ratio follows the same decaying trend at different actin concentrations for ADP-Arp2/3 complexes. Comparison of the branch renucleation ratio for different pulling forces for ADP (blue)- or ADP-BeFx (green)-Arp2/3 complex branch junctions, in the presence of the indicated concentration of G-actin and 200 µM ATP. Data at 0.3 and 1 µM actin for ADP-Arp2/3 complex branch junctions are from Ghasemi et al. (2024). Data at 1.5 µM actin are from Figure 4. For ADP-Arp2/3 complex branches, each point is from a single experiment with at least 30 analyzed branches. Continuous lines are the best fits of the branch renucleation ratio from Fig. 3. Dashed lines are the best fits of the branch renucleation ratio data at 0.3 and 1 µM with a rescaling factor compared with the fitted curve of the 1.5 µM experimental data, with the rescaling factor as the only free parameter. The branch renucleation ratio depends on the actin concentration as follows: $0.97.kon.([actin]−Cc)kon.([actin]−Cc)+koffATP−Arp2/3$ (Ghasemi et al., 2024). The renucleation factors estimated from the fits would give an estimate of the actin concentration of 0.6(±0.05) and 0.9(±0.2) µM for the experiment performed at 0.3 and 1 µM, respectively, in Ghasemi et al. (2024). (G) Branch renucleation at different actin concentrations. Branch renucleation ratio from ADP-BeFx-Arp2/3 branches (green) in the presence of 0.2 (n = 31 branches), 0.45 (n = 32 branches), 1.5 µM actin (n = 32 branches), 200 µM ATP, with an average pulling force of 2 pN. Branch renucleation ratio from ADP-Arp2/3 complex branch junctions exposed to 20 nM cortactin (gold), in the presence of 0.2 (n = 25 branches), 0.45 (n = 31 branches), 1.5 µM actin (n = 40 branches), 200 µM ATP, with an average pulling force of 2.2 pN. Black line and data points are ADP-Arp2/3 complex branch junctions without cortactin from Ghasemi et al. (2024), and were obtained for 1 pN pulling force. The dashed line corresponds to the theoretical renucleation ratio from the ADP-BeFx-Arp2/3 complex, with an actin binding rate to the barbed end of Arp2/3 assumed to be similar to the one determined for the ADP-Arp2/3 complex, k_on = 3.4 µM⁻¹.s⁻¹ (Ghasemi et al., 2024), in competition with the spontaneous dissociation of surviving ADP-BeFx-Arp2/3 complexes from mother filaments occurring at a rate of 4.6 10⁻³ s⁻¹ (Fig. 5). (H) Impact of ionic strength on branch renucleation ratio. Branch renucleation ratio from ADP-Arp2/3 branches in the presence of 0.5 µM actin and 200 µM ATP, with an average pulling force of 4.2 pN, for different KCl concentrations in the regular buffer. Individual data points are from single experiments (n > 30 branches for each experiment). The black data point at 81 mM KCl is the average from six experiments. Error bars are standard deviations. The dashed line indicates the linear regression of the individual data points.