Panel A shows a schematic diagram illustrating mother filament growth, branch nucleation, aging, and force-induced debranching. Panel B shows a line graph with x-axis Time (s) and y-axis Fraction of surviving branches. Panel C shows a schematic diagram illustrating mother filament growth, branch conversion, and force-induced debranching. Panel D shows a line graph with x-axis Time (s) and y-axis Fraction of surviving branches. Panel E shows fluorescence microscopy images of mother filaments, daughter filaments, and merged structures across steps. Panel F shows a scatter plot with x-axis Force (pN) and y-axis Debranching rate.
Stability of an Arp2/3 complex branch junction depends on its bound nucleotide. (A) Schematic of the debranching experiment using microfluidics to pull on ADP-Arp2/3 complex branches. Mother filaments are elongated from surface-anchored spectrin–actin seeds by flowing in 0.7 µM Alexa Fluor 488 (10%)–G-ATP-actin (magenta) for 5 min. Branches are nucleated by flowing in 0.4 µM Alexa Fluor 568 (10%)–G-ATP-actin (cyan), with VCA domains from N-WASP, and sheep Arp2/3 complex. They are then elongated and aged for 30 min by flowing in 0.2 µM Alexa Fluor 568 (10%)–G-ATP-actin alone. The dissociation of branches under constant pulling force is then monitored over time by flowing in 0.15 µM Alexa Fluor 568 (10%)–G-ATP-actin at different flow rates. The flowing solution applies a pulling force to the mother filaments and the branches. (B) Surviving fractions of ADP-Arp2/3 complex branches over time, using different flow rates to apply different constant forces using the procedure shown in A. Black curves are single-exponential fits (number of analyzed branches = 60 [0 pN], 18 [0.59 pN], 42 [1.57 pN], 25 [2.9 pN], 25 [3.62 pN], and 29 [5.21 pN]). Shaded areas are 95% confidence intervals. Vertical tick marks on the surviving curves indicate censoring events (see Materials and methods). (C) Schematic of the debranching experiment for ADP-BeFx-Arp2/3 complex branches. Steps 1 & 2 are similar to the ones in A. At step 3, branches are elongated for 6 min by flowing in 0.4 µM Alexa Fluor 568 (10%)–G-ATP-actin alone in a buffer containing 6 mM BeFx. At step 4, the dissociation of branches is observed over time in a regular buffer containing only 0.15 µM Alexa Fluor 568 (10%)–G-ATP-actin at different flow rates. (D) Surviving fractions of ADP-BeFx-Arp2/3 complex branches over time, using different flow rates to apply different constant forces using the procedure shown in C. Black curves are single-exponential fits (number of analyzed branches = 132 [0.69 pN], 100 [1.9 pN], 45 [3.04 pN], 100 [.22 pN], 60 [4.44 pN], 100 [5.12 pN], 60 [5.16 pN], and 106 [6.25 pN]). Shaded areas are 95% confidence intervals. Vertical tick marks on the surviving curves indicate censoring events. (E) Time-lapse images showing the different steps described in A, for branch formation and dissociation, with the actin mother filament in magenta, and actin branches in cyan. The scale bar is 5 µm. (F) Debranching rates as a function of constant pulling forces (log-linear representation), for ADP (blue)- and ADP-BeFx (green)-Arp2/3 complex branch junctions. 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 the 95% confidence intervals of the survival fractions from debranching experiments. For ADP-Arp2/3 complex branch junctions, data points at 0 pN are from experiments performed in open chambers, without any flow (2 replicates, Fig. S1, C and D). Data are obtained from experiments with at least 20 analyzed branches for each condition. Dashed lines are single-exponential fits of the experimental data points.