Panel A shows a schematic diagram illustrating branch growth, phosphate-buffer elongation, and force-induced debranching at different inorganic phosphate concentrations. Panel B shows a line graph with x-axis Time (seconds) and y-axis Fraction of surviving branches. Panel C shows a line graph with x-axis Inorganic phosphate concentration (millimolar) and y-axis Debranching rate (per second). Panel D shows a scatter plot with x-axis Force (piconewtons) and y-axis Debranching rate (per second). Panel E shows a schematic diagram comparing slow and fast debranching under high and low inorganic phosphate conditions. Panel F shows a line graph with x-axis Time (seconds) and y-axis Fraction of surviving branches.
Pi is in rapid equilibrium with the Arp2/3 complex at branch junctions. (A) Schematic of the debranching experiment for Arp2/3 complex branch junctions exposed to buffers containing different Pi concentrations. From step 3, branches, initiated as in Fig. 1 A, are constantly exposed to a phosphate buffer. (B) Surviving fractions of branches monitored over time by applying an average force of 3.3 (±0.2) pN, and exposing them to buffers of different Pi concentrations, containing 0.15 µM Alexa Fluor 568 (10%)–G-ATP-actin. Black curves are single-exponential fits (number of analyzed branches = 86 [0 mM Pi], 66 [5 mM Pi], 56 [10 mM Pi], 70 [25 mM Pi], and 60 [50 mM Pi]). Shaded areas are 95% confidence intervals. Vertical ticks on the surviving curves are times where censoring events occurred. (C) Debranching rates as a function of Pi concentration, obtained from the fits of the curves shown in B. Error bars are confidence intervals on the single-exponential fits, obtained by fitting the upper and lower bounds of 95% confidence interval curves. The line is the result of a hyperbolic fit yielding KD, the equilibrium dissociation constant of Pi from the Arp2/3 complex at branch junction. (D) Debranching rates as a function of constant pulling forces, for Arp2/3 complex branch junctions exposed to a 50 mM Pi buffer solution (green), and for ADP-BeFx-Arp2/3 complex branch junctions (light green). 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. For each force condition, data are from experiments with on average 100 analyzed branches for branches exposed to 50 mM Pi and 90 analyzed branches for ADP-BeFx-Arp2/3 complexes. The green dashed line is a single-exponential fit of the 50 mM Pi data. The blue dashed line is from Fig. 1 F for ADP-Arp2/3 complex branches. (E) Schematic of the debranching experiment of Arp2/3 complex branch junctions exposed to 50 mM Pi or regular buffer, with a fast flow switching between the two conditions (<1 s). (F) Surviving fraction of branches monitored over time by applying an average force of 2.3 (±0.3) pN and exposing them to either regular or 50 mM phosphate buffer, both containing 0.1 µM Alexa Fluor 568 (10%)–G-actin. Debranching rates obtained from single-exponential fits (not shown) of the different regimes of the curves are indicated (see also Fig. S3 D). 5 s before the buffer condition is rapidly changed by microfluidics, the acquisition frame rate is increased from 1 to 2 frames per second. Censoring events are indicated by vertical ticks. n = 62 analyzed branches.