Panel A shows a line graph of the fraction of surviving branches over time in a 2 millimolar sodium sulfate buffer with an average force of 3.67 plus or minus 0.3 piconewtons. The x-axis represents time in seconds, and the y-axis represents the fraction of surviving branches. Panel B shows a line graph comparing the fraction of surviving branches over time in a 50 millimolar sodium sulfate buffer and a regular buffer with an average force of 2.05 plus or minus 0.4 piconewtons. The x-axis represents time in seconds, and the y-axis represents the fraction of surviving branches. Panel C shows a line graph of normalized fluorescence intensity over time in a microfluidics assay with a flow rate of 10,000 nanoliters per minute. The x-axis represents time in seconds, and the y-axis represents normalized fluorescence intensity. Panel D shows a line graph of the fraction of surviving branches over time upon switching from a 50 millimolar phosphate buffer to a regular buffer. The x-axis represents time in seconds, and the y-axis represents the fraction of surviving branches.
Pi release rate from within the Arp2/3 complex at branch junctions. (A) ADP-Arp2/3 complex branches dissociate equally fast in Na2SO4 or regular buffer. Fraction of surviving ADP-Arp2/3 complex branch junctions exposed to a buffer with 0.15 µM actin and 0.8 mM Na2SO4, 23.4 mM NaF, and 50 mM KCl, with an average pulling force of 3.67 (±0.3) pN. The exponential fit (black line) yields a debranching rate of 7.9 10−3 (±0.3) s−1, in agreement with the expected rate for ADP-Arp2/3 complex branches (Fig 1 F). The acquisition time interval is 5 s. Number of analyzed branches = 100. (B) Fraction of surviving ADP-Arp2/3 complex branch junctions exposed to a buffer with 0.3 µM actin and either 50 mM KCl or 50 mM Na2SO4. The average force at the time of debranching is 2.05 pN. (n = 70 (50 mM KCl) and 60 (50 mM Na2SO4) branches analyzed). (C) Fast switching between two buffer conditions in microfluidics. Increase in fluorescence intensity upon flow switching in a microfluidics assay, with a flow rate of 10,000 nL/min. Depending on the size of the fluorescent compounds, either Alexa dye (blue, dashed line) or Alexa-labeled actin (green), the switching is typically between 0.5 and 1 s. Fluorescence images were recorded at 10 images per second in TIRF mode, with an evanescent penetration depth of 150 nm. All curves are positioned with time 0 when the normalized intensity reaches 0.5. (D) Estimating the Pi release rate of the Arp2/3 complex at branch junctions. Upon switching from 50 mM Pi buffer to regular buffer, the branch survival fraction decreases faster as a function of time. According to the reaction scheme presented in Fig. 8 A, Pi is released from the Arp2/3 complex at the branch junction, with a rate kPi rel. Debranching occurs at a rate kdeb., ADP-Pi or kdeb., ADP, for ADP-Pi- or ADP-Arp2/3 complex branches, respectively. The observed branch survival fraction thus evolves as (see Materials and methods) [(kdeb.,ADP - kdeb.,ADP-Pi).exp(-(kdeb.,ADP-Pi+kPi rel).t)-kPi rel.exp(-kdeb., ADP.t)]/(kdeb., ADP-kdeb.,ADP-Pi-kPi rel). The black line is the fit of this function to the experimental data (in blue, from Fig. 2 F), with three rates as free parameters. We obtained kdeb., ADP = 0.0143 s−1, kdeb., ADP-Pi = 0.00035 s−1 in reasonable agreement with the rate determined at 2.2 pN in separate experiments, and kPi rel = 0.21 s−1.