Panel A shows a line graph. The x-axis represents Glia Maturation Factor concentration in nanomolar, ranging from 0 to 2000 nanomolar. The y-axis represents the debranching rate in reciprocal seconds, ranging from 0.01 to 0.05 per second. The graph includes data points with error bars and a fitted curve indicating a binding affinity of Glia Maturation Factor for adenosine diphosphate–ARP2/3 complexes at branch junctions. Panel B shows a log-linear scatter plot of debranching rates versus force. The x-axis represents Force in piconewtons, ranging from 0 to 8 piconewtons. The y-axis represents the Debranching rate in reciprocal seconds. The graph includes data points with error bars and dashed lines representing single-exponential fits for different conditions. Panel C shows a scatter plot of branch renucleation ratios. The x-axis represents different conditions, including the state of the ARP2/3 complex, Glia Maturation Factor concentration, actin concentration, and adenosine triphosphate concentration. The y-axis represents the Branch renucleation ratio, ranging from 0 to 1. The graph includes small data points from single experiments and large data points representing average values for each condition.
GMF accelerates debranching in a nucleotide-dependent manner. (A) Debranching rate of ADP-Arp2/3 complex branch junctions exposed to various concentrations of GMF, from experiments performed at a constant average pulling force of 2.8 pN, in the presence of 0.15 µM actin and 200 µM ATP. Each data point is from a single experiment with at least 30 analyzed branches. Error bars are 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. The binding affinity of GMF for ADP-Arp2/3 complexes at branch junctions is 30 (±8) nM, as derived from the fitting of the saturation curve. (B) Debranching rate of ADP- or ADP-BeFx-Arp2/3 complex branch junctions, in the presence or absence of at least 500 nM GMF in solution, for different pulling forces (log-linear representation). Dashed lines are single-exponential fits of the data points for each condition. 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. Each data point is from a single experiment with at least 19 and 50 analyzed branches for ADP- and ADP-BeFx-Arp2/3 complex branch junctions in the presence of GMF, respectively. Data in the absence of GMF are from Fig. 3. To facilitate the comparison of the data, single-exponential fits are represented (dashed lines). (C) Branch renucleation ratio for ADP- or ADP-BeFx-Arp2/3 complex branch junctions exposed to an average pulling force of 2.5 pN, in the presence or absence of 0.5 µM actin and 1 µM GMF, and either 200 µM or 2 mM ATP in solution. Small data points are from single experiments with at least 30 analyzed branches. Large data points represent the average value for each condition (at least 2 replicates).