ADF7 and ADF10 differentially respond to pH in severing actin filaments and promoting actin monomer dissociation. (A and B) The effect of ADF7 and ADF10 on spontaneous actin polymerization at pH 6.5 and pH 8.0. Actin (3 μM, 10% pyrene-labeled) was polymerized in the presence of various concentrations of ADF7 and ADF10. (C) Plot of the actin polymerization rates in A and B. For each curve shown in A and B, the maximal slope is plotted. All reactions were repeated at least three times. The data are presented as mean ± SD. a.u., arbitrary units. (D) Time-lapse images of actin filaments. F-actin (50% rhodamine-labeled) at 250 nM was premixed with 100 nM ADF7 or ADF10 in TIRFM buffer at pH 6.5 or pH 8.0. The mixture was perfused into a chamber and photographed immediately for at least 200 time points at 2-s intervals. The yellow arrows indicate the severing events. Bar = 2 μm. (E) Quantification of actin-severing frequencies in the presence of 100 nM ADF7 or 100 nM ADF10. Average actin filament severing frequencies (number of breaks per unit length of actin filaments [μm] per second; breaks μm⁻¹ s⁻¹) were plotted. The mean values were indicated by black horizontal bars and the error values (SD) were indicated by vertical bars. Totally, 50 actin filaments were selected for measurements. The statistical analysis was performed with an unpaired two-tailed Student’s t test. ****P < 0.0001; ***P < 0.001. (F) Quantification of actin monomer dissociation rates in the presence of 100 nM ADF7 or 100 nM ADF10. Each data point represents monomer dissociation rate, defined as the number of dissociated actin monomers per second (subunits s⁻¹). The mean values were indicated by black horizontal bar and the error values (SD) were indicated by vertical bars. Totally, 50 actin filaments were selected for measurements. The statistical analysis was performed with an unpaired two-tailed Student’s t test. **P < 0.01; *P < 0.05.