ADF7-eGFP and ADF10-eGFP exhibit differential pH sensitivity in disassembling and severing actin filaments in vitro. (A) Schematic diagram showing the insertion of eGFP to generate eGFP fusion proteins of ADF7-eGFP and ADF10-eGFP. The black triangle indicates that eGFP is inserted after valine (V) in position 10. (B) SDS-PAGE analysis of purified recombinant ADF7-eGFP_V10 and ADF10-eGFP_V10 fusion proteins. (C) Time-lapse images of actin filaments. F-actin (50% rhodamine-labeled) at 250 nM was premixed with 100 nM ADF7-eGFP_V10 or ADF10-eGFP_V10 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. (D) Quantification of actin-severing frequencies in the presence of 100 nM ADF7-eGFP_V10 or 100 nM ADF10-eGFP_V10. Average actin filament severing frequencies (breaks μm⁻¹ s⁻¹) were plotted. The mean values are indicated by black horizontal bars, and the error values (SD) are indicated by vertical bars. In total, 50 actin filaments were selected for measurements. The statistical analysis was performed with an unpaired two-tailed Student’s t test. *P < 0.05; ****P < 0.0001. (E) Quantification of actin monomer dissociation rates in the presence of 100 nM ADF7 or 100 nM ADF10. Each data point represents a monomer dissociation rate (subunits s⁻¹). The mean values are indicated by black horizontal bars, and the error values (SD) are indicated by vertical bars. In total, 50 actin filaments were selected for measurements. The statistical analysis was performed with an unpaired two-tailed Student’s t test. *P < 0.05; ***P < 0.001. Source data are available for this figure: SourceData FS1.