ADF7 and ADF10 have differential responses to pH in disassembling actin filaments in vitro. (A) High-speed F-actin cosedimentation experiments in the presence of ADF7 and ADF10 at different pH. Actin filaments at 3 μM were incubated with 20 μM ADF7 or 20 μM ADF10 at different pH for 30 min at room temperature and the mixtures were subjected to analysis by SDS-PAGE. S, supernatant; P, pellet. (B) Quantification of the amount of actin in the supernatant. The amount of actin in the supernatant from A was plotted. The data are presented as mean ± SD. (C and D) The effect of ADF7 (C) or ADF10 (D) on promoting dilution-mediated actin depolymerization under different pHs. Preassembled actin filaments at 5 μM (50% NBD-labeled) were diluted to 200 nM in the presence of 200 nM ADF7 or 200 nM ADF10 at pH ranging from 5.5 to 8.0. (E and F) ADF7 and ADF10 shorten actin filaments. (E) Representative images of actin filaments. Preassembled actin filaments (50% rhodamine-labeled) at 250 nM were incubated with various concentrations of ADF7 or ADF10 at either pH 6.5 or pH 8.0 for 5 min, and the images were then captured by TIRF microscopy. Bar = 5 μm. (F) The lengths of actin filaments in the presence of ADF7 or ADF10 were measured and plotted. Data are presented as mean ± SD. The statistical analysis was performed with an unpaired two-tailed Student’s t test. ****P < 0.0001; ***P < 0.001; **P < 0.01; ns, no significant difference. (G) NBD-actin binding assay to determine the binding activity of ADF7 to G-actin at pH 8.0. The representative Kd for binding of ADF7 to ADP-actin is 1.15 μM. (H) NBD-actin binding assay to determine the binding activity of ADF7 to G-actin at pH 6.5. The representative Kd for binding of ADF7 to ADP-actin is 0.59 μM. (I) The average Kd for the binding of ADFs to NBD-ADP-actin. The average Kd (mean ± SD) for binding of ADF1, ADF7, or ADF10 to ADP-actin is presented. The experiment was repeated three times. Source data are available for this figure: SourceData F1.
