FIGURE 5.

CAP alone is sufficient to sever actin at acidic pH and rescues cofilin severing at neutral pH. A, time-lapsed images showing single actin filaments diluted into Assay Buffer at pH 6 (top series) or into 3 μm CAP at pH 6 (bottom series). Arrows indicate severing events. B, CAP scores as an actin severing factor at non-physiological, acidic pH but loses activity as the pH is raised to neutral. C, pyrene-actin polymerization seeded with CAP-severed F-actin treated at pH 6 (squares). Polymerization is inhibited by the addition of the barbed end-capping drug cytochalasin D (CytoD) (triangles), indicating that CAP-severed F-actin provides free barbed ends. Traces from one representative experiment are shown. D, quantification of filament severing rates at pH 7 in the presence of cofilin alone, CAP alone, or cofilin and CAP in combination. Neither cofilin nor CAP alone are sufficient to sever F-actin, but in combination (2 μm cofilin and 3 μm CAP), the two proteins sever F-actin at an accelerated rate. Single filament assays display the mean of at least three experiments ±S.D. Pyrene-actin assays were performed using 2 μm CAP during severing and 2 μm pyrene-actin and 300 nm cytochalasin D during polymerization. Error bars represent S.D. AU, arbitrary units.