FIGURE 4.

CAP accelerates cofilin severing activity without capping filament ends. A, cofilin severs actin filaments as expected (top series), and with the addition of CAP, both proteins sever actin filaments at an elevated rate (bottom series). Arrows indicate severing events. B, quantification of actin severing activity with increasing concentrations of CAP in the presence of 2 μm cofilin at pH 7.4. C, CAP scores in a pretreatment assay with single actin filaments, resulting in activity quantitatively similar to that seen when adding both CAP and cofilin simultaneously. D, severed ends can polymerize new actin. To Alexa Fluor 647-labeled actin filaments (panel a; pseudo-colored red), we added CAP and cofilin and tracked severing for 90 s. The image in panel b demonstrates a severing event at 45 s (arrow), producing a severed barbed end (panel c, lower arrow) and an unsevered barbed end (panel c, upper arrow). After rinsing and supplying Oregon Green-labeled G-actin, both the severed and unsevered barbed ends display the ability to nucleate new growth (panel d). E, unlabeled CAP/cofilin-severed filaments or unlabeled control filaments seeded pyrene-actin growth with CAP/cofilin-induced severing producing a decreased lag (green circles) relative to untreated control (squares), consistent with a greater number of nucleating ends. The addition of barbed end-capping drug cytochalasin D (CytoD) gave similar decreases in actin assembly rates in both control F-actin-nucleated and CAP/cofilin-treated F-actin-nucleated experiments (black circles and green triangles, respectively), consistent with a process dependent upon free barbed ends. Traces from one representative experiment are shown; bar graphs indicate mean values of at least three experiments ±S.D. Pyrene-actin assays were performed using 2 μm cofilin and 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.