PI(3,5)P2 antagonizes the activity of cortactin in branched actin regulation. (A and B) Synergistic activation of actin branching. (A) TIRF microscopy images of reactions containing actin (0.75 µM 33% Oregon Green labeled), 10 nM Arp2/3 complex, 50 nM GST-VCA, and the indicated amounts of cortactin and PI(3,5)P2- or PI(3,4)P2-liposomes. Bar, 3 µm. (B) Branch density plotted as a function of time. Error bars = SEM for ≥3 independent experiments. Branch density of cortactin + PI(3,5)P2-liposomes was significantly decreased (P < 0.05) as compared with that of the +cortactin only condition for each time point after 150 s, except for the 390- and 450-s time points. (C) and (D) Debranching. (C) Representative images of actin debranching over time. 3 µM G-actin was polymerized in the presence of 100 nM Arp2/3 and 600 nM GST-VCA for 8 min. At that time, buffer, 500 nM cortactin, 500 nM PI(3,5)P2, 500 nM cortactin + 500 nM PI(3,5)P2-liposomes, or 500 nM cortactin + 500 nM PI(3,4)P2-liposomes were added to individual reactions. Samples were incubated for an additional minute, as indicated, before the debranching reactions were stopped with 3 µM rhodamine-phalloidin and visualized. Bar, 3 µm. (D) Data points show percent branched filaments per field, calculated from three or four independent experiments. Error bars = SEM. * or #, P < 0.05; ** or ##, P < 0.01. Asterisks compare cortactin versus +buffer control and the # symbol compares +cortactin versus +cortactin + PI(3,5)P2 condition.
