Fig. 6.

PI(3)P/PI(4,5)P₂-containing liposomes stimulate actin polymerization by recruiting additional N-WASP and Arp2/3 complex to the liposome surface via Snx9. (A) Pyrene actin assay in response to GTPγS.Cdc42, PI(4,5)P₂, or PI(3)P/PI(4,5)P₂ from mock-depleted or toca-1–depleted extracts showing that PI(3)P/PI(4,5)P₂ stimulates actin polymerization independently of toca-1. (B) Western blot of toca-1 in mock- and toca-1–depleted extract used showing effective immunodepletion. (C) Pyrene actin assay in response to PI(3)P/PI(4,5)P₂ from mock-depleted extract, Snx9-depleted extract, and Snx9-depleted extract supplemented with 140 nM purified Snx9. (D) Western blot of Snx9 from mock- and Snx9-depleted extract showing effective immunodepletion. (E) Sedimentation of Snx9 from extracts with indicated liposome compositions showing that it binds preferentially to liposomes containing both PI(3)P and PI(4,5)P₂. (F) Quantification of Snx9 that is pelleted in the liposome compositions in E, adjusted for binding to control PC/PS liposomes alone, determined by Western blotting. *P = 0.037 and **P = 0.0022 are significant differences by t test. Data are representative of three independent experiments; error bars are SD. (G) Sedimentation of purified Snx9 with indicated liposome compositions showing that it binds preferentially to liposomes containing both PI(3)P and PI(4,5)P₂. (H) Liposome sedimentation assay showing the quantification of Snx9 in extract pelleted in PC/PI/PI(4,5)P₂ in the absence or presence of 10 μM BEZ-235, and in control PC/PS/PI(4,5)P₂ liposomes. *P = 0.037 and **P = 0.0061 indicate a significant and very significant difference in binding relative to PC/PI/PI(4,5)P₂ liposomes alone by t test. Data are representative of four experiments; error bars show SEM.