Figure 2.
Figure 2. Identifying the minimal machinery required for SNX9-regulated actin assembly. (A) Maximal activation of actin polymerization by liposomes containing 4% PI(4,5)P2 + 1% PI(3)P (with 48% PC and 47% PS) assayed by pyrene actin assay in the minimal reconstituted system (20 nM Arp2/3 complex, 500 nM Cdc42⋅GTP-γS, 100 nM N-WASP/WIP complex, 100 nM SNX9, and 1 µM actin, 65:35 pyrene actin) compared with 4% PI(4,5)P2 alone, actin alone, and activation by GST-VCA fragment from N-WASP as a positive control. Data show the mean of eight traces. AU, arbitrary units. (B) Maximal rates from two technical repeats each of four independent experiments showing the mean and SEM. The maximal rates were normalized against 100% activation by GST-VCA. Significance was tested using an ANOVA test with a Tukey's multiple comparison post-hoc test; actin versus PI(4,5)P2: P = 0.900; actin versus PI(4,5)P2/PI(3)P: ***, P = 0.001; actin versus −Cdc42: P = 0.9000. ns, not significant. (C) Direct observation of liposomes (4% PI(4,5)P2, 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing 50 nM Arp2/3 complex, 50 nM Cdc42⋅GTP-γS, 100 nM N-WASP–WIP complex, 100 nM SNX9, 8 µM unlabeled actin, and 0.3 µM Alexa Fluor 647–labeled actin. Actin asters form at the surface of highly curved liposomes only when all components are present. (D) Activation of Cdc42 is needed. Direct observation of liposome samples with a continuous size distribution from 50 nm to 5 µm (4% PI(4,5)P2, 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing Cdc42⋅GDP. (C and D) Bars, 3 µm. (E) Electron micrograph of actin asters after incubation of PI(4,5)P2/PI(3)P liposomes with the minimal purified system shows disordered and branched actin filaments. Bar, 100 nm. (F–I) All components of the purified system are required for efficient actin polymerization. No actin polymerization is seen with the minimal purified system minus each individual component: Cdc42⋅GTP-γS (F), SNX9 (G), N-WASP–WIP (H), or Arp2/3 complex (I). Bars, 6 µm.

Identifying the minimal machinery required for SNX9-regulated actin assembly. (A) Maximal activation of actin polymerization by liposomes containing 4% PI(4,5)P2 + 1% PI(3)P (with 48% PC and 47% PS) assayed by pyrene actin assay in the minimal reconstituted system (20 nM Arp2/3 complex, 500 nM Cdc42⋅GTP-γS, 100 nM N-WASP/WIP complex, 100 nM SNX9, and 1 µM actin, 65:35 pyrene actin) compared with 4% PI(4,5)P2 alone, actin alone, and activation by GST-VCA fragment from N-WASP as a positive control. Data show the mean of eight traces. AU, arbitrary units. (B) Maximal rates from two technical repeats each of four independent experiments showing the mean and SEM. The maximal rates were normalized against 100% activation by GST-VCA. Significance was tested using an ANOVA test with a Tukey's multiple comparison post-hoc test; actin versus PI(4,5)P2: P = 0.900; actin versus PI(4,5)P2/PI(3)P: ***, P = 0.001; actin versus −Cdc42: P = 0.9000. ns, not significant. (C) Direct observation of liposomes (4% PI(4,5)P2, 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing 50 nM Arp2/3 complex, 50 nM Cdc42⋅GTP-γS, 100 nM N-WASP–WIP complex, 100 nM SNX9, 8 µM unlabeled actin, and 0.3 µM Alexa Fluor 647–labeled actin. Actin asters form at the surface of highly curved liposomes only when all components are present. (D) Activation of Cdc42 is needed. Direct observation of liposome samples with a continuous size distribution from 50 nm to 5 µm (4% PI(4,5)P2, 1% PI(3)P, 65% PC, 30% PS; purple) in the presence of the minimal purified system containing Cdc42⋅GDP. (C and D) Bars, 3 µm. (E) Electron micrograph of actin asters after incubation of PI(4,5)P2/PI(3)P liposomes with the minimal purified system shows disordered and branched actin filaments. Bar, 100 nm. (F–I) All components of the purified system are required for efficient actin polymerization. No actin polymerization is seen with the minimal purified system minus each individual component: Cdc42⋅GTP-γS (F), SNX9 (G), N-WASP–WIP (H), or Arp2/3 complex (I). Bars, 6 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal