Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly
Figure 6
EspFU repeats synergistically activate actin assembly mediated by recombinant N-WASP/WIP complex in vitro.
(A) N-terminally His6-Flag-tagged N-WASP and His6-Myc-tagged WIP were co-expressed in insect cells, purified as a stoichiometric complex, resolved by SDS-PAGE, and stained with Coomassie blue. (B) Actin (2 µM) was polymerized in the presence of Arp2/3 complex, N-WASP/WIP complex, and the indicated concentrations of EspFU. F-actin fluorescence was measured in arbitrary units (AU). (C) Actin polymerization was examined in the presence of 20 nM Arp2/3 complex, 20 nM N-WASP/WIP complex, and the indicated concentrations of EspFU derivatives. Polymerization rates at half-maximal F-actin concentrations were measured relative to the rate of polymerization in control Arp2/3+N-WASP/WIP samples lacking EspFU. Curves were fit using Prism software. (D) Actin polymerization was measured as in (C), except that EspFU concentrations have been scaled to the number of repeats in each protein.