FIGURE 3.

Fimbrin inhibits Arp2/3 complex-mediated actin filament assembly. A, shown is a TIRF microscopy observation of a branched actin filament network (25 nm Arp2/3 complex + 100 nm GST-SpWsp1VCA) bundled by 250 nm Fim1. The arrow indicates a branch bundling onto the mother filament. The arrowhead indicates neighboring parallel branches bundling onto each other. Wedges indicate antiparallel bundling between branches. Seconds are indicated at the upper left. Bar, 5 μm. Corresponding supplemental Movie S9 is available online. B and C, spontaneous assembly of 20% pyrene-labeled 2.5 μm Mg-ATP-actin monomers. B, assembly of actin monomers with 25 nm Arp2/3 complex alone (thick curve) or Arp2/3 complex and 100 nm Wsp1 in the absence (●) or presence of 50 nm (□), 1 μm (♦), or 5 μm Fim1 (△). C, dependence of the initial assembly rate on concentration of Fim1 or tropomyosin Cdc8. D and E, TIRF microscopy observation of Arp2/3 complex-mediated branching. D, a plot of the branching rate (branches μm⁻¹ min⁻¹) by Arp2/3 complex and Wsp1 in the absence or presence of 250 nm Fim1. Values are the mean ± S.D. E, time-lapse micrographs of actin filament branching by 25 nm Arp2/3 complex and Wsp1 alone (top) or in the presence of 250 nm Fim1 (bottom). Time in seconds is indicated at the top left. Bar, 5 μm. Corresponding supplemental Movie S10 is available online.