Fig. 6. Actin polymerization in the absence or presence of preformed actin filaments. Actin polymerization is shown induced by Arp2/3 and VCA fragment (a) and wild-type (b) or basic mutant (c) N-WASP. All assays were performed with N-WASP or N-WASP mutants at 100 nM and Arp2/3 at 60 nM. F-actin was added to a final concentration of 300 nM. GTPS-loaded Cdc42 and PIP₂-containing vesicles were added at final concentrations of 500 nM and 1 µM, respectively. After pre-incubation for 5 minutes, actin was added (0.5 µM, 9% pyrene-labeled) and fluorescence was monitored. Pyrene fluorescence was converted to the amount of actin filaments. The rates of increase of actin filaments (the value obtained by differentiating the plot curve shown in a-c) are also shown: without Arp2/3 and N-WASP (d), VCA fragment (e), wild-type (f) and basic (g). (h) The barbed end concentration of actin filaments at points where polymerization is 80% complete (see Materials and Methods). Pre-existence of side of actin filament (gelsolin-capped filaments) increased the number of the barbed ends only in the case of wild-type N-WASP. (i) Depolymerization of gelsolin-capped actin filaments. Gelsolin-capped filaments were diluted to 0.1 µM in the presence of N-WASP (200 nM) or N-WASP (100 nM) with Cdc42 (500 nM), PIP₂ (1 µM vesicles) and Arp2/3 (60 nM) and the fluorescence was monitored.