Isogai et al., JCS 2015 Figure 4

<p><strong>Fig. 4. Full-length mDia1 cooperates with the Arp2/3 complex to form branched actin networks.</strong> (A) Representative Coomassie gels showing purified recombinant wild-type (WT) and mutant (MA) mDia1 (1.5 μg) (top) and WAVE2 (2 μg) (bottom). (B–E) mDia1 promotes both nucleation and elongation of linear actin filaments in the presence of profilin. (B,C) Representative frames extracted from TIRFm time-lapse imaging of actin polymerization at the indicated time and concentration of either mDia1 WT or its MA mutant. Profilin-bound actin (2.5 μM actin+5 μM profilin) was polymerized with the indicated concentration of mDia1 WT (B) or mDia1 MA (C). Scale bars: 10 μm. (D) At least ten filaments (thin lines) were tracked to determine filament length (mean±s.d., thick lines) versus time as described in the Materials and Methods. (E) Elongation rates were derived from D as described in the Materials and Methods. Scatter dot plots show average filament elongation rates. ****P<0.0001 (one-way ANOVA with Bonferroni’s multiple comparison test; n=10–12 filaments). (F) mDia1 accelerates polymerization of branched actin filaments induced by the Arp2/3 complex. The Arp2/3 complex (20 nM) activated by WAVE2 (25 nM) was used to stimulate polymerization of profilin–actin (5 μM and 2.5 μM, respectively), either alone or with increasing concentrations of mDia1 MA. Representative frames extracted from TIRFm time-lapse movies illustrate actin polymerization at the indicated time (s) and concentration of mDia1 MA. Scale bar: 10 μm. (G) mDia1 cooperates with the Arp2/3 complex in making branched actin filaments. The area filled with filaments was quantified as described in the Materials and Methods. A representative dose–response experiment carried out on the same day is shown.