Figure 2: Actin filaments stimulate the short pitch conformation weakly or not at all.

A. Cartoon depicting the short pitch crosslinking assay used to determine if actin filaments stimulate the short pitch conformation. BMOE (bismaleimidoethane) has a spacer arm of 8 Å. B. Analysis of crosslinking reactions by western blot with an anti-Arp3 antibody (top) or by Coomassie-staining of SDS-PAGE gels (bottom). Crosslinking reactions contained 1 μM ScArp2/3 dual cysteine complex with or without BMOE, LZ-VCA (leucine zipper N-WASP-VCA) or F-actin, as indicated. Reactions were quenched after 1 min for analysis. C. Quantification of the fraction of Arp2/3 complex with the short pitch crosslink for reaction conditions described in B. For all panels in this figure, bars represent the mean and standard deviation. n.s.: not significant, * p<0.05, ** p<0.005, **** p<0.0001 for sample compared with the reaction containing just BMOE. D. Binding isotherm showing fraction of Arp2/3 complex (45 nM total) bound versus concentration of actin filaments in copelleting assays. Each point indicates the mean (n = 5). Error bars: standard deviation. Data were fit as described in methods. We note that the affinity measured in these assays (~0.7 μM) is similar to affinities previously measured for S. cerevisiae, B. taurus and H. sapiens complexes using cosedimentation assays but ~30-400 fold tighter than affinities measured for S. cerevisiae or S. pombe complexes using fluorescence-based methods^2,12,28,52. The source of these discrepancies is currently unknown. At saturation, only ~60% of the complex is bound to filaments. While we cannot currently explain this observation, both crosslinked and uncrosslinked complexes eluted from gel filtration columns after the void volume in single gaussian peaks (Figure S7), indicating that the complex is not significantly dissociated or aggregated. E. Quantification of crosslinking reactions containing 1 μM ScArp2/3 dual cysteine complex with or without BMOE, 2.5 μM LZ-VCA (leucine zipper N-WASP-VCA), 5 μM Latrunculin B bound actin monomers and F-actin, as indicated. Reactions were quenched after 1 min for analysis. F. Quantification of crosslinking reactions containing 1 μM ScArp2/3 dual cysteine complex with or without BMOE, 2.5 μM LZ-VCA (leucine zipper N-WASP-VCA), 5 μM Latrunculin B bound budding yeast actin monomers and 15 μM budding yeast F-actin, as indicated. Reactions were quenched after 1 min for analysis. See also Figures S1, S2 and S7.