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. 2015 Jun 29;290(37):22494–22506. doi: 10.1074/jbc.M115.670166

FIGURE 1.

FIGURE 1.

INF2 depolymerizes actin to a limiting 1:4 INF2:actin complex. A, actin depolymerization assay using pyrene-labeled actin filaments (5 μm actin, 5% pyrene, prepolymerized overnight) and the indicated concentrations of INF2-FFC. Dead times between INF2 addition and data acquisition were ∼30–60 s. monomers (Mono) are 5 μm actin incubated overnight in the presence of 50 μm LatB. B, quantification of depolymerized actin based on plateau heights at 30 min from pyrene-actin assays in A. C, actin depolymerization assay similar to A, using 2 μm of the FFC constructs for mDia1, mDia2, FMNL3, and INF2. D, pyrene actin assays starting from actin monomers. Reactions contain 5 μm actin monomers (5% pyrene-labeled) alone, with 2 μm INF2-FFC, with 2 μm INF2-FFC and 10 μm profilin, or with 2 μm mDia1 FFC. E, high speed sedimentation assay with 5 μm actin (prepolymerized overnight) and 2 μm INF2-FFC ± 10 μm profilin, incubated for indicated times at 23 °C then ultracentrifuged for 20min at 4 °C. All lanes are from the same gel, with intervening lanes cropped. F, velocity analytical ultracentrifugation of 15 μm actin monomers (Actin), 7.5 μm INF2-FFC (INF2), or 15 μm actin mixed with 7.5 μm INF2-FFC (actin + INF2) in polymerizing conditions. Samples were preincubated for 60 min at 23 °C before starting the centrifugation. The actin sample also contained 30 μm LatB to maintain monomers. G, bar diagram of INF2 (1249 amino acids) showing domain structure (to scale with their lengths) and constructs used in this study. H, high speed sedimentation assay with indicated ratio of actin:CP and a final concentration of 1 μm actin. Samples were polymerized at 10× concentration for 1 h, then diluted 10×, and equilibrated 2 h before ultracentrifugation for 20 min at 4 °C. I, velocity analytical ultracentrifugation of 10 μm actin monomers mixed with varying ratios of capping protein in polymerizing conditions. Actin and capping protein were polymerized at 2× final concentration for 1 h at 23 °C, then diluted 2×, and equilibrated for 2 h at 20 °C before starting the centrifugation. The actin alone sample also contained 20 μm LatB to maintain monomers.