Figure 3.
IQGAP1(BAD) is a dimer that bundles actin but does not influence the rate of filament elongation. (A) IQGAP1(BAD) does not interfere with plus-end elongation in seeded actin assembly assays. Assays contain pre-polymerized (unlabeled) actin seeds, 0.5 µM actin monomers (5% pyrene-labeled), and 75 nM of each indicated IQGAP1 protein. Reactions with 10 nM Capping Protein (CP) or unlabeled seeds (alone) in the absence of IQGAP1 were used as polymerization negative controls. Values were averaged from n = 3 ± SD (shaded). (B) Single molecules of 488-labeled SNAP-IQGAP1, SNAP-IQGAP1(BAD), and SNAP-IQGAP1(160-end) subjected to step-photobleaching analysis. (C) Fluorescence intensity profiles of step photobleaching events for 1 nM SNAP-IQGAP1 proteins from reactions as in B. Red lines emphasize individual photobleaching steps. Scale, 1 µm. (D) Predictions and analysis of the oligomeric state of 488-SNAP-IQGAP1 proteins from photobleaching reactions in B and C (n = 300 molecules per protein, pooled from three replicates). (E) Representative two-channel montages depicting polymerization of single actin filaments (10% Alexa 647-label) with 75 nM of each 488-SNAP-labeled IQGAP1 over 75 s. Scale, 1 µm. (F) Kymographs and traces of elongating actin filaments as in E. Red lines and arrows indicate IQGAP1-induced pauses to filament elongation. Green lines and arrows denote IQGAP1-actin filament side-binding interactions. Scale: length, 3 µm; time, 100 s. (G and H) Percentage of actin filaments with 488-SNAP-IQGAP1 molecules on plus ends (G) or sides at 100 s in a single field of view (H) from reactions in E (n = 3 FOVs, dots). All error bars, SE unless otherwise noted. Statistics in G and H, ANOVA: (a) P ≤ 0.05 compared with 488-SNAP-IQGAP1; (b) P ≤ 0.05 compared with 488-SNAP-IQGAP1(BAD); ns, P ≥ 0.05 compared with 488-SNAP-IQGAP1. (I) Actin filament bundling (skewness) quantified at 90 s from TIRF reactions in E (n = 6–10 FOVs, dots). (J) Mean actin filament elongation rates from reactions as in E. Dots represent rates of individual actin filaments (n = 17 filaments from each of 3 independent replicates [different shades] per condition, n = 51 filaments measured in total per condition). Error bars, SD. Statistics in I and J, ANOVA: (a) P ≤ 0.05 compared with actin alone (no IQGAP1); (b) P ≤ 0.05 compared with untagged IQGAP1; ns, P ≥ 0.05. In all instances, the SNAP-tagged protein was not significantly different compared with the untagged protein. (K) Representative actin filament length-over-time plots for 75 nM 488-SNAP-IQGAP1 proteins. Both SNAP-IQGAP1 (blue) and SNAP-IQGAP1(160-end) (green) have noticeable pauses in filament elongation (red shading), whereas filaments polymerized with SNAP-IQGAP1(BAD) (purple) do not.

IQGAP1(BAD) is a dimer that bundles actin but does not influence the rate of filament elongation. (A) IQGAP1(BAD) does not interfere with plus-end elongation in seeded actin assembly assays. Assays contain pre-polymerized (unlabeled) actin seeds, 0.5 µM actin monomers (5% pyrene-labeled), and 75 nM of each indicated IQGAP1 protein. Reactions with 10 nM Capping Protein (CP) or unlabeled seeds (alone) in the absence of IQGAP1 were used as polymerization negative controls. Values were averaged from n = 3 ± SD (shaded). (B) Single molecules of 488-labeled SNAP-IQGAP1, SNAP-IQGAP1(BAD), and SNAP-IQGAP1(160-end) subjected to step-photobleaching analysis. (C) Fluorescence intensity profiles of step photobleaching events for 1 nM SNAP-IQGAP1 proteins from reactions as in B. Red lines emphasize individual photobleaching steps. Scale, 1 µm. (D) Predictions and analysis of the oligomeric state of 488-SNAP-IQGAP1 proteins from photobleaching reactions in B and C (n = 300 molecules per protein, pooled from three replicates). (E) Representative two-channel montages depicting polymerization of single actin filaments (10% Alexa 647-label) with 75 nM of each 488-SNAP-labeled IQGAP1 over 75 s. Scale, 1 µm. (F) Kymographs and traces of elongating actin filaments as in E. Red lines and arrows indicate IQGAP1-induced pauses to filament elongation. Green lines and arrows denote IQGAP1-actin filament side-binding interactions. Scale: length, 3 µm; time, 100 s. (G and H) Percentage of actin filaments with 488-SNAP-IQGAP1 molecules on plus ends (G) or sides at 100 s in a single field of view (H) from reactions in E (n = 3 FOVs, dots). All error bars, SE unless otherwise noted. Statistics in G and H, ANOVA: (a) P ≤ 0.05 compared with 488-SNAP-IQGAP1; (b) P ≤ 0.05 compared with 488-SNAP-IQGAP1(BAD); ns, P ≥ 0.05 compared with 488-SNAP-IQGAP1. (I) Actin filament bundling (skewness) quantified at 90 s from TIRF reactions in E (n = 6–10 FOVs, dots). (J) Mean actin filament elongation rates from reactions as in E. Dots represent rates of individual actin filaments (n = 17 filaments from each of 3 independent replicates [different shades] per condition, n = 51 filaments measured in total per condition). Error bars, SD. Statistics in I and J, ANOVA: (a) P ≤ 0.05 compared with actin alone (no IQGAP1); (b) P ≤ 0.05 compared with untagged IQGAP1; ns, P ≥ 0.05. In all instances, the SNAP-tagged protein was not significantly different compared with the untagged protein. (K) Representative actin filament length-over-time plots for 75 nM 488-SNAP-IQGAP1 proteins. Both SNAP-IQGAP1 (blue) and SNAP-IQGAP1(160-end) (green) have noticeable pauses in filament elongation (red shading), whereas filaments polymerized with SNAP-IQGAP1(BAD) (purple) do not.

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