Figure 1.
IQGAP1 reduces the mean actin filament elongation rate in vitro. (A) Schematic of IQGAP1 domains. Abbreviations: CHD, calponin homology domain; WW, WW domain; GRD, GAP-related domain; LBR, ligand binding region. (B) SDS-PAGE gel of purified IQGAP1. (C) Images from TIRF assays containing 1 μM actin monomers (20% Oregon Green [OG]-label) and noted concentrations of IQGAP1. Scale, 25 μm. (D) Image montages displaying the polymerization of single actin filaments in the absence or presence of 75 nM IQGAP1. Arrows mark actively growing ends (green) or IQGAP1-mediated pauses in actin filament elongation (pink). Scale, 2 µm. (E) Kymographs and traces of elongating actin filaments in the absence or presence of 75 nM IQGAP1. Red lines indicate pauses in elongation. Scale: length, 3 µm; time, 100 s. (F) Mean actin filament nucleation at noted concentrations of IQGAP1. Dots represent filament counts 200 s after initiation of reactions in C from n = 3 fields of view. (G) Mean actin filament elongation rates from TIRF reactions in C. Dots represent elongation rates of individual actin filaments (n = 75 filaments per condition; pooled from 3 independent trials). Error bars in F and G, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with control (0 nM IQGAP1); ns, P ≥ 0.05 control. (H) Representative actin filament length-over-time plots indicate that filaments polymerized in the presence of 75 nM IQGAP1 (teal) have pauses in filament elongation (red shading), whereas filaments polymerized without IQGAP1 (gray) do not. (I) Frequency distribution plots indicate the average duration (20.6 s) of IQGAP1-mediated pauses in actin filament elongation. Pauses were calculated from elongation rates measured in G (n = 31–70 pauses [331 total] measured from n = 75 filaments per condition). The R2 values for Gaussians ranged between 0.99 and 1.00, whereas the R2 values for non-pausing conditions fit using a non-linear fit ranged between 0.94 and 0.99. Source data are available for this figure: SourceData F1.

IQGAP1 reduces the mean actin filament elongation rate in vitro. (A) Schematic of IQGAP1 domains. Abbreviations: CHD, calponin homology domain; WW, WW domain; GRD, GAP-related domain; LBR, ligand binding region. (B) SDS-PAGE gel of purified IQGAP1. (C) Images from TIRF assays containing 1 μM actin monomers (20% Oregon Green [OG]-label) and noted concentrations of IQGAP1. Scale, 25 μm. (D) Image montages displaying the polymerization of single actin filaments in the absence or presence of 75 nM IQGAP1. Arrows mark actively growing ends (green) or IQGAP1-mediated pauses in actin filament elongation (pink). Scale, 2 µm. (E) Kymographs and traces of elongating actin filaments in the absence or presence of 75 nM IQGAP1. Red lines indicate pauses in elongation. Scale: length, 3 µm; time, 100 s. (F) Mean actin filament nucleation at noted concentrations of IQGAP1. Dots represent filament counts 200 s after initiation of reactions in C from n = 3 fields of view. (G) Mean actin filament elongation rates from TIRF reactions in C. Dots represent elongation rates of individual actin filaments (n = 75 filaments per condition; pooled from 3 independent trials). Error bars in F and G, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with control (0 nM IQGAP1); ns, P ≥ 0.05 control. (H) Representative actin filament length-over-time plots indicate that filaments polymerized in the presence of 75 nM IQGAP1 (teal) have pauses in filament elongation (red shading), whereas filaments polymerized without IQGAP1 (gray) do not. (I) Frequency distribution plots indicate the average duration (20.6 s) of IQGAP1-mediated pauses in actin filament elongation. Pauses were calculated from elongation rates measured in G (n = 31–70 pauses [331 total] measured from n = 75 filaments per condition). The R2 values for Gaussians ranged between 0.99 and 1.00, whereas the R2 values for non-pausing conditions fit using a non-linear fit ranged between 0.94 and 0.99. Source data are available for this figure: SourceData F1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal