Figure 4.
IQGAP1 and mDia1 can co-occupy plus ends and binding reduces formin-based elongation by displacing mDia1. (A) Schematic of formin (mDia1) constructs that bind (ΔDAD) or do not bind (FH1-C) to IQGAP1. Abbreviations: GBD, GTPase-binding domain; DID, Diaphanous inhibitory domain; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, Diaphanous autoregulatory domain. DD, dimerization domain. (B) Images from single-molecule TIRF of 1 nM 549-mDia1 constructs with 1 nM 488-IQGAP1. Arrows highlight examples of individual molecules of SNAP-IQGAP1 (green) or SNAP-mDia1 (pink) and colocalization of both proteins (white). Scale, 5 μm. (C) Quantification of colocalized mDia1-IQGAP1 molecules from reactions in B. Error bars, SE. Dots are percentages calculated for individual FOVs (n = 9 FOVs total, pooled from three replicates). Statistics, Student’s t test (two-tailed): (a) P ≤ 0.05 compared with mDia1(FH1-C). (D) Mean actin filament elongation rates from TIRF reactions containing 1 µM actin (10% Alexa 488 label), 5 µM profilin-1 (PFN1), 10 nM mDia1(FH1-C or ∆DAD), and 75 nM IQGAP1, as indicated. Reactions performed at least three times for each condition. Measurements from n = 24–105 filaments (dots). Horizontal lines indicate the mean elongation rate of filaments elongating alone (free ends; black), the mean filament elongation rate in the presence of IQGAP1 (blue) or the mean elongation rates stimulated by formin (e.g., ∆DAD growth with PFN1; pink). Error bars, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with control (actin alone); ns, P ≥ 0.05 compared with (actin alone). (E) Time-lapse montages from three-color TIRF reactions containing 1 µM actin (10% Alexa 647 label; gray) polymerizing in the presence of 5 μM PFN1, 1 nM 549-SNAP-mDia1(ΔDAD) (pink), and 1 nM 488-SNAP-IQGAP1 or 488-SNAP-IQGAP1(BAD) (green). Scale, 5 μm. Arrows indicate mDia1 (pink) and IQGAP1 (green) localization. (E′) Insets (boxes) from E show a zoomed in view of single molecules on or near filament ends. The asterisk in E marks an instance of likely IQGAP1-mediated formin displacement from the plus end. Scale, 1.5 µm. (F) Survival plots of the plus-end occupancy of 549-SNAP-mDia1(ΔDAD) molecules in the presence and absence of 488-labeled IQGAP1 proteins from reactions as in E. SNAP-mDia1 persists on ends for a longer time in reactions without IQGAP1 than in reactions with IQGAP1, as marked by dotted lines (n = 12–42 molecules per condition, as stated, from n = 3 independent reactions).

IQGAP1 and mDia1 can co-occupy plus ends and binding reduces formin-based elongation by displacing mDia1. (A) Schematic of formin (mDia1) constructs that bind (ΔDAD) or do not bind (FH1-C) to IQGAP1. Abbreviations: GBD, GTPase-binding domain; DID, Diaphanous inhibitory domain; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, Diaphanous autoregulatory domain. DD, dimerization domain. (B) Images from single-molecule TIRF of 1 nM 549-mDia1 constructs with 1 nM 488-IQGAP1. Arrows highlight examples of individual molecules of SNAP-IQGAP1 (green) or SNAP-mDia1 (pink) and colocalization of both proteins (white). Scale, 5 μm. (C) Quantification of colocalized mDia1-IQGAP1 molecules from reactions in B. Error bars, SE. Dots are percentages calculated for individual FOVs (n = 9 FOVs total, pooled from three replicates). Statistics, Student’s t test (two-tailed): (a) P ≤ 0.05 compared with mDia1(FH1-C). (D) Mean actin filament elongation rates from TIRF reactions containing 1 µM actin (10% Alexa 488 label), 5 µM profilin-1 (PFN1), 10 nM mDia1(FH1-C or ∆DAD), and 75 nM IQGAP1, as indicated. Reactions performed at least three times for each condition. Measurements from n = 24–105 filaments (dots). Horizontal lines indicate the mean elongation rate of filaments elongating alone (free ends; black), the mean filament elongation rate in the presence of IQGAP1 (blue) or the mean elongation rates stimulated by formin (e.g., ∆DAD growth with PFN1; pink). Error bars, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with control (actin alone); ns, P ≥ 0.05 compared with (actin alone). (E) Time-lapse montages from three-color TIRF reactions containing 1 µM actin (10% Alexa 647 label; gray) polymerizing in the presence of 5 μM PFN1, 1 nM 549-SNAP-mDia1(ΔDAD) (pink), and 1 nM 488-SNAP-IQGAP1 or 488-SNAP-IQGAP1(BAD) (green). Scale, 5 μm. Arrows indicate mDia1 (pink) and IQGAP1 (green) localization. (E′) Insets (boxes) from E show a zoomed in view of single molecules on or near filament ends. The asterisk in E marks an instance of likely IQGAP1-mediated formin displacement from the plus end. Scale, 1.5 µm. (F) Survival plots of the plus-end occupancy of 549-SNAP-mDia1(ΔDAD) molecules in the presence and absence of 488-labeled IQGAP1 proteins from reactions as in E. SNAP-mDia1 persists on ends for a longer time in reactions without IQGAP1 than in reactions with IQGAP1, as marked by dotted lines (n = 12–42 molecules per condition, as stated, from n = 3 independent reactions).

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