Figure S3.
Comparison of the actin assembly and IQGAP1-related activities of mDia1(FH1-C) and mDia1(∆DAD). (A) SDS-PAGE gel of untagged and SNAP-tagged formin proteins. (B and C) Bulk actin filament assembly assays comparing tagged and untagged formin constructs. Reactions contain 2 µM actin (5% pyrene-labeled), 5 µM profilin-1 (PFN1), 5 nM mDia1(FH1-C or ΔDAD), as indicated. Shaded values are SD from n = 3 assays. Curves in B and C are directly comparable (comparisons between FH1-C and ∆DAD and controls were performed on the same plates). Thus, the actin alone and actin and profilin control values are the same in each panel. (D) Mean actin filament elongation rates from TIRF reactions containing 1 µM actin monomers (10% Alexa 488 labeled) polymerizing alone or in the presence of 5 μM PFN1, 1 nM mDia1(FH1-C or ΔDAD), and 1 nM 549-SNAP-mDia1(FH1-C or ΔDAD) as indicated. Dots represent elongation rates of individual actin filaments (n = 17 filaments per reaction from three independent reactions; 51 total filaments per condition). Error bars, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with actin alone; ns, P ≥ 0.05 compared with actin alone. There was no significant difference in mean elongation when comparing untagged and SNAP-tagged proteins. (E) Images from single-molecule TIRF of 1 nM 549-mDia1 constructs with 1 nM 488-IQGAP1(BAD). Arrows highlight examples of individual molecules of SNAP-IQGAP1(BAD) (green) or SNAP-mDia1 (pink) and complexes of both proteins (white). Scale, 5 μm. (F) Quantification of colocalized mDia1-IQGAP1 molecules from reactions in E. Error bars, SE. Dots are percentages calculated for individual FOVs (n = 9 FOVs total, from three replicates). Statistics, Student’s t test (two-tailed): (a) P ≤ 0.05 compared with mDia1(FH1-C). (G) 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(FH1-C) (pink), and 1 nM 488-SNAP-IQGAP1 or 488-SNAP-IQGAP1(BAD) (green). Scale, 5 μm. Arrows indicate mDia1 (pink) and IQGAP1 (green) localization. (G′) Insets from G show a zoomed in view of single molecules present on or near filament ends. Scale, 1.5 µm. (H) Survival plots of the plus-end occupancy of 549-SNAP-mDia1(FH1-C) molecules in the presence and absence of 488-labeled IQGAP1 proteins from reactions in G. Reactions that contain IQGAP1 are shorter lived than those without IQGAP1 as marked by dotted lines (n = 18–47 molecules per condition [as stated], from n = 3 independent reactions).

Comparison of the actin assembly and IQGAP1-related activities of mDia1(FH1-C) and mDia1(∆DAD). (A) SDS-PAGE gel of untagged and SNAP-tagged formin proteins. (B and C) Bulk actin filament assembly assays comparing tagged and untagged formin constructs. Reactions contain 2 µM actin (5% pyrene-labeled), 5 µM profilin-1 (PFN1), 5 nM mDia1(FH1-C or ΔDAD), as indicated. Shaded values are SD from n = 3 assays. Curves in B and C are directly comparable (comparisons between FH1-C and ∆DAD and controls were performed on the same plates). Thus, the actin alone and actin and profilin control values are the same in each panel. (D) Mean actin filament elongation rates from TIRF reactions containing 1 µM actin monomers (10% Alexa 488 labeled) polymerizing alone or in the presence of 5 μM PFN1, 1 nM mDia1(FH1-C or ΔDAD), and 1 nM 549-SNAP-mDia1(FH1-C or ΔDAD) as indicated. Dots represent elongation rates of individual actin filaments (n = 17 filaments per reaction from three independent reactions; 51 total filaments per condition). Error bars, SE. Statistics, ANOVA: (a) P ≤ 0.05 compared with actin alone; ns, P ≥ 0.05 compared with actin alone. There was no significant difference in mean elongation when comparing untagged and SNAP-tagged proteins. (E) Images from single-molecule TIRF of 1 nM 549-mDia1 constructs with 1 nM 488-IQGAP1(BAD). Arrows highlight examples of individual molecules of SNAP-IQGAP1(BAD) (green) or SNAP-mDia1 (pink) and complexes of both proteins (white). Scale, 5 μm. (F) Quantification of colocalized mDia1-IQGAP1 molecules from reactions in E. Error bars, SE. Dots are percentages calculated for individual FOVs (n = 9 FOVs total, from three replicates). Statistics, Student’s t test (two-tailed): (a) P ≤ 0.05 compared with mDia1(FH1-C). (G) 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(FH1-C) (pink), and 1 nM 488-SNAP-IQGAP1 or 488-SNAP-IQGAP1(BAD) (green). Scale, 5 μm. Arrows indicate mDia1 (pink) and IQGAP1 (green) localization. (G′) Insets from G show a zoomed in view of single molecules present on or near filament ends. Scale, 1.5 µm. (H) Survival plots of the plus-end occupancy of 549-SNAP-mDia1(FH1-C) molecules in the presence and absence of 488-labeled IQGAP1 proteins from reactions in G. Reactions that contain IQGAP1 are shorter lived than those without IQGAP1 as marked by dotted lines (n = 18–47 molecules per condition [as stated], from n = 3 independent reactions).

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