Single-molecule imaging of IQGAP1 regulating actin filament dynamics

Abstract

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet there remains a limited understanding of how IQGAP proteins directly influence actin filament dynamics. To close this gap, we used single-molecule and single-filament total internal reflection fluorescence microscopy to observe IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results demonstrate that full-length human IQGAP1 forms dimers that stably bind to actin filament sides and transiently cap barbed ends. These interactions organize filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as an actin regulator in vivo and how it may be regulated in different biological settings.

FIGURE 1:

IQGAP1 transiently caps actin filament ends to inhibit barbed end growth. (A) Domain layouts for full-length IQGAP1 and fragments used in this study. Domains: CH, calponin homology; Repeats, six 50-amino-acid repeats; W, WW domain; IQ, four isoleucine–glutamine motifs; GRD, GAP-related domain; CT, C-terminal domain. Amino acid numbering and boundaries are from UniProt: P46940, PDB: 1 × 0H and structural work (Abel et al., 2015; LeCour et al., 2016). Binding partners of different domains are shown. (B) Representative images from open-flow TIRF microscopy assays 10 min after initiation of actin assembly. Reactions contain 1 µM G-actin (10% Oregon green–labeled, 0.5% biotin-labeled) and different concentrations of full-length IQGAP1. Scale bar, 10 μm. (C) Barbed end elongation rates for actin filaments in TIRF reactions as in B (n = 60 filaments, pooled from three independent trials for each condition). Mean and SD. Student’s t test was used to determine the statistical significance of differences between conditions (* p < 0.05). Inset graph: fraction of free growing barbed ends vs. concentration of IQGAP1 (nM) fitted with a hyperbolic binding curve to measure the equilibrium binding constant (K_d = 25 nM). Error bars, SEM. (D) Example traces of individual filament lengths over time (five each) for control reactions and reactions containing 20 nM IQGAP1, from the same reactions as in C. Note the increase in pause time (no growth) in the presence of 20 nM IQGAP1 (example pauses for one filament trace highlighted by magenta lines). (E) Duration of pauses in the presence of 20 nM IQGAP1 (blue histogram, red curve, n = 10 filaments, and 65 pause events) compared with control (gray histogram, black curve, n = 10 filaments, and 176 pause events). The mean barbed end pause time in the control reactions (lacking IQGAP1) was 5.1 s and in the presence of 20 nM IQGAP1 was 25.8 s. Fits were calculated from a single exponential equation. Inset: table listing IQGAP1 binding affinity, on rate, and off rate for the barbed end. (F) Representative quantitative Western blot (one of three independent trials) used to determine the concentration of endogenous IQGAP1 in U2OS cells. Blot was probed with anti-IQGAP1 antibody to compare the signal for endogenous IQGAP1 in the cell lysate lane to known quantities of purified 6His-IQGAP1. A standard curve was generated from the signals on the blot. The average cellular concentration of IQGAP1 (405 nM ± 112) was calculated from values obtained in three independent trials (482, 276, and 458 nM).

FIGURE 2:

IQGAP1 dimers bind stably to the sides of actin filaments. (A) Representative step photobleaching traces from single molecules of full-length 649-SNAP-IQGAP1. Plot shows fluorescence intensity over time. Inset shows montage of images for one of the molecules shown in the plot (molecule 1, magenta). (B) Fraction of 649-SNAP-IQGAP1 molecules (n = 157) that photobleached in one, two, or three steps (>3 photobleaching steps was never observed) from analysis as in A. Error bars, SEM. Observed fraction of photobleaching events (black) is compared with the predicted fraction of photobleaching events (based on SNAP-labeling efficiency [ Breitsprecher et al., 2012]) for different oligomeric states (color-coded symbols). (C) Representative time-lapse images and kymograph from TIRF reaction containing 2 nM 649-SNAP-IQGAP1, showing molecules (magenta) binding to an actin filament (cyan). Scale bar, 2 μm. Kymograph shows that 649-SNAP-IQGAP1 decoration is distributed along the filament over time. (D) Schematic showing experimental strategy to monitor 649-SNAP-IQGAP1 dissociation from filaments by mf-TIRF. Actin filaments with free barbed ends were polymerized from coverslip-anchored spectrin-actin seeds in the presence of 1 μM G-actin (15% Alexa-488-labeled) and 5 μM profilin and then capped at their barbed ends by flowing in 100 nM mouse capping protein (CP) for 1 min to prevent subsequent disassembly. Next, 0.5 nM 649-SNAP-IQGAP1 (without actin) was flowed in for 1 min to allow binding to filament sides, then buffer was flowed in (to remove free 649-SNAP-IQGAP1), and dissociation of 649-SNAP-IQGAP1 molecules was monitored over time. PE, pointed end; BE, barbed end. (E) Representative image and kymograph of 649-SNAP-IQGAP1 molecules (magenta) bound to an actin filament (cyan). Scale bar, 2 μm. Observed dwell times (n = 142 binding events) were plotted (dotted line), and an exponential fit (black line) was used to calculate the average dwell time of 16.8 min. (F) Observed dwell times of 549-SNAP-N-IQGAP1 molecules (n = 72 binding events) were plotted (dotted line), and an exponential fit (black line) was used to calculate the average dwell time of 2.8 min. (G) Observed dwell times of GST-549-SNAP-N-IQGAP1 molecules (n = 203 binding events) were plotted (dotted line), and an exponential fit (black line) was used to calculate the average dwell time of 6.8 min.

FIGURE 3:

Each half of IQGAP1 partially suppresses actin filament growth. (A) Representative images from open-flow TIRF microscopy assays 10 min after initiation of actin assembly. Reactions contain 1 µM G-actin (10% Oregon green–labeled, 0.5% biotin-labeled) and different concentrations of N-IQGAP1 or C-IQGAP1. Scale bar, 10 μm. (B) Barbed end growth rates for filaments in TIRF reactions as in A, comparing the effects of different concentrations of N-IQGAP1. Data pooled from three independent trials (number of filaments analyzed for each condition, left to right: 60, 40, 60, 60, 60, and 55). Mean and SD. Student’s t test was used to determine the statistical significance of differences between conditions (* p < 0.05). (C) Same as B, except for the testing variable concentrations of C-IQGAP1 (number of filaments analyzed for each condition, left to right: 60, 40, 60, 60, 60, 55, and 40). Gray shaded data show the combined effects of N-IQGAP1 and C-IQGAP1 (100 nM each) on barbed end elongation rate. (D) Comparison of concentration-dependent effects of full-length IQGAP1 (data from Figure 1C), N-IQGAP1 (data from B), and C-IQGAP1 (data from C) on barbed end growth rate. For each, the data were fitted to a single exponential decay curve. Error bars, SEM. Yellow dot highlights the combined effects of N-IQGAP1 and C-IQGAP1 (100 nM each). (E) Duration of pauses in barbed end growth for 50 nM N-IQGAP1 (red histogram, blue curve, n = 10 filaments and 139 pause events) and 50 nM C-IQGAP1 (green histogram, yellow curve, n = 10 filaments and 166 pause events) compared with control reactions (gray histogram, black curve, n = 10 filaments and 174 pause events). Fits were calculated from a single exponential equation.

FIGURE 4:

Dimerization of N-IQGAP1 promotes actin filament bundling. (A) Representative time-lapse images from open-flow TIRF microscopy reactions containing 2 μM F-actin (10% Oregon green–labeled) and 2 nM 649-SNAP-IQGAP1. Scale bar, 10 μm. (B) Representative time-lapse images from TIRF microscopy reactions containing 2 μM F-actin (10% Oregon green–labeled) grown to 5–10 µm and then 10 nM IQGAP1, N-IQGAP1, or C-IQGAP1 was flowed into the reaction chamber. Scale bar, 10 μm. IQGAP1 (or control buffer) was flowed in 300 s after initiation of actin assembly, when filaments had grown to lengths of 5–10 μm. (C) Change in bundle thickness over time for reactions as in B, determined by measuring the fluorescence intensity along a bundle and calculating the fluorescence density per unit length. Student’s t test was used to determine the statistical significance of the increase in fluorescence observed after time zero (* p < 0.05). (D) Bundle thickness was also assessed by measuring fluorescence intensity at FWHM of line segments drawn perpendicular to the bundle. The fluorescence intensity values were normalized to control (2 μM F-actin). Student’s t test was used to determine the statistical significance of differences between conditions (* p < 0.05). (E) Comparing monomeric vs dimeric N-IQGAP1 fragments bundling actin filaments by measuring fluorescence intensity at FWHM of a line segment drawn perpendicular to the bundle. The fluorescence intensity values were normalized to control (2 μM F-actin). Student’s t test was used to determine the statistical significance of differences between conditions (* p < 0.05).

FIGURE 5:

The monomeric N-terminal half of IQGAP1 strongly suppresses depolymerization at barbed ends. (A) Representative time-lapse images and kymographs of fluorescently labeled actin filaments (10% Oregon green–labeled actin) in mf-TIRF reactions, comparing depolymerization from barbed ends in the presence of 10 nM IQGAP1, N-IQGAP1, C-IQGAP1, or control buffer. Filaments anchored at their pointed ends were polymerized, and then IQGAP1, N-IQGAP1, or C-IQGAP1 (without actin monomers) was flowed in at time zero and depolymerization was monitored over time. Scale bar, 5 μm. (B) Barbed end depolymerization rates measured in the presence of different concentrations of IQGAP1, N-IQGAP1, and C-IQGAP1. Data pooled from three independent trials (number of filaments analyzed for each condition, left to right: 160, 93, 75, 68, 160, 315, 74, 221, 160, 59, 249, 107, and 103). Mean and SD. Student’s t test was used to determine the statistical significance of differences between conditions (* p < 0.05). (C) Graphs showing fraction of free depolymerizing barbed ends vs. concentration of IQGAP1, N-IQGAP1, or C-IQGAP1, in which a hyperbolic binding curve was fitted to the data to determine the equilibrium binding constant (K_d). Error bars, SEM. Note that N-IQGAP1 is nearly as potent as full-length IQGAP1 in suppressing depolymerization, whereas C-IQGAP1 is ∼300-fold weaker. (D) mf-TIRF experiment (and representative kymograph) showing arrest of barbed end depolymerization where a 649-SNAP-IQGAP1 molecule is bound to the side of a filament. Left panel shows experimental scheme. Actin filaments with free barbed ends were first polymerized from coverslip-anchored spectrin-actin seeds in the presence of 1 μM G-actin (15% Alexa-488–labeled) and 5 μM profilin. Next, 0.4 nM 649-SNAP-IQGAP1 (without actin) was flowed in for 4 min to allow binding to filament sides, then buffer alone was flowed in, and depolymerization was monitored. PE, pointed end; BE, barbed end. The example kymograph shows depolymerization halting (white arrow) where the 649-SNAP-IQGAP1 molecule is bound to the filament side. Select time points from the merged kymograph (t = 0, 3, 4, 10 min) highlight barbed end depolymerization (cyan arrowhead) halting at 649-SNAP-IQGAP1 molecule (magenta arrowhead). Scale bar = 2 µm.

FIGURE 6:

Working model for IQGAP1 regulatory activities on actin filament dynamics and spatial organization. Top panel shows domain layout of full-length IQGAP1. Bottom panel shows working model for how IQGAP1 dimers directly control actin filament growth, bundling, and stabilization, with each activity highlighted in red. The N-terminal half of IQGAP1 binds tightly to filament sides using its CH domain and plays a central role in stabilizing filaments. Dimerization of the N-terminal half is mediated by the W-IQ region of IQGAP1, which is required for bundling but not stabilization. C-terminal domains in IQGAP1 transiently cap the barbed end, attenuating filament elongation. Importantly, the C-terminal domains work in close coordination with the N-terminal side-binding half of IQGAP1 to achieve full inhibition of elongation. The C-terminal (CT) domain of IQGAP1 binds to the formin Dia1, as well as CLIP-170 and adenomatous polyposis coli (APC), which directly collaborate with Dia1 to promote actin assembly (Fukata et al., 2002; Watanabe et al., 2004; Brandt et al., 2007; Lewkowicz et al., 2008; Okada et al., 2010; Breitsprecher et al., 2012; Henty-Ridilla et al., 2016). Thus, IQGAP1 may have additional regulatory roles in controlling formin- and APC-mediated actin assembly, but it is not clear how these suggested roles of the C-terminal half are coordinated with its transient capping effects on the barbed end.