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, 88 (2), 1387-402

Real-time Measurements of Actin Filament Polymerization by Total Internal Reflection Fluorescence Microscopy

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Real-time Measurements of Actin Filament Polymerization by Total Internal Reflection Fluorescence Microscopy

Jeffrey R Kuhn et al. Biophys J.

Abstract

Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 microM(-1) s(-1) and the dissociation rate constant is 0.89 s(-1). At the pointed end the association and dissociation rate constants are 0.56 microM(-1) s(-1) and 0.19 s(-1). When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s(-1) and from pointed ends at 0.16 s(-1) regardless of buffer nucleotide.

Figures

FIGURE 1
FIGURE 1
Diagram of the inverted microscope modified for total internal reflection fluorescence illumination. L, laser; FO, fiber optic; LS, laser shutter; BE, 5× beam expander; APS, active phase scrambler; GM, Gimbal-mounted mirror; M, mirror; FL, 100-mM plano-convex focusing lens; P, 10-mm right-angle prism; C, flow chamber; BB, beam block; AL, mercury arc lamp; AS, arc-lamp shutter; FT, automated filter cube turret; OL, 1.4-N.A., 60× objective lens; CCD, cooled charge-coupled device camera.
FIGURE 2
FIGURE 2
Imaging the time course of the polymerization of Mg-ATP-actin. Conditions: 1.5 μM (30% Oregon green 488 labeled) Mg-ATP-actin, 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 100 mM DTT, 0.2 mM ATP, 50 μM CaCl2, 15 mM glucose, 20 μg/ml catalase, 100 μg/ml glucose oxidase, 0.5% methylcellulose at 25°C. (A) Labeled actin was mixed with polymerization buffer, placed in a flow chamber previously coated with 0.2 μM NEM-inactivated myosin and blocked with 1% BSA. Growing barbed ends of four filaments are marked as ad. Numbers are seconds after adding polymerization buffer. Scale bar is 20 μm. (B) Linear kymographs of the growing filaments marked in panel A. Intensities were sampled at 1-pixel increments along each filament center (vertical axis) for several consecutive frames (horizontal axis) starting from the pointed end (bottom). Barbed-end growth occasionally stalled (a and c). Arrowheads indicate low speckle contrast. Length scale bar (L) is 10 μm and timescale bar (T) is 1000 s. (C) Lengths of 13 filaments as a function of time (solid lines) expressed in subunits, calculated using 370 subunits/μm (Huxley and Brown, 1967). Dashed lines are linear fits of each filament growth trace. Arrowhead indicates filament pause. The average slope was 3.9 ± 0.4 s−1. (D) Change in length of 13 filaments as a function of time. Individual growth traces were combined by subtracting each filament's starting length (based on the linear fit) from total length and start time from total time for each data point. Length measurements for stalled barbed ends were disregarded. A single rate of 3.974 ± 0.010 sub·s−1 (solid line) was assigned to the entire sequence (N = 925) by linear regression. (E) Instantaneous growth rates of filament shown in panel C. A Gaussian smoothing window was applied to each filament's length measurement and the change in smoothed length per change in time is shown. Rates near zero indicate filament growth stalls. Markings (a and c) correspond to stalling filaments shown in panel B. (F) Distribution of smoothed instantaneous growth rates for all length measurements with stalled sections removed. Rates were binned into 0.5 s−1 increments and plotted as a histogram of observation frequency for each binned rate (shaded bars). A Gaussian distribution (solid line) corresponding to the average measured instantaneous rate of 4.0 and ± SD of 1.5 s−1 (N = 925) is shown for comparison.
FIGURE 3
FIGURE 3
Drift correction reveals NEM-myosin attachment points useful as fiducial marks to separate barbed from pointed ends. Conditions: same buffer as Fig. 2. Actin filaments with segments of different fluorescence intensity were created by loading the chamber sequentially with polymerizing buffer containing 5 μM 25% labeled actin, followed by 5 μM 50% labeled actin, followed by 0.175 μM 25% labeled actin. (A) Image of a field at 10 s after addition of the final actin concentration, showing sequentially labeled filaments. Dim segments of the filaments are pointed ends, bright segments are barbed ends. Annealing created filaments with multiple bright segments. Bright spots of fluorescent material from the actin sample (arrows) remained stationary relative to each other. (B) The intensity of each image in the sequence (25 frames; 2 h total) was normalized and the sequence was combined into a single image with a maximum intensity projection. In this life-history image, both filaments and the bright spots moved as the stage drifted. (C) Maximum intensity projection. Several bright spots were tracked with subpixel accuracy and the average positions were used to estimate the drift at each time point. Each image was shifted in the reverse direction to compensate for the drift. Several filaments exhibited constriction points (arrows) around which the filaments rotated over time. Labeled fiducial marks (the boundaries of segments differing in intensity) did not move with respect to these constriction points over 2 h. Scale bar is 20 μm.
FIGURE 4
FIGURE 4
Residual analysis of length as a function of time data is used to separate affixed and sliding filaments. Conditions: 2 μM, 30% labeled Mg-ATP-actin was polymerized and the resulting sequence was corrected for drift and combined with a maximum intensity projection. Barbed and pointed ends were separated during length measurements by transection with a line drawn across a constriction site. Change in length as a function of change in time for (A and B) barbed ends and (C and D) pointed ends. Growth rates were obtained by robust linear regression. (E and F) Residuals of the barbed end (□, dashed line, measured length minus fitted length), negative residuals of the pointed end (•, solid line, fitted minus measured length), and residuals of total filament length (×, dotted line, measured minus fitted total length). (A, C, and E) An example of a filament fixed at its constriction point with no correlation between barbed and pointed end residuals. (B, D, and F) An example of a filament sliding over its constriction point with correlated barbed end and pointed end residuals.
FIGURE 5
FIGURE 5
Dependence of polymerization rates on actin concentration and fraction of labeling. Conditions: Mg-ATP-actin in same buffer as Fig. 2. (A and B) Dependence of elongation rates at (A) the barbed end and (B) pointed end on the concentration of 30% labeled actin (formula image). Eight to 15 filaments fixed at their constriction points were selected at each concentration. Barbed and pointed end lengths of each filament were measured over 10–60 frames, and rates were obtained by averaging smoothed instantaneous growth rates for every measurement. Three trials using two different actin preparations are shown. Rate constants (□, solid line) for trial 1 of formula image (•, dashed line) for trial 2 of formula image and (○, dashed-dotted line) for trial 3 of formula image were obtained by linear regression. Standard distributions of instantaneous rate measurements are shown for trial 3. Similar distributions were seen for trial 1 and trial 2. Trials 2 and 3 were from the same preparation on separate days. (C and D) Dependence of elongation rates at the (C) barbed end and (D) pointed end on the mol fraction of labeled actin. Mg-ATP-actin with 10–50% of the molecules labeled was polymerized at (○, dashed line) 1 μM and (•, solid line) 2.5 μM total actin and average smoothed instantaneous growth rates were fitted with linear regression.
FIGURE 6
FIGURE 6
Direct measurement of subunit dissociation. Conditions: 1.5 μM Mg-ATP-actin (25% fraction label) in the same buffer as Fig. 2 was polymerized on the slide for 10 min, followed by incubation with 0.18 μM (25% label) Mg-ATP-actin for 22 min to maintain filament length during phosphate release and followed by 1.5 μM (50% label) Mg-ATP-actin for 1–2 min to mark barbed ends. Filaments were depolymerized in either ATP or ADP buffer with 5 μM vitamin D binding protein to sequester free actin monomers. Barbed ends of marked filaments depolymerized faster than pointed ends. Unmarked filament ends were categorized as barbed or pointed based on their average depolymerization rate. Smoothed instantaneous growth rates (see Fig. 2) of marked and unmarked filaments were combined, binned into 0.5 s−1 increments, and plotted as a histogram (shaded bars) of observation frequency for the (A and C) barbed and (B and D) pointed end. Gaussian distributions (solid lines) of equivalent mean and standard deviation are shown for comparison. (A and B) Depolymerization of Mg-ADP-actin filament (A) barbed ends and (B) pointed ends in ATP buffer. Barbed ends depolymerized at 1.23 ± 2.88 s−1 (mean ± SD) and pointed ends at 0.06 ± 1.89 s−1. (C and D) Depolymerization of Mg-ADP-actin filament (C) barbed ends and (D) pointed ends in ADP buffer. Rates were 1.52 ± SD of 2.86 s−1 at the barbed end and 0.26 ± 2.20 s−1 at the pointed end.
FIGURE 7
FIGURE 7
Treadmilling of subunits through actin filaments at low concentrations of monomers. Conditions: Mg-ATP-actin in same buffer as Fig. 2. Labeled ends were created by growing filaments with sequential washes of 5 μM 50% labeled actin, followed by 5 μM, 35% labeled actin and observed in the presence of 0.15 μM, 25% labeled actin. (A) Life histories of four filaments are shown as a sequence of images, with time given in seconds in the horizontal direction. (MIP) The right panel shows a maximum intensity projection of the sequence. Dashed lines show the position of a stationary fiducial mark for each filament. Filaments are oriented with shrinking pointed ends (bright fluorescence) to the bottom and growing barbed ends to the top. Length scale bar (L) at left is 10 μm. (B) Each filament is presented as a “straightened” kymograph, where filament intensity was sampled at equally spaced intervals along its length (vertical direction) for each frame in the sequence (horizontal direction, time increasing toward bottom). Vertical dimensions are the same as the scale bar in panel A. Total width of kymograph represents 7500 s.
FIGURE 8
FIGURE 8
Dependence of elongation rates on actin concentration. (A and B) Behavior of (A) barbed ends and (B) pointed ends at low concentrations of Mg-ATP-actin. Conditions: 5 μM Mg-ATP-actin with 30% fraction label in the same polymerization buffer as Fig. 2. A single fraction label was used in each experiment. After polymerization of seeds, the chamber was washed with polymerization buffer containing from 0.025 to 0.2 μM total actin monomer with the same fraction labeled. The plots show rates as a function of the concentration of unlabeled actin for (○) all the low actin concentrations tested. Rate constants of formula image were obtained by linear regression. Mg-ATP-actin depolymerization rates (•) are shown for comparison. (C and D) Global summary of the polymerization rates at (C) barbed ends and (D) pointed ends as a function of unlabeled actin concentration for low (×) and high (○) actin concentrations. Rate constants of formula image were obtained by linear regression. The linear regression is the solid line and the upper and lower 95% confidence intervals are dashed lines.
FIGURE 9
FIGURE 9
Reactions for Mg-actin polymerization and nucleotide cycling. Schematic for Mg-ATP-, Mg-ADP-Pi-, Mg-ADP-, and nucleotide free actin binding to filament barbed and pointed ends at pH 7, including nucleotide hydrolysis, phosphate release, and nucleotide exchange. Nucleotide content of internal subunits is ignored. Numbers denote rate constants measured here (bold) or previously (1, Blanchoin and Pollard, 2002; 2, Carlier and Pantaloni, 1988; 3, Melki et al., 1996; 4, Wanger and Wegner, 1987; 5, Pollard, 1986; 6, Selden et al., 1999 ; 7, De La Cruz and Pollard, 1995). Kr,p indicate equilibrium constants for reactants r and products p; k+r,p, association rate; k−r,p, dissociation rate; ?, unknown rate constants; Ø, highly unfavorable reactions; *, calculated rate or equilibrium constants.

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