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. 2010 Sep 22;99(6):1818-26.
doi: 10.1016/j.bpj.2010.06.066.

The stepping pattern of myosin X is adapted for processive motility on bundled actin

Benjamin L Ricca  1 , Ronald S Rock
Affiliations

The stepping pattern of myosin X is adapted for processive motility on bundled actin

Benjamin L Ricca et al. Biophys J. .

Abstract

Myosin X is a molecular motor that is adapted to select bundled actin filaments over single actin filaments for processive motility. Its unique form of motility suggests that myosin X's stepping mechanism takes advantage of the arrangement of actin filaments and the additional target binding sites found within a bundle. Here we use fluorescence imaging with one-nanometer accuracy to show that myosin X takes steps of ∼18 nm along a fascin-actin bundle. This step-size is well short of the 36-nm step-size observed in myosin V and myosin VI that corresponds to the actin pseudohelical repeat distance. Myosin X is able to walk along bundles with this step-size if it straddles two actin filaments, but would be quickly forced to spiral into the constrained interior of the bundle if it were to use only a single actin filament. We also demonstrate that myosin X takes many sideways steps as it walks along a bundle, suggesting that it can switch actin filament pairs within the bundle as it walks. Sideways steps to the left or the right occur on bundles with equal frequency, suggesting a degree of lateral flexibility such that the motor's working stroke does not bias it to the left or to the right. On single actin filaments, we find a broad mixture of 10-20-nm steps, which again falls short of the 36-nm actin repeat. Moreover, the motor leans to the right as it walks along single filaments, which may require myosin X to adopt strained configurations. As a control, we also tracked myosin V stepping along actin filaments and fascin-actin bundles. We find that myosin V follows a narrower path on both structures, walking primarily along one surface of an actin filament and following a single filament within a bundle while occasionally switching to neighboring filaments. Together, these results delineate some of the structural features of the motor and the track that allow myosin X to recognize actin filament bundles.

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Figures

Figure 1
Figure 1
Myosin X takes sidesteps as it walks along a fascin-actin bundle. (a) Myosin X HMM labeled with a quantum dot (magenta) at its tail was imaged walking along surface-attached tetramethylrhodamine-phalloidin-labeled fascin-actin bundles (or single filaments). The actin image and the predominant direction of motion of the quantum dot defined the position (blue) and positive direction (magenta arrow) of the x axis, with the y axis perpendicular (red). Our coordinate system is defined for an observer looking down on the top of the actin, with the coverslip underneath. We verified the arrangement of our coordinate system by translating the microscope stage in known directions in the laboratory frame while tracking objects observed on the camera. (b) Example position trace of one motor walking along a bundle. The motor is moving predominantly from left to right while taking some side steps, up and down. We rotate all measured trajectories to show the motor moving from the origin along the +x direction. This coordinate rotation is derived from the actin filament image as described in the Methods. (c) Plot of the x position (open squares) and y position (open triangles) versus time of the motor shown in panel b (see Fig. S5 for more examples). The step-fitting algorithm (28) was used to pick the steps independently in x (blue trace) and in y (red trace). Each position is given a unique color (from black to magenta) in panels b and c. Some x steps occur concurrently with a y step (gray vertical lines, solid arrow) resulting in a single step oblique to both the x and y axes. Multiple, successive y steps with no corresponding x step are also observed (double-headed arrow). At ∼200 s and again at ∼275 s, the quantum dot blinked to low intensity, and these frames were filtered out as described in text (see Methods). (d) Myosin V walks straight along an actin filament. An example FIONA position-trace of myosin V, labeled at the tail as for myosin X, walking along a single actin filament is shown. Over these short lengths, we do not expect to see significant evidence of the slow spiraling with >1 μm pitch observed by Ali et al. (30). Note the lack of direct sidesteps. Myosin V walked with a step-size of 37.2 ± 0.9 nm (SE, n = 259).
Figure 2
Figure 2
Myosin X steps short of the 36-nm pseudohelical repeat of actin taking 17.5-nm steps along fascin-actin bundles. (a) Plot of runlength over time (gray circles) for the same example event as in Fig. 1b. The runlength trace is remarkably similar to the x trace in Fig. 1c, because the x direction is the primary contributor to runlength. Steps (black trace) were picked from runlength traces using the step-fitting algorithm (28). (b) Step-size histogram of myosin X on fascin-actin bundles (n = 239 steps). The histogram was fit to the sum of two Gaussians (black trace), centered at −12.8 ± 2.5 nm (σ = 4.4 ± 1.5 nm) and 17.5 ± 1.9 nm (σ = 11.1 ± 2.0 nm). (c) Step-size histogram of myosin X on single actin filaments (n = 327 steps; picked from plots of runlength over time traces. See Fig. S7). The histogram was fit to the sum of two Gaussians (black trace), centered at −16.0 ± 2.0 nm (σ = 6.1 ± 1.3 nm) and 16.4 ± 1.8 nm (σ = 12.6 ± 2.0 nm). Errors were obtained by curve fits to 200 bootstrap-sampled histograms with the same bin parameters.
Figure 3
Figure 3
Myosin X takes a spiraling path along single actin filaments. (Upper left, violet), example position trace of an event on a single actin filament. (Upper right, green) Example position trace of an event on a fascin-actin bundle. Each event is moving predominantly in the forward direction (from left to right, as plotted; see Fig. S7 for more examples). The position traces on single actin filaments show local trends of movement forward and to the right (upper left to lower right, as plotted). Local clusters of points within position traces on bundled actin are uncorrelated. Large axes, histograms of the local slope calculated from position traces of events on fascin-actin bundles (green, n = 6675 slopes) and on single actin filaments (violet, n = 6466 slopes). Each slope was calculated using a sliding window of 25 consecutive points in a position trace. Along fascin-actin bundles, the motor shows no asymmetry, with the distribution of slopes centered at 0 (gray dashed line). There is no statistically significant difference between the positive slopes and the absolute value of the negative slopes (p > 0.9 by Wilcoxon rank-sum test). On single actin filaments, the distribution of slopes is asymmetric ∼0. The difference between the positive slopes and the absolute value of the negative slopes is statistically significant (p < 10−34 by Wilcoxon rank-sum test). This shoulder of negative slopes in the distribution demonstrates that the motor is moving to the right locally as it moves forward over short distances, suggesting it turns with the actin helix over the course of a few small steps. The difference between the overall bundle and single filament local slope distributions is statistically significant (p < 10−20 by Wilcoxon rank-sum test). These conclusions do not change when long runs are omitted (>200 points) or the sliding window size is changed (10 or 50 points). The spread in the y position data (∼50 nm) is approximately twice the sum of the actin filament radius (4 nm), the radius of the quantum dot (∼10 nm), and the distance from the binding domain to the C-terminal end of the coiled-coil (∼10 nm or more), and this spread agrees with a recent measurement for the twirling radius of a similarly sized myosin constructs on single actin filaments (41).
Figure 4
Figure 4
Myosin X meanders symmetrically along fascin-actin bundles. (a) Scatter plot of the x and y components for all steps on fascin-actin bundles (n = 379). Steps occur either along the x axis (solid diamonds, n = 161), along the y axis (open circles, n = 135), or in one of the four Cartesian quadrants (open triangles, n = 83). Quadrants are noted with Roman numerals. (b) Histogram of y step-sizes with no corresponding x step-size (n = 135; 68 positive y step-sizes, 67 negative y step-sizes). (c) Histogram of x step-sizes with no corresponding y step-size (n = 161; 137 positive x step-sizes, 24 negative x step-sizes). (d) (Top) Histogram of x step-sizes of concurrent steps with a positive y step-size (n = 45; Quadrants I and II, n = 37 and n = 8, respectively). (Bottom) Histogram of x step-sizes of concurrent steps with a negative y step-size (n = 38; Quadrants III and IV, n = 7 and n = 31, respectively). (e) (Left) Histogram of y step-sizes of concurrent steps with a negative x step-size (n = 15; Quadrants II and III, n = 8 and n = 7, respectively). (Right) Histogram of y step-sizes of concurrent steps with a positive x step-size (n = 68; Quadrants I and IV, n = 37 and n = 31, respectively). Note that the motor takes steps to the left or right (up or down, as plotted) at nearly equal frequencies.
Figure 5
Figure 5
Myosin X steps in all directions from a common intermediate. On a fascin-actin bundle, the dwell time of myosin X before a step in any direction is part of the same single-exponential distribution. (Black solid trace) Survivor function of dwell times preceding steps in any direction (n = 363 dwells, 9.2 ± 0.5 s, mean ± SE). (Cyan solid trace) Positive x steps (along the x axis; n = 122 dwells, 8.5 ± 0.8 s). (Cyan dashed trace) Negative x steps (n = 22 dwells, 11.2 ± 2.6 s). (Gray solid trace) Positive y steps (along the y axis; n = 59 dwells, 8.1 ± 1.4 s). (Gray dashed trace) Negative y steps (n = 58 dwells, 10.6 ± 1.4 s). (Red trace) Quadrant I steps (n = 45 dwells, 7.2 ± 1.1 s). (Blue trace) Quadrant II steps (n = 9 dwells, 10.9 ± 3.9 s). (Magenta trace) Quadrant III steps (n = 8 dwells, 7.1 ± 2.1 s). (Green trace) Quadrant IV steps (n = 40 dwells, 9.7 ± 1.8 s). We detected no significant statistical differences among these nine distributions by a Wilcoxon rank-sum test at the α = 0.5 level.
Figure 6
Figure 6
Model of potential stepping pattern of myosin X on a fascin-actin bundle. Myosin X walks toward the barbed end of actin (away from the observer). (a) A possible configuration for myosin X to bind to actin in a two-headed fashion, with the rear head (blue) to the right of the motor's center, the forward head (red) to the left of the motor's center, and the quantum dot (gold) at the approximate center-of-mass. If the rear head detaches and lands on the bright blue actin subunit 36 nm away from the head's starting position, the motor takes a straight and forward step of 18 nm. However, the rear head can also reach a number of other binding sites indicated by, but not limited to, the light-blue actin subunits, resulting in oblique forward steps or sideways steps to the left. The forward head may also detach instead of the rear head. If it lands on the bright-red actin subunit, the motor takes a straight backward step, but it could land on one of the light-red actin subunits, resulting in an oblique backward step or a sideways step to the right. (b) After the motor steps from its starting position in a to the solid-blue subunit in a, it could adopt a binding configuration as shown in b. Here, the pattern of positions of reachable subunits is the mirror image of that shown in a because the rear head is now to the left of the motor's center.
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