Axial rotation of sliding actin filaments revealed by single-fluorophore imaging

Abstract

In the actomyosin motor, myosin slides along an actin filament that has a helical structure with a pitch of approximately 72 nm. Whether myosin precisely follows this helical track is an unanswered question bearing directly on the motor mechanism. Here, axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface was visualized through fluorescence polarization imaging of individual tetramethylrhodamine fluorophores sparsely bound to the filaments. The filaments underwent one revolution per sliding distance of approximately 1 microm, which is much greater than the 72 nm pitch. Thus, myosin does not "walk" on the helical array of actin protomers; rather it "runs," skipping many protomers. Possible mechanisms involving sequential interaction of myosin with successive actin protomers are ruled out at least for the preparation described here in which the actin filaments ran rather slowly compared with other in vitro systems. The result also indicates that each "kick" of myosin is primarily along the axis of the actin filament. The successful, real-time observation of the changes in the orientation of a single fluorophore opens the possibility of detecting a conformational change(s) of a single protein molecule at the moment it functions.

Figure 1

Measurement of axial rotation through fluorescence-polarization imaging. An actin filament was sparsely labeled with fluorophores with their transition moment (shown as a thick bar) at ≈45° from the filament axis. Fluorescence was excited with circularly polarized light. The vertically polarized component of the fluorescence was projected onto the upper half of the detector plane, and the horizontal component onto the lower half, through a dual-view apparatus (6). Filament rotation will result in an alternate appearance of each fluorophore between the V and H images.

Figure 2

Axial rotation detected in polarized fluorescence images. (A) Snapshots, at 33-ms intervals, of fluorescence from individual fluorophores on an actin filament sliding from bottom right toward top left. V, vertically polarized fluorescence; H, horizontally polarized fluorescence; numbers indicate time in seconds. Each image was spatially averaged over 3 × 3 pixels (0.66 × 0.66 μm²), and corrected for shading (due mainly to inhomogeneous excitation). (B) Time courses of the polarized spot intensities. The peak intensity of the trailing spot in A (starting at bottom right at 0 s) was measured for V and H, from which the total intensity, I = V + H (solid line), and polarization, p = (V − H)/(V + H) (○) were calculated. Because the signal-to-noise ratio was not high, the peak intensities, V and H, were not corrected for the background intensity, which varied to some extent from sample to sample and amounted to, on the average, one-third of the spot intensity; the total intensity beyond 3.1 s (also beyond 1.7 s in E) represents the background intensity. The inclusion of the background intensity resulted in smaller values of p, but our primary concern is its time dependence. Broken line indicates the displacement of the spot peak from the position at time 0. If the absorption and emission moments (assumed parallel) of the dye make an angle θ with the filament axis, and if the filament rotates at an angular velocity of ω around its axis, which lies at 45° with V and H axes, the total intensity and polarization for circularly polarized excitation are given by I = cos²θ + sin²θsin²ωt and p = sin2θsinωt/I (without correction for the large collection angle of the objective). Note that the theoretical I for a single fluorophore takes maximal values at times t when p passes either positive or negative peaks. The drop of the total intensity at ≈3.1 s resulted from photobleaching of the dye. (C) Trace of the trailing spot in A. (•), Spot positions where p > 0; (○), spot positions where p < 0. Arrow indicates the direction of sliding. The frame enclosing the trace represents the same area as each image in A. (D–F) Control experiment in the absence of ATP. The fluctuation of the spot intensity in this sample serves as a measure of the precision in A–C. The dye was photobleached at ≈1.7 s.

Figure 3

The longest rotation record observed. See Fig. 2 for symbols. Between 6.7 and 10.7 s (−9 and −15 μm in B), the microscope stage was moved manually to keep the moving spot in the field of view.

Figure 4

The relationship between axial rotation and forward translocation. The sliding speed (abscissa) was estimated from the slope of the displacement record (broken lines in Figs. 2B and 3A). The rotational rate (ordinate) was calculated as the number of revolutions in a record divided by the total duration determined by photobleaching. The size of the symbols is proportional to the duration of the record. The average of the durations is 5.6 s, which is approximately twice the average lifetime of all single fluorophores (long-lived spots were selected for the analysis of rotation). Vertical bars indicate the uncertainty in the revolution count. In estimating the revolution count, peaks with a small amplitude or short duration (0.1 s or less) were judged as noises and ignored. In Fig. 3A, for example, the revolution count during the 14-s period was estimated to be between 27 and 29.