Myosin VI is a processive motor with a large step size

Abstract

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.

Figure 1

Processivity assays. (A) Motility of GFP-tagged myosin VI on fascin-actin bundles as seen by total internal reflection fluorescence. Two separate runs are shown. Frame width, 1.5 μm. (B) Run-length distribution for single, fluorescently labeled myosin VI molecules. Exponential curves were fit to the data by using only runs that lasted longer than 1 s, all of which were ≥0.3 μm. n = 36 measurements, histogram bin widths are 0.14 μm. (C) Actin filament landing rates as a function of myosin VI density. First-power (reduced χ² = 2.45) and second-power (reduced χ² = 17.4) fits are shown. (D) Fraction of filaments that moved more than their length before dissociating, as a function of motor density. Fits describing single-molecule motility (reduced χ² = 2.2) and double molecule motility (reduced χ² = 8.9) are shown. Error bars are SE obtained from counting statistics.

Figure 2

Processive stepping by myosin VI observed by the dual-beam optical trap. (A) Dual-bead optical trap scheme. An actin filament is held between two 1-μm polystyrene beads. Bead positions are recorded with single nm resolution over 10 kHz. For fixed trap measurements (B and C) the optical load increases with displacement. (B) Sample trace of stepping behavior at 2 mM ATP with a fixed trap. Dwell periods at low load (<1.75 pN) fit single exponential statistics, with a mean stepping rate of 9.1 ± 0.6 s⁻¹ (n = 234). This rate is similar to the 8- to 9-s⁻¹ steady-state ATPase rate for single-headed constructs (9). (C) Oscillatory behavior observed at 2 mM ATP and high load (>2.5 pN).

Figure 3

Force feedback measurements. Constant separation between the bead center and the trap center was maintained by a feedback loop for the left-hand bead (see Fig. 2A). Forward steps indicate positive displacements of the left-hand bead out of its trap, and do not reflect the polarity of the actin filament. (A) Sample force-feedback trace of myosin VI stepping under 1.7 pN of load, and 2 mM ATP. Black trace, bead position; red trace, trap position. (B) Histogram of measured myosin VI step sizes. Forward steps were 30 ± 12 nm (n = 615), and backsteps were −13 ± 8 nm (n = 114). Both the |mean| and variances of the forward and backward steps differed significantly (t probability and F probability < 0.0001). Therefore the backsteps observed here arise from a different process than the oscillations observed at high load in Fig. 2 B and C (where backward and forward transitions are of equal magnitude). (C) Sample force-feedback trace of myosin V stepping under 1 pN of load and 2 mM ATP, using the same dual-bead trapping setup described for myosin VI (see text). (D) Step size histogram of myosin V. The narrow distribution of myosin V step sizes near 36 nm (35 ± 6 nm, n = 131) is in agreement with published results (15). This indicates that the broad distribution of myosin VI steps around 30 nm is not due to the geometry used, and the myosin VI step size is significantly less than 36 nm (t probability < 0.0001).

Figure 4

Model of myosin VI stepping. Myosin V and VI molecules are shown in gray, and the actin filament is shown in color. Knobs on the actin filament indicate stereospecific myosin-binding sites (although the actual binding site is located between two actin monomers, binding is shown on only one monomer for simplicity). Both motors are bound to actin subunits facing directly right. Myosin V (Lower), having six light chains, spans the 36-nm actin pseudorepeat to within ± 1 binding site (green and blue-green). Myosin VI (Upper), having only one light chain and the unique insert (square), cannot. Instead, the bound myosin VI head swings the unbound head to the left side of the actin filament as suggested by the transition from the ADP-bound state to the rigor state in electron microscopy structures (7). The actin subunits shown in red indicate the preferred binding sites of the unbound myosin VI head toward both the pointed end (≈27 nm) and the barbed end (≈11 nm) of actin, corresponding to the modal values seen in the optical trapping experiments. The spread of the color from red to blue reflects the step size histogram. Therefore, nearby actin subunits shown in purple are somewhat less accessible, but steps to these subunits can occur. Subunits shown in blue are relatively inaccessible because their myosin VI-binding sites are on either the right side or the underside of the actin filament. The measured step sizes of myosin VI toward both the pointed end (modal value, 27 nm) and the barbed end (modal value, −11 nm) of actin are a direct result of the accessibility of binding sites on the left side of the actin filament to the unbound myosin VI head.