Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles

Abstract

Myosin Va (myoVa) is a processive, actin-based molecular motor essential for intracellular cargo transport. When a cargo is transported by an ensemble of myoVa motors, each motor faces significant physical barriers and directional challenges created by the complex actin cytoskeleton, a network of actin filaments and actin bundles. The principles that govern the interaction of multiple motors attached to the same cargo are still poorly understood. To understand the mechanical interactions between multiple motors, we developed a simple in vitro model in which two individual myoVa motors labeled with different-colored Qdots are linked via a third Qdot that acts as a cargo. The velocity of this two-motor complex was reduced by 27% as compared to a single motor, whereas run length was increased by only 37%, much less than expected from multimotor transport models. Therefore, at low ATP, which allowed us to identify individual motor steps, we investigated the intermotor dynamics within the two-motor complex. The randomness of stepping leads to a buildup of tension in the linkage between motors-which in turn slows down the leading motor-and increases the frequency of backward steps and the detachment rate. We establish a direct relationship between the velocity reduction and the distribution of intermotor distances. The analysis of run lengths and dwell times for the two-motor complex, which has only one motor engaged with the actin track, reveals that half of the runs are terminated by almost simultaneous detachment of both motors. This finding challenges the assumptions of conventional multimotor models based on consecutive motor detachment. Similar, but even more drastic, results were observed with two-motor complexes on actin bundles, which showed a run length that was even shorter than that of a single motor.

Figure 1

(A) Illustration of a two-myoVa motor complex on an actin filament. (B) Sequential images of a two-motor complex labeled with two different-colored Qdots (red and green spots) moving along a TRITC-labeled (green) actin filament in 1 mM MgATP. Scale bar (yellow line), 500 nm. The elapsed time is shown in the top right corner of each image. (C) Two-dimensional movement path of a two-motor complex traveling along an actin filament in 2 μM MgATP. The spatial position of the heads (red and green dots; see Materials and Methods) is shown for each image frame. (D) Illustration of a two-motor complex on a fascin (yellow ovals) cross-linked actin bundle. Motors within a complex can travel either on the same or on different filaments in the bundle. (E) Traces as in (C) for a complex on an actin bundle showing both motors traveling on the same filament, followed by one motor (red) switching to another filament. (F) Traces similar to those in (E), but highlighting that a motor can tap between binding sites on different actin filaments, as shown previously (19). The lateral distance between the two motors within the complex can be as large as 100 nm (double-headed arrow). To see this figure in color, go online.

Figure 2

Velocity and run-length comparisons between single motors and two-motor complexes at saturating (1 mM) MgATP. (A) The velocity of a two-motor complex was significantly reduced for two-motor complexes on actin filaments (left) and on actin bundles (right) (^∗p < 0.05, statistically significant). (B) The run length of a two-motor complex on an actin filament was significantly longer than that of a single myoVa motor (^∗p < 0.05, statistically significant). In contrast, the run length of a two-motor complex was not significantly different than that of a single motor on an actin bundle (p > 0.05). To see this figure in color, go online.

Figure 3

(A) Movement trajectories of labeled heads on the leading (green) and trailing motors (red) on an actin filament at low ATP (2 μM MgATP). The intermotor distance fluctuates between nearly 0 nm (inset, blue rectangle) and 172 nm (blue double-headed arrow). (B) Distribution of intermotor distances during trajectories (black) and before a backward step taken by the leading motor (blue). Negative values indicate that the formerly trailing motor had passed the formerly leading motor. (Insets) Illustrations of a two-motor complex show the possible short and long intermotor distances based on the known structural dimensions of the motor components (see text for details). (C) Step-size distribution for a single motor (black) compared to that for the leading (green) and trailing (red) motors within a complex. Steps by motors in the complex show a much wider distribution. To see this figure in color, go online.

Figure 4

Stepping rates and intermotor time delays for two-motor complexes traveling at 2 μM MgATP on single actin filaments (A and B) or on actin bundles (C and D). (A) Stepping rates of the two motors in a complex as a function of intermotor distance. Rates associated with positive intermotor distances represent those of the leading motor and rates associated with negative intermotor distances reflect those of the trailing motor. Trends in the forward stepping rates (black circles) and backward stepping rates (red circles) are highlighted by lines fit by eye. Dashed lines show the average stepping rates of the leading/trailing motor. As a reference, the stepping rate of a single myoVa motor is shown (blue line, mean ± SE). (B) Time delays between steps of the leading and those of the trailing motor. t_T (squares) represents the average time between a forward step of the leading motor and the next forward step of the trailing motor as a function of the intermotor distance (Δx) measured immediately after the step of the leading motor (inset). t_L (triangles) shows the average delay between a step of the trailing motor and the next step of the leading motor. The difference between the two delays indicates a distance-dependent degree of coordination between the two motors. (C and D) Data are equivalent to those in (A) and (B), respectively, but apply to two-motor complexes running on actin bundles. To see this figure in color, go online.

Figure 5

Termination rate as a function of intermotor distance for the two-motor complex traveling on actin filaments (black) or bundles (red). Both termination rates increase sharply at intermotor distances >80 nm. See Fig. S6 for distributions of all data points and the number of terminations at a given intermotor distance, which were used to calculate the termination rates. To see this figure in color, go online.

Figure 6

(A) Simple kinetic model of a two-motor complex that is used to relate the run-length enhancement to the engagement state of the motors within the complex (see main text for details). We start our analysis in state 2, in which both motors are engaged to the actin filament. The transition from state 2 into state 1 (rate k₂₁) occurs when either the trailing or the leading motor detaches, leaving a single engaged motor. In state 1, the tethered, detached motor can reengage (rate k₁₂) to state 2, or the two-motor complex can totally detach (state 0), terminating the run. (B) The alternative kinetic scheme includes two pathways to run termination. In addition to the states and transitions shown in (A), a two-motor complex in state 2 can undergo a transition (rate k₂₀) into state 1^∗ in which one motor is engaged but rapidly detaches, leading to run termination in state 0. Approximately 50% of run terminations follow this pathway where state 1^∗ is a short-lived state.

Figure 7

Trajectory of a two-motor complex on a single actin filament at 2μM MgATP. Detachment and reengagement of the trailing motor can be seen. The run terminated with the detachment of the leading motor, followed by the trailing motor. The bar on top marks periods with one (magenta) or two (blue) engaged motors. To see this figure in color, go online.

Figure 8

(A) Dwell-time intervals with one or two engaged motors. All traces were truncated 6 s into the first two-engaged-motor interval. (B and C) Survival plots of initial (green) and intermediate (purple) two-engaged-motor intervals and intermediate single-motor intervals (blue). The dashed line shows an exponential fit $\left(\text{exp}\left(-t/\tau \right)\right)$ with the following parameters: two engaged motors, τ = 9.0 ± 1.0 s (mean ± SE; N = 84); single engaged motor, τ = 4.3 ± 0.7 s (mean ± SE; N = 39). (D) Survival plot (red) of single-motor intervals ending in run terminations. The distribution is fitted with the sum (black solid line) of a fast and a slow exponential $\left(R_S\text{exp}\left(-k_{1}t\right)+\left(1-R_{\text{s}}\right)\text{exp}\left(-k_{2}t\right)\right)$ using the maximum-likelihood method, resulting in R_s = 0.52 ± 0.1, k₁ = 1.6 ± 0.6 s⁻¹ and k₂ = 0.33 ± 0.1s⁻¹ (mean ± SE, N = 54). The dashed line shows the contribution of the slow component alone. To see this figure in color, go online.