We used an in vitro system to test whether switching of polymer bead cargos at microtubule-actin filament intersections can be regulated solely via the number of engaged myosin V and dynein motors through a tug-of-war mechanism. We measured the forces produced by the motors along their respective tracks with an optical trap and found for both myosin V and dynein that these forces scaled with motor number (). When the density of myosin V on the bead was high relative to dynein so that the ratio of maximum myosin V force along actin was greater than the maximum force of dynein along the MT, the beads most often continued along the AF or switched onto it after encountering a MT-AF intersection. Similarly, when the maximum dynein force was greater than the myosin V force, the beads primarily continued along the MT or switched onto it at an intersection. When the forces of the two motors were approximately equal, the beads stopped at the MT-AF intersection, left the intersection on their starting filament, or switched onto the other filament with about equal probability ( and ). These results are consistent with a straightforward model of track switching by beads in vitro in which the maximum force exerted by the motors determines the outcome at the intersection, a “tug of war” between the two types of motors ().
Figure 6 Cartoon of tug-of-war scenarios in which number of motors control cargo switching at MT-AF intersections. (A) At myosin V:dynein maximum force ratio of 0.5 (1 myosin V per 4 dyneins), the cargo mostly exits on the MT. (B) At a force ratio of 1 (1 myosin (more ...)
Pausing of beads at the intersections, rotation of beads about an axis normal to the glass surface, and bending of the filaments or deflection of the bead trajectory from a straight path indicate that beads containing dynein-dynactin and myosin V interact with both filaments simultaneously at the intersections. The likelihood of pausing, passing through the intersections, or switching to the crossing track is fairly independent of whether an AF or MT is the starting filament or which filament type is closer to the glass. This independence of the geometry at the intersection seemed surprising at first but is most easily explained by the simultaneous interaction with both filaments. When a bead enters the intersection from an underpass (the filament closer to the glass), the overlying crossing filament is an obstacle, so simultaneous interaction is obligatory. When the bead enters from the overpass (the filament farther from the glass), though, the dual interaction suggests that the bead wobbles enough as it translocates that it comes into contact with the crossing filament below. We speculate that once both motor types on a bead are engaged with both filaments, the initial conditions become irrelevant.
The optical trap force measurements led to estimates of the number of myosin V and dynein motors actively engaged with their respective filaments at the various loading concentrations. It had been shown previously that stall force scales with the number of actively engaged kinesin-1 and dynein motors [20
]. We found that this behavior is true for myosin V, as well (). Based on maximum force measurements (), we estimate that beads incubated with the lowest ratio of myosin V to dynein motors used had ~1 myosin V motor and ~4 dynein motors engaged at the time of maximum force production at the intersection. These beads always exited on the MT (). Beads incubated with the highest ratio of myosin V to dynein motors used had ~3-4 myosin V motors and ~2 dynein motors engaged at the intersections and exited on the AFs 90-100% of the time (). Thus, the probability of switching filaments can be tuned by pairing 1-4 myosin V motors and 1-4 dynein motors on a cargo.
These low motor numbers agree with estimates for the number of actively engaged motors driving cargos in vivo. In vivo
optical trapping of motile lipid droplets in Drosophila
embryos and evidence from cryo-electron microscopy (cryo-EM) indicate that the number of engaged MT motors may typically range between 1 and 5 [17
]. Endosomes purified from Dictyostelium
cells display forces consistent with the engagement of 1 kinesin and 4-8 dyneins [36
]. Photobleaching and quantitative western blotting of vesicles containing GFP-dynamitin purified from mouse brain combined with mathematical modeling of the in vitro
motility of these purified vesicles provide an estimate for the number of engaged motors in a similar range [A.G. Hendricks, J.L. Ross, E. Perlson, H.W. Schroeder III, M. Tokito, and E.L.F. Holzbaur, unpublished data]. Less information is available on the number of myosin V motors driving cargos, but indirect statistical estimates suggest that 1 – 2 myosin V's may be engaged in transporting Xenopus
The simple statistical model fitted to the data ( and S8
) is similar to that of Müller, et al. [40
]. In their work, they applied statistical distributions of engaged motors to simulate dynein and kinesin pulling a cargo in opposite directions along the MT axis. Due to stochastic changes in the number of engaged motors and the force dependence of the unbinding rate, the formerly predominant motors are forcibly detached and the direction of transport is reversed.
The situation in our experiments differs from the scenario modeled by Müller et al. because the two motor types, myosin V and dynein, simultaneously engage only near a filament intersection at which point they can exert sideways forces on one another that are perpendicular to their direction of travel. In vitro
studies show that myosin V and kinesin-1 are relatively resistant to sideways deflection. Forces that would stall the motor(s) from moving forward often failed to detach the motor(s) when applied in the sideways direction [22
]. We also observed that beads were more likely to detach from both MTs and AFs if we applied alternating forward and backward loads near the stall force than alternating left and right sideways loads. Nevertheless, the probabilities of switching onto the crossing filaments were closely related to the relative axial stall forces, indicating that sideways detachment forces scale with motor number as do the maximum axial forces.
Once the maximum numbers of motors available for engagement with the filaments (1-4 myosin Vs or dyneins) and the unitary stall force ratio (Fs myosin V
= 2) are set, the only parameters that are adjustable in fitting the model to the data are the engagement ratios, pmyosin V
, for the two motors. The engagement ratio is similar to the duty ratio of a single motor head, but here it is the proportion of time the double-headed molecule stays attached under load. For myosin V, the optical trap data allowed an estimate of pmyosin V
of ~0.7 from the ratio of average stall force during bead-actin interactions and the maximum force ( inset). For dynein, setting pdynein
to 0.85, provided the best fit to the data. Interestingly, the value of 0.85 for pdynein
is very close to the value of 0.86 calculated by dividing the dynein on rate constant by the sum of the on (1.6 s-1
) and off (0.27 s-1
) rate constants found by Müller et al. [40
] to fit the bidirectional switching of lipid droplets in Drosophila
What are the physical processes that lead to a bead pausing at the intersection and then suddenly exiting along one of the filaments? Beads pause at intersections, especially when the forces applied by the dynein and myosin V motors are nearly equal. During these pauses, the occasional rotation of the bead around an axis perpendicular to the plane of the slide suggests that a torque is applied by the concurrent action of the two motor types. As the bead rotates, the bound motors will be affected differentially by forward, backward, and sideways forces, based on their attachment points on the bead relative to the axis of rotation. These forces may affect the kinetics of motor attachment/detachment, in a tug-of-war that is expected to continue until there is a clear difference in the relative forces generated by engaged motors of each type. Pausing time at intersections correlates with balance of forces, and observed rotations > 360° indicate that this tug-of-war is sometimes prolonged. However, once the moment of selection is reached, the bead exits the intersection along the filament track of the dominant class of motor.
In the cell, the transfer of cargos from the microtubule to the actin network and back may occur via a coordinated mechanism in which the MT motors disengage from their track(s), and subsequently, the AF motors engage their track(s) and vice versa
. However, evidence from studies on mouse melanocytes, neurons, macrophages, and Xenopus
melanophores points toward a tug-of-war mechanism in which both motor classes, the MT motors and the AF motors, simultaneously engage and compete for their respective tracks [7
]. In both cases, regulation of cargo switching could occur via control of motor-cargo attachment, motor activity, and/or modification of the tracks [17
]. Our experimental data shows that alteration of the maximum number of engaged motors, Nmyosin V
, alone permits control of cargo switching at MT-AF intersections in vitro
. The data fit a straightforward model that provides a framework for understanding one possible mechanism for transfer of cargos between the MT and AF cytoskeletal networks, a crucial step in many cellular functions such as endocytosis and secretion.