Opposite-polarity motors activate one another to trigger cargo transport in live cells

Myosin Va (myoV) and myosin VI (myoVI) are processive molecular motors that transport cargo in opposite directions on actin tracks. Because these motors may bind to the same cargo in vivo, we developed an in vitro "tug of war" to characterize the stepping dynamics of single quantum-dot-labeled myoV and myoVI motors linked to a common cargo. MyoV dominates its myoVI partner 79% of the time. Regardless of which motor wins, its stepping rate slows due to the resistive load of the losing motor (myoV, 2.1 pN; myoVI, 1.4 pN). Interestingly, the losing motor steps backward in synchrony with the winning motor. With ADP present, myoVI acts as an anchor to prevent myoV from stepping forward. This model system emphasizes the physical communication between opposing motors bound to a common cargo and highlights the potential for modulating this interaction by changes in the cell's ionic milieu.intracellular transport | motility assay | processivity | single molecule biophysics I ntracellular cargo transport relies on at least two classes of myosin molecular motors to navigate the actin cytoskeleton. Class V (myoV) and class VI (myoVI) myosins are double-headed motors that transport cargo in opposite directions along polarized actin filament tracks (1, 2). These motors convert the energy of ATP hydrolysis into force and motion and in doing so step processively on actin in a hand-over-hand fashion. With the plusends of actin filaments oriented toward the cell periphery, myoV transport contributes to exocytosis, whereas myoVI transport is critical to endocytosis (1, 2). Ensembles of molecular motors share the responsibility for transporting a single vesicle. For example, approximately 60 myoV motors coat a melanosome, although a smaller number is likely to be engaged with the track at any given time (3). Transport can also be bidirectional when oppositely directed motors are operative, as for axonal transport by the microtubule-based motors, kinesin and dynein (4, 5). With myoV and myoVI colocalizing to organelles (6, 7), these motors may engage in a virtual tug of war. If so, how do these oppositely directed myosin motors mechanically interact so that cargo is efficiently delivered to its destination?Individual motors within a transport ensemble may coordinate, cooperate, or mechanically impede one another, but determining by which mode they operate is complicated by the cargo geometry, the number and directionality of engaged motors, and the physical challenges presented by the dense actin cytoskeleton. Therefore, investigators have designed in vitro experiments to limit the number of coupled motors so that mechanistic modeling efforts were tractable (8-11). For these models, the velocity and direction of cargo transport generated by ensembles of antagonistic motors depended solely on the relative number of motors pulling in either direction (12, 13). The mechanical interactions between motors and the resultant stepping dynamics of each motor within the ensemble were beyond the predictive capacity of these models. Therefore, ...

Section: In-phasementioning

confidence: 55%

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

Safer³

et al. 2011

“…T he active intracellular transport of a large variety of cargos is fundamental for many cellular processes. In such transport, molecular motors bound to the cargo usually work together in teams (1)(2)(3). These teams can consist of one or several types of motor, with varying mechanical properties, including velocity, directionality, and processivity (2)(3)(4)(5).…”

mentioning

confidence: 99%

Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors

Furuta

Toyoshima

Amino

et al. 2012

216

223

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

“…Quantitative analyses of motor numbers in living cells have shown that a few motors of each type bind to the same organelle, where they coordinate and control each other's activity (3,6,7). The number of bound motors is remarkably similar in different systems, usually ranging from one to five motors of each type (7)(8)(9)(10), although in a few cases larger numbers were also reported (11,12).…”

mentioning

confidence: 96%

“…It is usually bidirectional, which is thought to help motors to bring their cargo to the right destination (3). Intracellular transport is mediated by motor proteins belonging to the kinesins and dyneins that bind to the organelles and hydrolyze ATP to move their cargo along the microtubules (MTs) (4).…”

mentioning

confidence: 99%

Transient binding of dynein controls bidirectional long-range motility of early endosomes

Schuster

Lipowsky

Assmann³

et al. 2011

141

258

In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3-based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the longrange motility behavior of the organelles. membrane trafficking | modeling