Myosin Va and Myosin VI Engage in a “Tug of War” on Actin Tracks while Transporting Cargoes in vitro

Abstract

Myosin Va (myoVa) and myosin VI (myoVI) are processive molecular motors that transport cargo in opposite directions on actin tracks. Since myoVa and myoVI may colocalize to the same cargo in vivo, these motors may undergo a tug of war. Therefore, we sought to characterize the stepping dynamics of single myoVa and myoVI motors in vitro as they mechanically interact when linked together by a Qdot cargo. Expressed myoVa-HMM with an N-terminal biotin tag were labeled with streptavidin-Qdots (565nm) while expressed dimerized myoVI-HMM were Qdot(655nm)-labeled on an exchanged calmodulin. The effective tug of war on actin filament tracks (25mM KCl, 2mM ATP, 22°C) was observed in TIRF with 6nm resolution, allowing individual steps to be detected. MyoVa won ∼80% of the time and regardless of which motor won, its stepping rate was reduced ∼50% below its unloaded value due to the resistive load of the opposing motor. Interestingly, as the winning motor stepped forwards (myoVa, 73nm; myoVI, 56nm) the opposing motor stepped backwards (myoVa, 68nm; myoVI, 65nm) at the same rate, although myoVI appeared to be dragged at times. Why does myoVa dominate when its stall force is similar to myoVI? Given the probability that both myoVa and myoVI take occasional backsteps and experience a 2-3-fold reduction in stepping rate when winning, we estimate based on optical trapping data (Altman et al., 2004; Kad et al., 2008) that myoVa exerts a 50% greater resistive load compared to myoVI, providing a potential advantage to myoVa. Differences in the length of the myoVa and myoVI constructs could lead to each motor experiencing different vectorial force components, the potential that this may influence the outcome of the tug of war is being investigated.