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Direct observation of the mechanochemical coupling in myosin Va during processive movement

Abstract

Myosin Va transports intracellular cargoes along actin filaments in cells1. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis2. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded3,4,5,6,7,8,9,10. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated4,5,11. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.

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Figure 1: Imaging deac-aminoADP and Alexa-Fluor-568–MyoV-HMM.
Figure 2: Correlation between the movement of MyoV-HMM and the binding/dissociation of deac-aminonucleotide.
Figure 3: Histogram of lifetimes of deac-aminonucleotide association and dissociation.
Figure 4: A scheme of the tight coupling pathway of myosin Va.

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Acknowledgements

We thank F. Zhang, A. Smith and G. Reid for technical assistance; E. Yokoi for the TIRF illuminator to combine two optical cables; C. Fanghella for technical help in the calculation of the number of photons; and C. Weaver for help with Metamorph. We are appreciative of the critical comments on the manuscript made by P. J. Knight and E. Homsher. M.R.W. was supported by the Medical Research Council, UK. J.R.S. and T.S. were supported by the National Heart, Lung and Blood Intramural Program. H.D.W. and E.F. were supported by NIH EB00209 and a postdoctoral fellowship from the American Heart Association.

Author Contributions Single molecule motility experiments and data analysis were performed by T.S. and kinetic experiments data by E.F. Deac-aminoATP/ADP were provided by M.R.W. T.S., J.R.S. and H.D.W. participated in the conception of the experiment. T.S. wrote the first draft of the manuscript and all authors participated in producing the final version. All authors participated in discussion and interpretation of the data.

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Correspondence to James R. Sellers.

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Sakamoto, T., Webb, M., Forgacs, E. et al. Direct observation of the mechanochemical coupling in myosin Va during processive movement. Nature 455, 128–132 (2008). https://doi.org/10.1038/nature07188

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