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Video imaging of walking myosin V by high-speed atomic force microscopy

Abstract

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative ‘visual evidence’ for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action.

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Figure 1: Directly visualized walking M5-HMM.
Figure 2: AFM images of M5-HMM in ADP and under the nucleotide-free condition.
Figure 3: Foot stomp and L-head conformations in different nucleotide conditions.

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Acknowledgements

We thank T. Uchihashi and H. Yamashita for technical assistance and discussion, and J. Sellers for critical reading of the draft and for comments. This work was supported by CREST/JST, Special Coordination Funds for Promoting Science and Technology (Effective Promotion of Joint Research with Industry, Academia, and Government) from JST, a Grant-in-Aid for Basic Research (S) from JSPS, and Knowledge Cluster/MEXT–Japan.

Author information

Authors and Affiliations

Authors

Contributions

N.K. performed the HS-AFM experiments and data analysis and wrote the first draft of the manuscript. D.Y. and N.K. developed the lipid-bilayer-based assay system. R.I. participated in the sample preparations in the early stage of this study. T.A. and N.K. developed the HS-AFM apparatus. T.A. designed the experiment and prepared the final manuscript.

Corresponding author

Correspondence to Toshio Ando.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1, Supplementary Figures 1-6 with legends, legends for Supplementary Movies 1-6 and additional references. (PDF 2052 kb)

Supplementary Movie 1

This file contains high-speed AFM movies showing unidirectional processive movement of M5-HMM - see Supplementary Information file for full legend. (MOV 1895 kb)

Supplementary Movie 2

This file contains high-speed AFM movies showing hand-over-hand movement of M5-HMM - see Supplementary Information file for full legend. (MOV 987 kb)

Supplementary Movie 3

This file contains high-speed AFM movie of two-head bound M5-HMM observed in 50 μM ADP - see Supplementary Information file for full legend. (MOV 1082 kb)

Supplementary Movie 4

This file contains high-speed AFM movies showing unwinding of short coiled-coil tail of M5-HMM - see Supplementary Information file for full legend. (MOV 1404 kb)

Supplementary Movie 5

This file contains High-speed AFM movies of two-head bound M5-HMM observed in the NF condition- see Supplementary Information file for full legend. (MOV 3665 kb)

Supplementary Movie 6

This file contains high-speed AFM movie showing transition between the straight and sharp bend conformations of the L-head - see Supplementary Information file for full legend. (MOV 1579 kb)

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Kodera, N., Yamamoto, D., Ishikawa, R. et al. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010). https://doi.org/10.1038/nature09450

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