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Engineering myosins for long-range transport on actin filaments

Abstract

Cytoskeletal motors act as cargo transporters in cells1 and may be harnessed for directed transport applications in molecular detection and diagnostic devices2. High processivity, the ability to take many steps along a track before dissociating3, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins4,5 are dimeric and use internal tension to coordinate the detachment cycles of the two heads6,7,8. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s−1.

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Figure 1: Multimerization effects on engineered myosin VI processivity and stepping behaviour.
Figure 2: Effects of increased flexibility on myosin VI constructs with short lever arms.
Figure 3: Characterization of engineered myosin XI motors.
Figure 4: Characterization of design targets for combining processivity with other desirable characteristics.

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Acknowledgements

The authors thank M.W. Elting and K. Aathavan for valuable insights and discussions, R. Cooke for providing full-length skeletal muscle myosin, S. Sutton for providing purified actin, and A.J. Lam for native PAGE advice and assistance. This work was supported by a Pew Scholars Award and a National Institutes of Health (NIH) grant (DP2 OD004690 to Z.B.). T.D.S. was supported by a Stanford Graduate Fellowship, a Ruth L. Kirschstein NRSA Graduate Training Program in Biotechnology (NIH grant 5T32GM008412 awarded to Stanford University) and a Siebel Scholars award. P.L. was supported by a Stanford Interdisciplinary Graduate Fellowship (SIGF) and the Natural Sciences and Engineering Research Council of Canada (award NSERC PGS-D3).

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Authors and Affiliations

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Contributions

T.D.S. and Z.B. designed the myosin constructs. T.D.S. and L.C. performed experiments on the bidirectional design target. T.D.S. and P.L. performed gold nanoparticle tracking experiments. T.D.S. performed all other experiments with assistance from M.N. and L.C., and analysed the experimental data. Z.B. conceived and supervised the project. T.D.S. and Z.B. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Zev Bryant.

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The authors declare no competing financial interests.

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Schindler, T., Chen, L., Lebel, P. et al. Engineering myosins for long-range transport on actin filaments. Nature Nanotech 9, 33–38 (2014). https://doi.org/10.1038/nnano.2013.229

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