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Single-molecule reconstitution of mRNA transport by a class V myosin

Nature Structural & Molecular Biology volume 20, pages 952–957 (2013)Cite this article

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Abstract

Molecular motors are instrumental in mRNA localization, which provides spatial and temporal control of protein expression and function. To obtain mechanistic insight into how a class V myosin transports mRNA, we performed single-molecule in vitro assays on messenger ribonucleoprotein (mRNP) complexes reconstituted from purified proteins and a localizing mRNA found in budding yeast. mRNA is required to form a stable, processive transport complex on actin—an elegant mechanism to ensure that only cargo-bound motors are motile. Increasing the number of localizing elements ('zip codes') on the mRNA, or configuring the track to resemble actin cables, enhanced run length and event frequency. In multi–zip-code mRNPs, motor separation distance varied during a run, thus showing the dynamic nature of the transport complex. Building the complexity of single-molecule in vitro assays is necessary to understand how these complexes function within cells.

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Figure 1: The frequency of mRNP processive runs increases with zip code number.
Figure 2: The Myo4p globular tail stabilizes the mRNP, and the recruitment of multiple motors to ASH1 is concentration dependent.
Figure 3: Metal-shadowed images show variable recruitment of motors.
Figure 4: Comparison of mRNP movement on single actin filaments versus bundles.
Figure 5: The spacing between two moving motor complexes coupled by mRNA varies.

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Acknowledgements

The authors thank M. Daniels for EM support and use of the National Heart, Lung, and Blood Institute Electron Microscopy Core Facility. We also thank G. Kennedy for technical assistance, D. Warshaw for use of the TIRF microscope, E. Krementsova for protein expression and M. Sckolnick, A. Hodges, H. Lu and S. Lowey for helpful discussions. This work was supported by funds from the US National Institutes of Health (GM078097 to K.M.T.).

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Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont, USA

    Thomas E Sladewski, Carol S Bookwalter & Kathleen M Trybus

  2. Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

    Myoung-Soon Hong

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  1. Thomas E Sladewski
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  2. Carol S Bookwalter
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  4. Kathleen M Trybus
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Contributions

T.E.S. and K.M.T. designed experiments. T.E.S. conducted experiments and analyzed the data. C.S.B. created RNA constructs and cloned and expressed the proteins. M.-S.H. performed rotary shadowing EM. T.E.S. and K.M.T. wrote the manuscript.

Corresponding author

Correspondence to Kathleen M Trybus.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–2, and Supplementary Videos 1–4 (PDF 471 kb)

Supplementary Video 1

No movement of mRNPs containing an ASH1 mRNA construct without zipcode elements on yeast actin–tropomyosin tracks. mRNA, green. Actin, red. Conditions: 140 mM KCl, 1mM MgATP. Movie time, 17s. Image width, 7 μm. (AVI 2031 kb)

Supplementary Video 2

Movement of mRNPs containing the native four zipcode ASH1 mRNA construct on yeast actin–tropomyosin tracks. mRNA, green. Actin, red. Conditions: 140 mM KCl, 1mM MgATP. Movie time, 17 s. Image width, 7μm. (AVI 2031 kb)

Supplementary Video 3

Movement of mRNPs containing an ASH1 mRNA construct with a single (E1) zipcode element on yeast actin–tropomyosin tracks. Run frequency is 1/3 that of native ASH1. mRNA, green. Actin, red. Conditions: 140 mM KCl, 1mM MgATP. Movie time, 17 s. Image width, 7 μm. (AVI 2039 kb)

Supplementary Video 4

Movement of mRNPs containing an ASH1 mRNA construct with eight zipcode elements on yeast actin–tropomyosin tracks. Run frequency is 2-fold higher than with the native ASH1. mRNA, green. Actin, red. Conditions: 140 mM KCl, 1mM MgATP. Movie time, 17s. Image width, 7 μm. (AVI 2031 kb)

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Sladewski, T., Bookwalter, C., Hong, MS. et al. Single-molecule reconstitution of mRNA transport by a class V myosin. Nat Struct Mol Biol 20, 952–957 (2013). https://doi.org/10.1038/nsmb.2614

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