A single myosin head moves along an actin filament with regular steps of 5.3 nanometres

Abstract

Actomyosin, a complex of actin filaments and myosin motor proteins, is responsible for force generation during muscle contraction. To resolve the individual mechanical events of force generation by actomyosin, we have developed a new instrument with which we can capture and directly manipulate individual myosin subfragment-1 molecules using a scanning probe. Single subfragment-1 molecules can be visualized by using a fluorescent label. The data that we obtain using this technique are consistent with myosin moving along an actin filament with single mechanical steps of approximately 5.3 nanometres; groups of two to five rapid steps in succession often produce displacements of 11 to 30 nanometres. This multiple stepping is produced by a single myosin head during just one biochemical cycle of ATP hydrolysis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Direct capture and manipulation of a single S1 molecule by a scanning probe.
Figure 2: Displacements caused by single S1 molecules captured on the tip of the scanning probe.
Figure 3: Steps in the rising phase of displacement records on an expanded timescale.
Figure 4: Statistical analysis of stepping motion.

References

  1. 1

    Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. Atomic stucture of the actin: DNase I complex. Nature 347, 37–44 (1990).

    ADS  CAS  Article  Google Scholar 

  2. 2

    Rayment, I. et al. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261, 50–58 (1993).

    ADS  CAS  Article  Google Scholar 

  3. 3

    Huxley, H. E. The mechanism of muscular contraction. Science 164, 1355–1366 (1969).

    ADS  Article  Google Scholar 

  4. 4

    Huxley, A. F. & Simmons, R. M. Proposed mechanism of force generation in striated muscle. Nature 233, 533–538 (1971).

    ADS  CAS  Article  Google Scholar 

  5. 5

    Spudich, J. A. How molecular motors work. Nature 372, 515–518 (1994).

    ADS  CAS  Article  Google Scholar 

  6. 6

    Rayment, I. et al. Structure of the actin-myosin complex and its implications for muscle contraction. Science 261, 58–65 (1993).

    ADS  CAS  Article  Google Scholar 

  7. 7

    Fisher, A. J. et al. X-ray structures of myosin motor domain of Dictyostelium discoideum complexed with MgADP·BeFxand MgADP·AlF4. Biochemistry 34, 8960–8972 (1995).

    CAS  Article  Google Scholar 

  8. 8

    Cooke, R. Actomyosin interaction in striated muscle. Physiol. Rev. 77, 671–697 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Howard, J. Molecular motors: structural adaptations to cellular functions. Nature 389, 561–567 (1997).

    ADS  CAS  Article  Google Scholar 

  10. 10

    Kishino, A. & Yanagida, T. Force measurements by micromanipulation of a single actin filament by glass needles. Nature 334, 74–76 (1988).

    ADS  CAS  Article  Google Scholar 

  11. 11

    Finer, J. T., Simmons, R. M. & Spudich, J. A. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368, 113–119 (1994).

    ADS  CAS  Article  Google Scholar 

  12. 12

    Ishijima, A., Doi, T., Sakurada, K. & Yanagida, T. Sub-piconewton force fluctuations of actomyosin in vitro. Nature 352, 301–306 (1991).

    ADS  CAS  Article  Google Scholar 

  13. 13

    Ishijima, A. et al. Single-molecule analysis of the actomyosin motor using nano-manipulation. Biochem. Biophys. Res. Commun. 199, 1057–1063 (1994).

    CAS  Article  Google Scholar 

  14. 14

    Miyata, H. et al. Stepwise motion of an actin filament over a small number of heavy meromyosin molecules is revealed in an in vitro motility assay. J. Biochem. (Tokyo) 115, 644–647 (1994).

    CAS  Article  Google Scholar 

  15. 15

    Molloy, J. E., Burns, J. E., Kendrick-Jones, J., Tregear, R. T. & White, D. C. S. Movement and force produced by a single myosin head. Nature 378, 209–212 (1995).

    ADS  CAS  Article  Google Scholar 

  16. 16

    Guilford, W. H. et al. Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap. Biophys. J. 72, 1006–1021 (1997).

    ADS  CAS  Article  Google Scholar 

  17. 17

    Mehta, A. D., Finer, J. T. & Spudich, J. A. Detection of single-molecule interaction using correlated thermal diffusion. Proc. Natl Acad. Sci. USA 94, 7927–7931 (1997).

    ADS  CAS  Article  Google Scholar 

  18. 18

    Ishijima, A. et al. Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces. Biophys. J. 70, 383–400 (1996).

    ADS  CAS  Article  Google Scholar 

  19. 19

    Ishijima, A. et al. Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92, 161–171 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Tanaka, H., Ishijima, A., Honda, M., Saito, K. & Yanagida, T. Orientation dependence of displacements by a single one-headed myosin relative to the actin filament. Biophys. J. 75, 1886–1894 (1998).

    CAS  Article  Google Scholar 

  21. 21

    Yanagida, T., Arata, T. & Oosawa, F. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366–369 (1985).

    ADS  CAS  Article  Google Scholar 

  22. 22

    Harada, Y., Sakurada, K., Aoki, T., Thomas, D. D. & Yanagida, T. Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay. J. Mol. Biol. 216, 49–68 (1990).

    CAS  Article  Google Scholar 

  23. 23

    Higuchi, H. & Goldman, Y. E. Sliding distance between actin and myosin filaments per ATP molecule hydrolysed in skinned muscle fibres. Nature 352, 352–354 (1991).

    ADS  CAS  Article  Google Scholar 

  24. 24

    Lombardi, V., Piazzesi, G. & Linari, M. Rapid regeneration of the actin-myosin power stroke in contracting muscle. Nature 355, 638–641 (1992).

    ADS  CAS  Article  Google Scholar 

  25. 25

    Kitano, M., Hamabe, T., Maeda, S. & Okabe, T. Growth of large tetrapod-like ZnO crystals. J. Crystal Growth 102, 965–973 (1990).

    ADS  CAS  Article  Google Scholar 

  26. 26

    Kado, H., Yokoyama, K. & Tohda, T. Atomic force microscopy using ZnO whisker tip. Rev. Sci. Instrum. 63, 3330–3332 (1992).

    ADS  CAS  Article  Google Scholar 

  27. 27

    Tokunaga, M., Kitamura, K., Saito, K., Iwane, A. H. & Yanagida, T. Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy. Biochem. Biophys. Res. Commun. 235, 47–53 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Funatsu, T., Harada, Y., Tokunaga, M., Saito, K. & Yanagida, T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555–559 (1995).

    ADS  CAS  Article  Google Scholar 

  29. 29

    Huxley, A. F. & Tideswell, S. Filament compliance and tension transients in muscle. J. Muscle Res. Cell Motil. 17, 507–511 (1996).

    CAS  Article  Google Scholar 

  30. 30

    Woledge, R. C., Curtin, N. A. & Homsher, R. Energetic Aspects of Muscle Contraction Ch. 3 (Academic, London, (1985)).

    Google Scholar 

  31. 31

    Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993).

    ADS  CAS  Article  Google Scholar 

  32. 32

    Harada, Y., Noguchi, A., Kishino, A. & Yanagida, T. Sliding movement of single actin filaments on one-headed myosin filaments. Nature 326, 805–808 (1987).

    ADS  CAS  Article  Google Scholar 

  33. 33

    Iwane, A. H., Kitamura, K., Yokunaga, M. & Yanagida, T. Myosin subfragment-1 is fully equipped with factors essential for motor function. Biochem. Biophys. Res. Commun. 230, 76–80 (1997).

    CAS  Article  Google Scholar 

  34. 34

    Bagshaw, C. R. Muscle Contraction (Chapman & Hall, London, (1993)).

    Google Scholar 

  35. 35

    Kojima, H., Muto, E., Higuchi, H. & Yanagida, T. Mechanics of single kinesin molecules measured by optical trapping nanometry. Biophys. J. 73, 2012–2022 (1997).

    CAS  Article  Google Scholar 

  36. 36

    Schnitzer, M. J. & Block, S. M. Kinesin hydrolyses one ATP per 8-nm step. Nature 388, 386–390 (1997).

    ADS  CAS  Article  Google Scholar 

  37. 37

    Yasuda, R., Noji, H., Kinosita, K. J & Yoshida, M. F1-ATPase is a highly efficient molecular motor that rotates with discrete 120° steps. Cell 93, 1117–1124 (1998).

    CAS  Article  Google Scholar 

  38. 38

    Huxley, H. E. & Brown, W. The low-angle x-ray diagram of vertebrate striated muscle and its behavior during contraction and rigor. J. Mol. Biol. 30, 383–434 (1967).

    CAS  Article  Google Scholar 

  39. 39

    Astumian, R. D. Thermodynamics and kinetics of a brownian motor. Science 276, 917–922 (1997).

    CAS  Article  Google Scholar 

  40. 40

    Egelman, E. H., Francis, N. & DeRosier, D. J. F-actin is a helix with a random variable twist. Nature 298, 131–135 (1982).

    ADS  CAS  Article  Google Scholar 

  41. 41

    Yanagida, T., Nakase, M., Nishiyama, K. & Oosawa, F. Direct observation of motion of single F-actin filaments in the presence of myosin. Nature 307, 58–60 (1984).

    ADS  CAS  Article  Google Scholar 

  42. 42

    Lymn, R. W. & Taylor, E. W. Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10, 4617–4624 (1971).

    CAS  Article  Google Scholar 

  43. 43

    Klibanov, A. M. Enzyme memory. What is remembered and why? Nature 374, 596 (1995).

    ADS  CAS  Article  Google Scholar 

  44. 44

    Margossian, S. S. & Lowey, S. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 85, 55–71 (1982).

    CAS  Article  Google Scholar 

  45. 45

    Spudich, J. A. & Watt, S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. Biol. Chem. 246, 4866–4871 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Craig, S. W., Lancashire, C. L. & Cooper, J. A. Preparation of smooth muscle α-actinin. Methods Enzymol. 85, 316–321 (1982).

    CAS  Article  Google Scholar 

  47. 47

    Tokunaga, M., Aoki, T., Hiroshima, M., Kitamura, K. & Yanagida, T. Subpiconewton intermolecular force microscopy. Biochem. Biophys. Res. Commun. 231, 566–569 (1997).

    CAS  Article  Google Scholar 

  48. 48

    Saito, K., Tokunaga, M., Iwane, A. H. & Yanagida, T. Dual color microscopy of single fluorophores bound to myosin interacting with fluorescently-labelled actin using anti-Stokes fluorescence. J. Microsc. 188, 255–263 (1997).

    CAS  Article  Google Scholar 

  49. 49

    Bevington, P. R. & Robinson, D. K. Data Reduction and Error Analysis for the Physical Sciences (McGraw-Hill, New York, (1992)).

    Google Scholar 

  50. 50

    Wand, M. P. & Jones, M. C. Kernel Smoothing (Chapman & Hall, London, (1995)).

    Google Scholar 

Download references

Acknowledgements

We thank T. Funatsu, K. Saito, H. Higuchi, A. Ishijima and H. Kojima for technical suggestions; Y. Ishii and other colleagues of the ERATO project and Osaka University for valuable discussions; A. F. Huxley, Y. E. Goldman, F. Brozovich, C. R. Bagshaw, J. E. Molloy, A. D. Mehta, R. D. Vale and J. West for critically reading the manuscript; S. Kimura for instructions on preparing α-actinin; M. Taniguchi for advice on statistical analysis; and M. Kitano, H. Kado and H. Ogawa for the gift of the ZnO whiskers. This work was partially supported by JSPS Research Fellowships for Young Scientists (K.K.).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Toshio Yanagida.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kitamura, K., Tokunaga, M., Iwane, A. et al. A single myosin head moves along an actin filament with regular steps of 5.3 nanometres. Nature 397, 129–134 (1999). https://doi.org/10.1038/16403

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing