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Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution

Abstract

VISUALIZATION of single actin filaments by fluorescence microscopy1led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level2-5. The ability to manipulate actin filaments with a microneedle6,7 or an optical trap8 combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosins9. To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to previously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface10-12, our method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of many types of enzymes and biomolecules.

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References

  1. Yanagida, T., Nakase, M., Nishiyama, K. & Oosawa, F. Nature 307, 58–60 (1984).

    Article  CAS  ADS  Google Scholar 

  2. Kron, S. J. & Spudich, J. A. Proc. natn. Acad. Sci. U.S.A. 83, 6272–6276 (1986).

    Article  CAS  ADS  Google Scholar 

  3. Toyoshima, Y. Y. et al. Nature 328, 536–539 (1987).

    Article  CAS  ADS  Google Scholar 

  4. Harada, Y., Noguchi, A., Kishino, A. & Yanagida, T. Nature 326, 805–808 (1987).

    Article  CAS  ADS  Google Scholar 

  5. Kishino, A. & Yanagida, T. Nature 334, 74–76 (1988).

    Article  CAS  ADS  Google Scholar 

  6. Ishijima, A., Doi, T., Sakurada, K. & Yanagida, T. Nature 352, 301–306 (1991).

    Article  CAS  ADS  Google Scholar 

  7. Ishijima, A. et al. Biochem. biophys. Res. Commun. 199, 1057–1063 (1994).

    Article  CAS  Google Scholar 

  8. Finer, J. T., Simmons, R. M. & Spudich, J. A. Nature 368, 113–119 (1994).

    Article  CAS  ADS  Google Scholar 

  9. Sowerby, A. J., Seehra, C. K., Lee, M. & Bagshaw, C. R. J. molec. Biol. 234, 114–123 (1993).

    Article  CAS  Google Scholar 

  10. Betzig, E. & Chichester, R. J. Science 262, 1422–1425 (1993).

    Article  CAS  ADS  Google Scholar 

  11. Trautman, J. K., Macklin, J. J. & Betzig, E. Nature 369, 40–42 (1994).

    Article  CAS  ADS  Google Scholar 

  12. Ishikawa, M., Hirano, K., Hayakawa, T., Hosoi, S. & Brenner, S. Jap. J. appl. Phys. 33, 1571–1576 (1994).

    Article  CAS  ADS  Google Scholar 

  13. Harada, Y. & Yanagida, T. Cell Motil. Cytoskel. 10, 71–76 (1988).

    Article  CAS  Google Scholar 

  14. Southwick, P. L. et al. Cytometry 11, 418–430 (1990).

    Article  CAS  Google Scholar 

  15. Axelrod, D. Meth. Cell Biol. 30, 245–270 (1989).

    Article  CAS  Google Scholar 

  16. Yanagida, T., Arata, T. & Oosawa, F. Nature 316, 366–369 (1985).

    Article  CAS  ADS  Google Scholar 

  17. Huxley, H. E. J. biol. Chem. 265, 8347–8350 (1990).

    CAS  PubMed  Google Scholar 

  18. Yanagida, T., Harada, Y. & Ishijima, A. Trends biochem. Sci. 18, 319–324 (1993).

    Article  CAS  Google Scholar 

  19. Bagshaw, C. R. in Muscle Contraction 2nd edn (Chapman & Hall, London, 1993).

    Book  Google Scholar 

  20. Spudich, J. A. Nature 372, 515–518 (1994).

    Article  CAS  ADS  Google Scholar 

  21. Harada, Y., Sakurada, K., Aoki, T., Thomas, D. D. & Yanagida, T. J. molec. Biol. 216, 49–68 (1990).

    Article  CAS  Google Scholar 

  22. Lowey, S., Waller, G. S. & Trybus, M. K. Nature 365, 454–456 (1993).

    Article  CAS  ADS  Google Scholar 

  23. Lindberg, M. & Mosbach, K. Eur. J. Biochem. 53, 481–486 (1975).

    Article  CAS  Google Scholar 

  24. Kodama, T., Fukui, K. & Kometani, K. J. Biochem. (Tokyo) 99, 1465–1472 (1986).

    Article  CAS  Google Scholar 

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Funatsu, T., Harada, Y., Tokunaga, M. et al. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555–559 (1995). https://doi.org/10.1038/374555a0

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