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

Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Springer Journals

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

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References (24)

Publisher
Springer Journals
Copyright
Copyright © 1995 by Nature Publishing Group
Subject
Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
ISSN
0028-0836
eISSN
1476-4687
DOI
10.1038/374555a0
Publisher site
See Article on Publisher Site

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.

Journal

Nature – Springer Journals

Published: Apr 6, 1995

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