Abstract
Myosins are mechano-enzymes that convert the chemical energy of ATP hydrolysis into mechanical work. They are involved in diverse biological functions including muscle contraction, cell migration, cell division, hearing, and vision. All myosins have an N-terminal globular domain, or “head” that binds actin, hydrolyses ATP, and produces force and movement. The C-terminal “tail” region is highly divergent amongst myosin types, and this part of the molecule is responsible for determining the cellular role of each myosin. Many myosins bind to cell membranes. Their membrane-binding domains vary, specifying which lipid each myosin binds to. To directly observe the movement and localisation of individual myosins within the living cell, we have developed methods to visualise single fluorescently labelled molecules, track them in space and time, and gather a sufficient number of individual observations so that we can draw statistically valid conclusions about their biochemical and biophysical behaviour. Specifically, we can use this approach to determine the affinity of the myosin for different binding partners, and the nature of the movements that the myosins undergo, whether they cluster into larger molecular complexes and so forth. Here, we describe methods to visualise individual myosins as they move around inside live mammalian cells, using myosin-10 and myosin-6 as examples for this type of approach.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sellers, J. R. (2000) Myosins: a diverse superfamily. Biochim. Biophys. Acta 17, 3–22.
Oliver, T. S., Berg, J. S., and Cheney, R. E. (1999) Tails of unconventional myosins. Cellular and Molecular Live Sciences 56, 243–257.
Peckham, M and Knight, P.J. (2009) When a predicted coiled coil is really a single alpha helix, in myosins and other proteins. Soft Matter 5, 2493–2503.
Berg, J. S., and Cheney, R. E. (2002) Myosin-X is an unconventional myosin that undergoes intrafilapodial motility. Nat. Cell Biol. 4, 246–250.
Axelrod, D. (1992) Total Internal Reflection Fluorescence. Plenum Press, New York.
Mashanov, G. I., Tacon, D., Knight, A. E., Peckham, M., and Molloy, J. E. (2003) Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. Methods 29, 142–152.
Mashanov, G. I., Tacon, D., Peckham, M., and Molloy, J. E. (2004) The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts. J. Biol. Chem. 279, 15274–15280.
Yanagida, T., Ishii, Y. (2003) Stochastic processes in nano-biomachines revealed by single molecule detection BioSystems 71, 233–244
Mashanov, G. I., and Molloy, J. E. (2007) Automatic detection of single fluorophores in live cells. Biophys. J. 92, 2199–2211.
Hern, J. A., Baig, A. H., Mashanov, G. I., Birdsall, B., Corrie, J. E. T., et al. (2010) Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. PNAS 107, 2693–2698.
Friedman, L.J., Chung J., and Gelles, J. (2006) Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence. Biophys. J. 91, 1023–1031.
Kubitscheck, U., Kuckmann, O., Kues, T., and Peters, R. (2000) Imaging and tracking of single GFP molecules in solution. Biophys. J. 78, 2170–2179.
Thompson, R.E., Larson, D.R., and Webb, W. W. (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783.
Pierce, D. W., Hom-Booher N., Vale R. D. (1997) Imaging individual green fluorescent proteins. Nature 388, 338.
Sako, Y., Minoghchi, S., and Yanagida, T. (2000) Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168–172.
Mashanov, G. I., Nenasheva, T. A., and Molloy, J. E. (2006) Cell biochemistry studied by single-molecule imaging. Biochem. Soc. Trans. 34, 983–988.
Saxton, M. J. (1997) Single-Particle Tracking: The Distribution of Diffusion Coefficients. Biophys. J. 72, 1744–1753.
Douglass, A. D., and Vale, R. D. (2005) Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signalling molecules in T-cells. Cell 121, 937–950.
Belyantseva, I. A., Boger, E.T., Naz, S., Frolenkov, G. I., Sellers, J, R, Ahmed, Z,M, et al. (2005) Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia. Nat. Cell Biol. 7, 148–157.
Mashanov, G. I., Nobles, M., Harmer, S. C., Molloy, J. E., and Tinker, A. (2010) Direct observation of individual KCNQ1 potassium channels reveals their distinctive diffusive behavior. J. Biol. Chem. 285, 3664–3675.
Acknowledgments
The authors would like to thank Dr. Tom Cater and Laura Knipe (National Institute for Medical Research, London, UK) for cell culture support. T.A. Nenasheva was supported by Physiological Society (London) junior fellowship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Nenasheva, T.A., Mashanov, G.I., Peckham, M., Molloy, J.E. (2011). Imaging Individual Myosin Molecules Within Living Cells. In: Mashanov, G., Batters, C. (eds) Single Molecule Enzymology. Methods in Molecular Biology, vol 778. Humana Press. https://doi.org/10.1007/978-1-61779-261-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-61779-261-8_9
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-260-1
Online ISBN: 978-1-61779-261-8
eBook Packages: Springer Protocols