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Imaging Individual Myosin Molecules Within Living Cells

  • Tatiana A. Nenasheva
  • Gregory I. Mashanov
  • Michelle Peckham
  • Justin E. Molloy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 778)

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.

Key words

Total Internal Reflection Fluorescence Microscopy Single-molecule detection Single-particle tracking Myosin Cell motility 

Notes

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.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Tatiana A. Nenasheva
    • 1
  • Gregory I. Mashanov
    • 2
  • Michelle Peckham
    • 3
  • Justin E. Molloy
    • 1
  1. 1.MRC National Institute for Medical ResearchLondonUK
  2. 2.Division of Physical BiochemistryNational Institute for Medical ResearchLondonUK
  3. 3.School of Biomedical ScienceLeeds UniversityLeedsUK

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