Advertisement

Snapshots of Kinesin Motors on Microtubule Tracks

  • Franck J. Fourniol
  • Carolyn A. MooresEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 778)

Abstract

Kinesin motors couple ATP hydrolysis to movement along microtubules, which act both as tracks and as activators of kinesin ATPase activity. Cryo-electron microscopy and image processing enables generation of three-dimensional snapshots of kinesin motors on their tracks at different stages of their ATPase cycle, and can reveal their motor mechanisms at secondary structure resolution. Here, we describe in detail the methods and conditions employed in our lab to prepare high-quality frozen-hydrated samples, which yield structural insights into kinesin motor mechanisms.

Key words

Kinesin Microtubules Co-sedimentation assay Negative stain electron microscopy Cryo-electron microscopy 

Notes

Acknowledgments

The authors would like to thank past and present members of the Moores Lab (Andy Bodey, Carsten Peters, Christina Hoey, and Kanwal Zehra) for helpful discussions. We are grateful to Kanwal Zehra and Natasha Lukoyanova for providing us with constructive comments on the manuscript. We thank the Wellcome Trust, the Birth Defects Foundation, the Ecole Normale Supérieure, and the French Ministère de la Recherche for funding.

References

  1. 1.
    Hirokawa, N. (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519–526PubMedCrossRefGoogle Scholar
  2. 2.
    Wittmann, T., Hyman, A., and Desai, A. (2001) The spindle: A dynamic assembly of microtubules and motors. Nat. Cell Biol. 3, 28–34CrossRefGoogle Scholar
  3. 3.
    Vale, R.D., and Milligan, R.A. (2000) The way things move: looking under the hood of molecular motor proteins. Science 288, 88–95PubMedCrossRefGoogle Scholar
  4. 4.
    Cross, R.A. (2004) The kinetic mechanism of kinesin. TRENDS in Biochem. Sci. 29, 301–309CrossRefGoogle Scholar
  5. 5.
    Dubochet, J., Adrian, M., Chang, J.J., Homo, L.C., Lepault, J., McDowall, A.W., and Schultz, P. (1988) Cryo-electron microscopy of vitrified specimens. Q Rev. Biophys. 21, 129–228PubMedCrossRefGoogle Scholar
  6. 6.
    Kikkawa, M., and Hirokawa, N. (2006) High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations. EMBO J. 25, 4187–4197PubMedCrossRefGoogle Scholar
  7. 7.
    Hirose, K., Akimura, E., Akiba, T., Endow, S.A., and Amos, L.A. (2006) Large conformational changes in a kinesin motor catalyzed by interaction with microtubules. Mol. Cell 23, 913–923PubMedCrossRefGoogle Scholar
  8. 8.
    Sindelar, C.V., and Downing, K.H. (2007) The beginning of kinesin’s force-generating cycle visualized at 9-Å resolution. J. Cell Biol. 177, 377–385PubMedCrossRefGoogle Scholar
  9. 9.
    Bodey, A.J., Kikkawa, M., and Moores, C.A. (2009) 9  Å structure of a microtubule-bound mitotic motor. J. Mol. Biol. 388, 218–224PubMedCrossRefGoogle Scholar
  10. 10.
    Sindelar, C.V., and Downing, K.H. (2010) An atomic-level mechanism for activation of the kinesin molecular motors. PNAS 107, 4111–4116.PubMedCrossRefGoogle Scholar
  11. 11.
    Amos, L.A., and Hirose, K. (2007) Studying the structure of microtubules by electron microscopy. Methods Mol. Med. 137, 65–91PubMedCrossRefGoogle Scholar
  12. 12.
    Moores, C. (2008) Studying Microtubules by Electron Microscopy. Methods in Cell Biol. 88, 299–317CrossRefGoogle Scholar
  13. 13.
    Iancu, C.V., Tivol, W.F., Schooler, J.B., Dias, D.P., Henderson, G.P., Murphy, G.E., Wright, E.R., Li, Z., Yu, Z., Briegel, A., Gan, L., He, Y., and Jensen, G.J. (2006) Electron cryotomography sample preparation using the Vitrobot. Nat. Protoc. 1, 2813–2819PubMedCrossRefGoogle Scholar
  14. 14.
    Grassucci, R.A., Taylor, D.J., and Frank, J. (2007) Preparation of macromolecular complexes for cryo-electron microscopy. Nat. Protoc. 2, 32393246PubMedCrossRefGoogle Scholar
  15. 15.
    Grassucci, R.A., Taylor, D.J., and Frank, J. (2008) Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat. Protoc. 3, 330–339PubMedCrossRefGoogle Scholar
  16. 16.
    Metlagel, Z., Kikkawa, Y.S., and Kikkawa, M. (2007) Ruby-Helix: An implementation of helical image processing based on object-oriented scripting language. J. Struc. Biol. 157, 95–105CrossRefGoogle Scholar
  17. 17.
    Shaikh, T.R., Gao, H., Baxter, W.T., Asturias, F.J., Boisset, N., Leith, A., and Frank, J. (2008) SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat. Protoc. 3, 1941–1974PubMedCrossRefGoogle Scholar
  18. 18.
    Grigorieff, N. (2007) FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117125PubMedCrossRefGoogle Scholar
  19. 19.
    Ray, S., Meyhofer, E., Milligan, R.A., and Howard, J. (1993) Kinesin follows the microtubule’s protofilament axis. J. Cell Biol. 121, 1083–1093PubMedCrossRefGoogle Scholar
  20. 20.
    Li, H., DeRosier, D.J., Nicholson, W.V., Nogales, E., and Downing, K.H. (2002) Microtubule structure at 8  Å resolution. Structure 10, 1317–1328PubMedCrossRefGoogle Scholar
  21. 21.
    Chretien, D., Metoz, F., Verde, F., Karsenti, E., and Wade, R.H. (1992) Lattice defects in microtubules: Protofilament numbers vary within individual microtubules. J. Cell Biol. 117, 1031–1040PubMedCrossRefGoogle Scholar
  22. 22.
    Konishi, Y., and Setou, M. (2009) Tubulin tyrosination navigates the kinesin-1 motor domain to axons. Nat. Neurosci. 12, 559–567PubMedCrossRefGoogle Scholar
  23. 23.
    Tilney, L.G., Bryan, J., Bush, D.J., Fujiwara, K., Mooseker, M.S., Murphy, D.B., and Snyder, D.H. (1973) Microtubules: evidence for 13 protofilaments. J. Cell Biol. 59, 267–275PubMedCrossRefGoogle Scholar
  24. 24.
    McIntosh, J.R., Morphew, M.K., Grissom, P.M., Gilbert, S.P., and Hoenger, A. (2009) Lattice structure of cytoplasmic microtubules in a cultured Mammalian cell. J. Mol. Biol. 394, 177–182PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Institute of Structural Molecular BiologyBirkbeck CollegeLondonUK

Personalised recommendations