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Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro

Nature Cell Biology volume 8, pages 470–476 (2006)Cite this article

Abstract

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5–513–5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5–513–5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.

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Figure 1: Schematic representation of the experimental geometry (not to scale).
Figure 2: Single Eg5–513–5His dimers are processive.
Figure 3: Eg5 velocity as a function of ATP concentration and force with global model fits.
Figure 4: Eg5 randomness as a function of ATP and force.

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Acknowledgements

This work was supported by grants to S.M.B. and S.P.G. from the National Institutes of Health (NIH). M.T.V. was supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. P.M.F. was supported by a predoctoral fellowship from the National Science Foundation (NSF) and a Lieberman fellowship. We thank W. Greenleaf, N. Guydosh and J. Burney for careful reading of the manuscript, and J. Shaevitz, C. Asbury and Block laboratory members for helpful discussions.

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Author notes

  1. Megan T. Valentine and Polly M. Fordyce: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Stanford University, Stanford, 94305, CA, USA

    Megan T. Valentine & Steven M. Block

  2. Department of Physics, Stanford University, Stanford, 94305, CA, USA

    Polly M. Fordyce

  3. Applied Physics, Stanford University, Stanford, 94305, CA, USA

    Steven M. Block

  4. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, 15260, PA, USA

    Troy C. Krzysiak & Susan P. Gilbert

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  1. Megan T. Valentine
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Correspondence to Steven M. Block.

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Valentine, M., Fordyce, P., Krzysiak, T. et al. Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro. Nat Cell Biol 8, 470–476 (2006). https://doi.org/10.1038/ncb1394

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