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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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References

  1. Kashina, A., Rogers, G. & Scholey, J. M. The bimC family of kinesins: essential bipolar mitotic motors driving centrosome separation. Biochim. Biophys. Acta. 1357, 257–271 (1997).

    Article  CAS  Google Scholar 

  2. Sawin, K. E., LeGuellec, K., Philippe, M. & Mitchison, T. J. Mitotic spindle organization by a plus-end-directed microtubule motor. Nature 359, 540–543 (1992).

    Article  CAS  Google Scholar 

  3. Mayer, T. U. et al. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–974 (1999).

    Article  CAS  Google Scholar 

  4. Miyamoto, D. T., Perlman, Z. E., Burbank, K. S., Groen, A. C. & Mitchison, T. J. The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles. J. Cell Biol. 167, 813–818 (2004).

    Article  CAS  Google Scholar 

  5. Kapoor, T. M. & Mitchison, T. J. Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix. J. Cell Biol. 154, 1125–1133 (2001).

    Article  CAS  Google Scholar 

  6. Kwok, B. H., Yang, J. G. & Kapoor, T. M. The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5. Curr. Biol. 14, 1783–1788 (2004).

    Article  CAS  Google Scholar 

  7. Kapitein, L. C. et al. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 435, 114–118 (2005).

    Article  CAS  Google Scholar 

  8. Sharp, D. J. et al. The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles. J. Cell Biol. 144, 125–138 (1999).

    Article  CAS  Google Scholar 

  9. Block, S. M., Goldstein, L. S. & Schnapp, B. J. Bead movement by single kinesin molecules studied with optical tweezers. Nature 348, 348–352 (1990).

    Article  CAS  Google Scholar 

  10. Hackney, D. D. Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains. Nature 377, 448–450 (1995).

    Article  CAS  Google Scholar 

  11. Crevel, I. M., Lockhart, A. & Cross, R. A. Kinetic evidence for low chemical processivity in ncd and Eg5. J. Mol. Biol. 273, 160–170 (1997).

    Article  CAS  Google Scholar 

  12. Kamei, T., Kakuta, S. & Higuchi, H. Biased binding of single molecules and continuous movement of multiple molecules of truncated single-headed kinesin. Biophys. J. 88, 2068–2077 (2005).

    Article  CAS  Google Scholar 

  13. deCastro, M. J., Fondecave, R. M., Clarke, L. A., Schmidt, C. F. & Stewart, R. J. Working strokes by single molecules of the kinesin-related microtubule motor ncd. Nature Cell Biol. 2, 724–729 (2000).

    Article  CAS  Google Scholar 

  14. Turner, J. et al. Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker. J. Biol. Chem. 276, 25496–25502 (2001).

    Article  CAS  Google Scholar 

  15. Uyeda, T. Q., Kron, S. J. & Spudich, J. A. Myosin step size. Estimation from slow sliding movement of actin over low densities of heavy meromyosin. J. Mol. Biol. 214, 699–710 (1990).

    Article  CAS  Google Scholar 

  16. Vale, R. D. et al. Direct observation of single kinesin molecules moving along microtubules. Nature 380, 451–453 (1996).

    Article  CAS  Google Scholar 

  17. Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993).

    Article  CAS  Google Scholar 

  18. Svoboda, K. & Block, S. M. Force and velocity measured for single kinesin molecules. Cell 77, 773–784 (1994).

    Article  CAS  Google Scholar 

  19. Block, S. M., Asbury, C. L., Shaevitz, J. W. & Lang, M. J. Probing the kinesin reaction cycle with a 2D optical force clamp. Proc. Natl Acad. Sci. USA 100, 2351–2356 (2003).

    Article  CAS  Google Scholar 

  20. Schnitzer, M. J., Visscher, K. & Block, S. M. Force production by single kinesin motors. Nature Cell Biol. 2, 718–723 (2000).

    Article  CAS  Google Scholar 

  21. Endow, S. A. & Waligora, K. W. Determinants of kinesin motor polarity. Science 281, 1200–1202 (1998).

    Article  CAS  Google Scholar 

  22. Thorn, K. S., Ubersax, J. A. & Vale, R. D. Engineering the processive run length of the kinesin motor. J. Cell Biol. 151, 1093–1100 (2000).

    Article  CAS  Google Scholar 

  23. Sindelar, C. V. et al. Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy. Nature Struct. Mol. Biol. 9, 844–848 (2002).

    CAS  Google Scholar 

  24. Rice, S. et al. A structural change in the kinesin motor protein that drives motility. Nature 402, 778–784 (1999).

    Article  CAS  Google Scholar 

  25. Schnitzer, M. J. & Block, S. M. Kinesin hydrolyses one ATP per 8-nm step. Nature 388, 386–390 (1997).

    Article  CAS  Google Scholar 

  26. Schnitzer, M. J. & Block, S. M. Statistical kinetics of processive enzymes. Cold Spring Harb. Symp. Quant. Biol. 60, 793–802 (1995).

    Article  CAS  Google Scholar 

  27. Shaevitz, J. W., Block, S. M. & Schnitzer, M. J. Statistical kinetics of macromolecular dynamics. Biophys. J. 89, 2277–2285 (2005).

    Article  CAS  Google Scholar 

  28. Rosenfeld, S. S., Xing, J., Jefferson, G. M. & King, P. H. Docking and rolling — a model of how the mitotic motor Eg5 works. J. Biol. Chem. 280, 35684–35695 (2005).

    Article  CAS  Google Scholar 

  29. Crevel, I. M., Alonso, M. C. & Cross, R. A. Monastrol stabilises an attached low-friction mode of Eg5. Curr. Biol. 14, R411–R412 (2004).

    Article  CAS  Google Scholar 

  30. Block, S. M. Nanometres and piconewtons: the macromolecular mechanics of kinesin. Trends Cell Biol. 5, 169–175 (1995).

    Article  CAS  Google Scholar 

  31. Visscher, K., Schnitzer, M. J. & Block, S. M. Single kinesin molecules studied with a molecular force clamp. Nature 400, 184–189 (1999).

    Article  CAS  Google Scholar 

  32. Lang, M. J., Fordyce, P. M., Engh, A. M., Neuman, K. C. & Block, S. M. Simultaneous, coincident optical trapping and single-molecule fluorescence. Nature Methods 1, 133–139 (2004).

    Article  CAS  Google Scholar 

  33. Lang, M. J., Asbury, C. L., Shaevitz, J. W. & Block, S. M. An automated two-dimensional optical force clamp for single molecule studies. Biophys. J. 83, 491–501 (2002).

    Article  CAS  Google Scholar 

  34. Neuman, K. C. & Block, S. M. Optical trapping. Rev. Sci. Instrum. 75, 2787–2809 (2004).

    Article  CAS  Google Scholar 

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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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