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Working strokes by single molecules of the kinesin-related microtubule motor ncd

Abstract

The ncd protein is a dimeric, ATP-powered motor that belongs to the kinesin family of microtubule motor proteins. Here we resolve single mechanochemical cycles of recombinant, dimeric, full-length ncd, using optical-tweezers-based instrumentation and a three-bead, suspended-microtubule assay. Under conditions of limiting ATP, isolated and transient microtubule-binding events exhibit exponentially distributed and ATP-concentration-dependent lifetimes. These events do not involve consecutive steps along the microtubule, quantitatively confirming that ncd is non-processive. At low loads, a single motor molecule produces ATP-triggered working strokes of about 9 nm, which occur at the ends of binding events.

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Figure 1: Single-molecule binding events generated by full-length ncd in a three-bead assay.
Figure 2: Histograms of attachment durations.
Figure 3: Ensemble-averaged ncd-binding events.
Figure 4: Signal-bead positions for the ensemble-averaged 2 μM data set.
Figure 5: Possible mechanism of ncd movement.

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References

  1. Goldstein, L. S. & Philp, A. V. The road less traveled: emerging principles of kinesin motor utilization. Annu. Rev. Cell Dev. Biol. 15, 141–183 (1999).

    Article  CAS  Google Scholar 

  2. Howard, J., Hudspeth, A. J. & Vale, R. D. Movement of microtubules by single kinesin molecules . Nature 342, 154–158 (1989).

    Article  CAS  Google Scholar 

  3. Case, R. B., Pierce, D. W., Hom-Booher, N., Hart, C. L. & Vale, R. D. The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain. Cell 90, 959–966 ( 1997).

    Article  CAS  Google Scholar 

  4. Stewart, R. J., Semerjian, J. & Schmidt, C. F. Highly processive motility is not a general feature of the kinesins. Eur. Biophys. J. 27, 353 –360 (1998).

    Article  CAS  Google Scholar 

  5. Okada, Y. & Hirokawa, N. A processive single-headed motor: kinesin superfamily protein KIF1A. Science 283, 1152–1157 (1999).

    Article  CAS  Google Scholar 

  6. Scholey, J. M. Kinesin-II, a membrane traffic motor in axons, axonemes, and spindles. J. Cell Biol. 133, 1–4 (1996).

    Article  CAS  Google Scholar 

  7. Endow, S. A., Chandra, R., Komma, D. J., Yamamoto, A. H. & Salmon, E. D. Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis. J. Cell Sci. 107, 859– 867 (1994).

    CAS  PubMed  Google Scholar 

  8. Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W. & Scholey, J. M. Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos. Nature Cell Biol. 1, 51–54 (1999).

    Article  CAS  Google Scholar 

  9. Sablin, E. P., Kull, F. J., Cooke, R., Vale, R. D. & Fletterick, R. J. Crystal structure of the motor domain of the kinesin-related motor ncd [see comments]. Nature 380, 555 –559 (1996).

    Article  CAS  Google Scholar 

  10. Walker, R. A., Salmon, E. D. & Endow, S. A. The Drosophila claret segregation protein is a minus-end directed motor molecule. Nature 347, 780–782 (1990).

    Article  CAS  Google Scholar 

  11. McDonald, H. B., Stewart, R. J. & Goldstein, L. S. The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor. Cell 63, 1159–1165 (1990).

    Article  CAS  Google Scholar 

  12. deCastro, M. J., Ho, C. H. & Stewart, R. J. Motility of dimeric ncd on a metal-chelating surfactant: evidence that ncd is not processive. Biochemistry 38 , 5076–5081 (1999).

    Article  CAS  Google Scholar 

  13. Henningsen, U. & Schliwa, M. Reversal in the direction of movement of a molecular motor [see comments]. Nature 389, 93–96 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Foster, K. A. & Gilbert, S. P. Kinetic studies of dimeric Ncd: evidence that Ncd is not processive. Biochemistry 39 , 1784–1791 (2000).

    Article  CAS  Google Scholar 

  16. Pechatnikova, E. & Taylor, E. W. Kinetics processivity and the direction of motion of Ncd. Biophys. J. 77, 1003–1016 (1999).

    Article  CAS  Google Scholar 

  17. Sablin, E. P. et al. Direction determination in the minus-end-directed kinesin motor ncd. Nature 395, 813– 816 (1998).

    Article  CAS  Google Scholar 

  18. Arnal, I., Metoz, F., DeBonis, S. & Wade, R. H. Three-dimensional structure of functional motor proteins on microtubules. Curr. Biol. 6, 1265–1270 (1996).

    Article  CAS  Google Scholar 

  19. Hirose, K., Cross, R. A. & Amos, L. A. Nucleotide-dependent structural changes in dimeric NCD molecules complexed to microtubules. J. Mol. Biol. 278, 389–400 (1998).

    Article  CAS  Google Scholar 

  20. Hoenger, A. & Milligan, R. A. Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry . J. Mol. Biol. 265, 553– 564 (1997).

    Article  CAS  Google Scholar 

  21. Finer, J. T., Simmons, R. M. & Spudich, J. A. Single myosin molecule mechanics: piconewton forces and nanometre steps [see comments]. Nature 368, 113–119 (1994).

    Article  CAS  Google Scholar 

  22. Gittes, F. & Schmidt, C. F. Interference model for back-focal-plane displacement detection in optical tweezers. Optics Lett. 23, 7–9 (1998).

    Article  CAS  Google Scholar 

  23. Allersma, M. W., Gittes, F., deCastro, M. J., Stewart, R. J. & Schmidt, C. F. Two-dimensional tracking of ncd motility by back focal plane interferometry. Biophys. J. 74, 1074–1085 (1998).

    Article  CAS  Google Scholar 

  24. Veigel, C. et al. The motor protein myosin-I produces its working stroke in two steps [see comments]. Nature 398, 530 –533 (1999).

    Article  CAS  Google Scholar 

  25. Gittes, F. & Schmidt, C. F. Signals and noise in micromechanical measurements. Methods Cell Biol. 55, 129 –156 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Foster, K. A., Correia, J. J. & Gilbert, S. P. Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains. J. Biol. Chem. 273, 35307–35318 (1998).

    Article  CAS  Google Scholar 

  29. Huxley, A. F. Muscle structure and theories of contraction. Prog. Biophys. Biophys. Chem. 7, 255–318 (1957).

    Article  CAS  Google Scholar 

  30. Whittaker, M. et al. A 35-A movement of smooth muscle myosin on ADP release [see comments]. Nature 378, 748– 751 (1995).

    Article  CAS  Google Scholar 

  31. McDonald, H. B. & Goldstein, L. S. Identification and characterization of a gene encoding a kinesin-like protein in Drosophila. Cell 61, 991–1000 (1990).

    Article  CAS  Google Scholar 

  32. Karabay, A. & Walker, R. A. The Ncd tail domain promotes microtubule assembly and stability. Biochem. Biophys. Res. Commun. 258, 39–43 (1999).

    Article  CAS  Google Scholar 

  33. Buckheit, J. D. & Donoho, D. L. in Wavelets and Statistics (eds Oppenheim, G. & Antoniadis, A.) Ch. 5, 55–81 (Springer, New York, 1995).

    Book  Google Scholar 

  34. Veigel, C., Bartoo, M. L., White, D. C., Sparrow, J. C. & Molloy, J. E. The stiffness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers transducer. Biophys. J. 75, 1424–1438 (1998).

    Article  CAS  Google Scholar 

  35. Gittes, F., Mickey, M., Nettleton, J. & Howard, J. Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape. J. Cell Biol. 120, 923–934 (1993).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Shoham and D. Warren for discussion of data analysis and wavelet filtering; and M. Allersma, W. Moehler and F. Gittes for help with experiments and data analysis. We also thank the Rowland Institute for Science for generous technical support. This work was supported by a predoctoral fellowship from the Whitaker Foundation (to M.J.d.), and grants from the NSF (BIR9512699 and CTS-9624907) and the NIH (1R55GM55679-01).

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Correspondence to Christoph F. Schmidt or Russell J. Stewart.

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Correspondence should be addressed to R. J. S. or C. F. S. Requests for materials should be addressed to R. J. S.

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deCastro, M., Fondecave, R., Clarke, L. et al. Working strokes by single molecules of the kinesin-related microtubule motor ncd. Nat Cell Biol 2, 724–729 (2000). https://doi.org/10.1038/35036357

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