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Processive bidirectional motion of dynein–dynactin complexes in vitro

Nature Cell Biology volume 8, pages 562–570 (2006)Cite this article

Abstract

Cytoplasmic dynein is the primary molecular motor responsible for transport of vesicles, organelles, proteins and RNA cargoes from the periphery of the cell towards the nucleus along the microtubule cytoskeleton of eukaryotic cells. Dynactin, a large multi-subunit activator of dynein, docks cargo to the motor and may enhance dynein processivity. Here, we show that individual fluorescently labelled dynein–dynactin complexes exhibit bidirectional and processive motility towards both the plus and minus ends of microtubules. The dependence of this activity on substrate ATP concentration, nucleotide analogues and inhibitors suggests that bidirectional motility is an active energy-transduction property of dynein–dynactin motor mechano-chemistry. The unique motility characteristics observed may reflect the flexibility of the dynein structure that leads to an enhanced ability to navigate around obstacles in the cell.

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Figure 1: Purification and characterization of dynein–dynactin–GFP from GFP–dynamitin mouse brains.
Figure 2: Example traces of motion exhibited by dynein–dynactin–GFP as visualized by TIRF.
Figure 3: Histograms of velocities of individual dynein–dynactin–GFP complexes exhibiting processive runs.
Figure 4: ATP dependence of average velocity and the effects of dynein inhibitors.
Figure 5: Possible models for the bidirectional motility observed for dynein–dynactin.

References

  1. Paschal, B. M. & Vallee, R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature 330, 181–183 (1987).

    Article  CAS  Google Scholar 

  2. Burgess, S. A., Walker, M. L., Sakakibara, H., Knight, P. J. & Oiwa, K. Dynein structure and power stroke. Nature 421, 715–718 (2003).

    Article  CAS  Google Scholar 

  3. Ogawa, K. Four ATP-binding sites in the midregion of the β heavy chain of dynein. Nature 352, 643–645 (1991).

    Article  CAS  Google Scholar 

  4. Gibbons, I. R. et al. Photosensitized cleavage of dynein heavy chains: Cleavage at the 'V1 site' by irradiation at 365 nm in the presence of ATP and vanadate. J. Biol. Chem. 262, 2780–2786 (1987).

    CAS  PubMed  Google Scholar 

  5. Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y. Y. & Sutoh, K. Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. Biochemistry 43, 11266–11274 (2004).

    Article  CAS  Google Scholar 

  6. Takahashi, Y., Edamatsu, M. Toyoshima, Y. Y. Multiple ATP-hydrolyzing sites that potentially function in cytoplasmic dynein. Proc. Natl Acad. Sci. USA 101, 12865–12869 (2004).

    Article  CAS  Google Scholar 

  7. Schroer, T. A. Dynactin. Annu. Rev. Cell Dev. Biol. 20, 759–779 (2004).

    Article  CAS  Google Scholar 

  8. Schroer, T. A. & Sheetz, M. P. Two activators of microtubule-based vesicle transport. J. Cell Biol. 115, 1309–1318 (1991).

    Article  CAS  Google Scholar 

  9. Waterman-Storer, C. M. et al. The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport. Proc. Natl Acad. Sci. USA 94, 12180–12185 (1997).

    Article  CAS  Google Scholar 

  10. Levy, J. R. et al. A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation. J. Cell Biol. 172, 733–745 (2006).

    Article  CAS  Google Scholar 

  11. Puls, I. et al. Mutant dynactin in motor neuron disease. Nature Genet. 33, 455–456 (2003).

    Article  CAS  Google Scholar 

  12. King, S. J. & Schroer, T. A. Dynactin increases the processivity of the cytoplasmic dynein motor. Nature Cell Biol. 2, 20–24 (2000).

    Article  CAS  Google Scholar 

  13. Wang, Z. & Sheetz, M. P. One-dimensional diffusion on microtubules of particles coated with cytoplasmic dynein and immunoglobulins. Cell Struct. Funct. 24, 373–383 (1999).

    Article  CAS  Google Scholar 

  14. Ishii, Y., Nishiyama, M. & Yanagida, T. Mechano-chemical coupling of molecular motors revealed by single molecule measurements. Curr. Protein Pept. Sci. 5, 81–87 (2004).

    Article  CAS  Google Scholar 

  15. Kon, T., Mogami, T., Ohkura, R., Nishiura, M. & Sutoh, K. ATP hydrolysis cycle-dependent tail motions in cytoplasmic dynein. Nature Struct. Mol. Biol. 12, 513–519 (2005).

    Article  CAS  Google Scholar 

  16. Mallik, R., Carter, B. C., Lex, S. A., King, S. J. & Gross, S. P. . Cytoplasmic dynein functions as a gear in response to load. Nature 427, 649–652 (2004).

    Article  CAS  Google Scholar 

  17. LaMonte, B. H. et al. Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration. Neuron 34, 715–727 (2002).

    Article  CAS  Google Scholar 

  18. Bingham, J. B., King, S. J. & Schroer, T. A. Purification of dynactin and dynein from brain tissue. Methods Enzymol. 298, 171–184 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Schafer, D. A., Gill, S. R., Cooper, J. A., Heuser, J. E. & Schroer, T. A. Ultrastructural analysis of the dynactin complex: An actin-related protein is a component of a filament that resembles F-actin. J. Cell Biol. 126, 403–412 (1994).

    Article  CAS  Google Scholar 

  21. Euteneuer, U., Koonce, M. P., Pfister, K. K. & Schliwa, M. An ATPase with properties expected for the organelle motor of the giant amoeba, Reticulomyxa. Nature 332, 176–178 (1988).

    Article  CAS  Google Scholar 

  22. Mallik, R., Petrov, D., Lex, S. A., King, S. J. & Gross, S. P. Building complexity: An in vitro study of cytoplasmic dynein with in vivo implications. Curr. Biol. 15, 2075–2085 (2005).

    Article  CAS  Google Scholar 

  23. Wang, Z., Khan, S. & Sheetz, M. P. Single cytoplasmic dynein molecule movements: Characterization and comparison with kinesin. Biophys. J. 69, 2011–2023 (1995).

    Article  CAS  Google Scholar 

  24. Wang, Z. & Sheetz, M. P. The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity. Biophys. J. 78, 1955–1964 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Shimizu, T., Toyoshima, Y. Y., Edamatsu, M. & Vale, R. D. Comparison of the motile and enzymatic properties of two microtubule minus-end-directed motors, ncd and cytoplasmic dynein. Biochemistry 34, 1575–1582 (1995).

    Article  CAS  Google Scholar 

  27. Hersch, G. L., Burton, R. E., Bolon, D. N., Baker, T. A. & Sauer, R. T. Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: Allosteric control of a protein machine. Cell 121, 1017–1027 (2005).

    Article  CAS  Google Scholar 

  28. Mancini, E. J. et al. Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell 118, 743–755 (2004).

    Article  CAS  Google Scholar 

  29. Holzbaur, E. L. & Johnson, K. A. Microtubules accelerate ADP release by dynein. Biochemistry 28, 7010–7016 (1989).

    Article  CAS  Google Scholar 

  30. Culver-Hanlon, T. L., Lex, S. A., Stephens, A. D., Quintyne, N. J. & King, S. J. A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules. Nature Cell Biol. 8, 264–270 (2006).

    Article  CAS  Google Scholar 

  31. Waterman-Storer, C. M., Karki, S. & Holzbaur, E. L. F. The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). Proc. Natl Acad. Sci. USA 92, 1634–1638 (1995).

    Article  CAS  Google Scholar 

  32. Deacon, S. W. et al. Dynactin is required for bidirectional organelle transport. J. Cell Biol. 160, 297–301 (2003).

    Article  CAS  Google Scholar 

  33. Lasek, R. J. & Brady, S. T. Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP–PNP. Nature 316, 645–647 (1985).

    Article  CAS  Google Scholar 

  34. Murray, J. W., Bananis, E. & Wolkoff, A. W. Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: An oscillatory bidirectional process. Mol. Biol. Cell 11, 419–433 (2000).

    Article  CAS  Google Scholar 

  35. Vale, R. D., Reese, T. S. & Sheetz, M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39–50 (1985).

    Article  CAS  Google Scholar 

  36. He, Y. et al. Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments. J. Cell Biol. 168, 697–703 (2005).

    Article  CAS  Google Scholar 

  37. Ling, S.-C., Fahrner, P. S., Greenough, W. T. & Gelfand, V. I. Transport of Drosophila fragile X mental retardation protein-containing ribonucleoprotein granules by kinesin-1 and cytoplasmic dynein. Proc. Natl Acad. Sci. USA 101, 17428–17433 (2004).

    Article  CAS  Google Scholar 

  38. Nan, X., Sims, P. A., Chen, P. & Xie, X. S. Observation of individual microtubule motor steps in living cells with endocytosed quantum dots. J. Phys. Chem. B 109, 24220–24224 (2005).

    Article  CAS  Google Scholar 

  39. Gross, S. P. et al. Interactions and regulation of molecular motors in Xenopus melanophores. J. Cell Biol. 156, 855–865 (2002).

    Article  CAS  Google Scholar 

  40. Karki, S. & Holzbaur, E. L. F. Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex. J. Biol. Chem. 270, 28806–28811 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by a National Institutes of Health (NIH) project program grant (P01–AR–051174) to the Pennsylvania Muscle Institute, by an ALS Association grant to E.L.F.H., and J.L.R. is supported by an NIH NRSA grant (1F32GM075754–01). The authors also thank A. Loh, R. Kudaravalli, H. Pham, W. Zhou and M. Tokito.

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  1. Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, 19104, PA, USA

    Jennifer L. Ross, Karen Wallace, Henry Shuman, Yale E. Goldman & Erika L.F. Holzbaur

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Correspondence to Erika L.F. Holzbaur.

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Ross, J., Wallace, K., Shuman, H. et al. Processive bidirectional motion of dynein–dynactin complexes in vitro. Nat Cell Biol 8, 562–570 (2006). https://doi.org/10.1038/ncb1421

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