Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Cargo adaptors regulate stepping and force generation of mammalian dynein–dynactin

Abstract

Cytoplasmic dynein is an ATP-driven motor that transports intracellular cargos along microtubules. Dynein adopts an inactive conformation when not attached to a cargo, and motility is activated when dynein assembles with dynactin and a cargo adaptor. It was unclear how active dynein–dynactin complexes step along microtubules and transport cargos under tension. Using single-molecule imaging, we showed that dynein–dynactin advances by taking 8 to 32-nm steps toward the microtubule minus end with frequent sideways and backward steps. Multiple dyneins collectively bear a large amount of tension because the backward stepping rate of dynein is insensitive to load. Recruitment of two dyneins to dynactin increases the force generation and the likelihood of winning against kinesin in a tug-of-war but does not directly affect velocity. Instead, velocity is determined by cargo adaptors and tail–tail interactions between two closely packed dyneins. Our results show that cargo adaptors modulate dynein motility and force generation for a wide range of cellular functions.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: DDR hydrolyzes ATP and moves faster than DDB.
Fig. 2: The stepping behavior of mammalian dynein–dynactin in unloaded conditions.
Fig. 3: Velocity and stepping of mammalian dynein–dynactin under load.
Fig. 4: Tail–tail interactions increase the velocity of dynein–dynactin.
Fig. 5: Dynein–dynactin multiplicity increases total force generation.
Fig. 6: Recruitment of a second dynein shifts the balance toward the MT minus end in a tug-of-war.

Data availability

All data that support the conclusions are available from the authors on request.

Code availability

Code used in this paper is available from the corresponding author upon request.

References

  1. Roberts, A. J., Kon, T., Knight, P. J., Sutoh, K. & Burgess, S. A. Functions and mechanics of dynein motor proteins. Nat. Rev. Mol. Cell Biol. 14, 713–726 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Reck-Peterson, S. L., Redwine, W. B., Vale, R. D. & Carter, A. P. The cytoplasmic dynein transport machinery and its many cargoes. Nat. Rev. Mol Cell Biol. 19, 382–398 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Carter, A. P., Cho, C., Jin, L. & Vale, R. D. Crystal structure of the dynein motor domain. Science 331, 1159–1165 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kon, T. et al. The 2.8 Å crystal structure of the dynein motor domain. Nature 484, 345–350 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Schmidt, H., Gleave, E. S. & Carter, A. P. Insights into dynein motor domain function from a 3.3-Å crystal structure. Nat. Struct. Mol. Biol. 19, 492–497 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhang, K. et al. Cryo-EM reveals how human cytoplasmic dynein is auto-inhibited and activated. Cell 169, 1303–1314 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Can, S., Lacey, S., Gur, M., Carter, A. P. & Yildiz, A. Directionality of dynein is controlled by the angle and length of its stalk. Nature 566, 407–410 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. 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  PubMed  Google Scholar 

  9. McKenney, R. J., Vershinin, M., Kunwar, A., Vallee, R. B. & Gross, S. P. LIS1 and NudE induce a persistent dynein force-producing state. Cell 141, 304–314 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rai, A. K., Rai, A., Ramaiya, A. J., Jha, R. & Mallik, R. Molecular adaptations allow dynein to generate large collective forces inside cells. Cell 152, 172–182 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Torisawa, T. et al. Autoinhibition and cooperative activation mechanisms of cytoplasmic dynein. Nat. Cell Biol. 16, 1118–1124 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Moughamian, A. J. & Holzbaur, E. L. Dynactin is required for transport initiation from the distal axon. Neuron 74, 331–343 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schlager, M. A. et al. Bicaudal D family adaptor proteins control the velocity of dynein-based movements. Cell Rep. 8, 1248–1256 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Trokter, M., Mucke, N. & Surrey, T. Reconstitution of the human cytoplasmic dynein complex. Proc. Natl Acad. Sci. USA 109, 20895–20900 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Toropova, K., Mladenov, M. & Roberts, A. J. Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements. Nat. Struct. Mol. Biol. 24, 461–468 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Urnavicius, L. et al. The structure of the dynactin complex and its interaction with dynein. Science 347, 1441–1446 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chowdhury, S., Ketcham, S. A., Schroer, T. A. & Lander, G. C. Structural organization of the dynein–dynactin complex bound to microtubules. Nat. Struct. Mol Biol. 22, 345–347 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Grotjahn, D. A. et al. Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility. Nat. Struct. Mol. Biol. 25, 203–207 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Urnavicius, L. et al. Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature 554, 202–206 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Splinter, D. et al. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures. Mol. Biol. Cell 23, 4226–4241 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. McKenney, R. J., Huynh, W., Tanenbaum, M. E., Bhabha, G. & Vale, R. D. Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes. Science 345, 337–341 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schlager, M. A., Hoang, H. T., Urnavicius, L., Bullock, S. L. & Carter, A. P. In vitro reconstitution of a highly processive recombinant human dynein complex. EMBO J. 33, 1855–1868 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Belyy, V. et al. The mammalian dynein-dynactin complex is a strong opponent to kinesin in a tug-of-war competition. Nat. Cell Biol. 18, 1018–1024 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ross, J. L., Wallace, K., Shuman, H., Goldman, Y. E. & Holzbaur, E. L. Processive bidirectional motion of dynein-dynactin complexes in vitro. Nat. Cell. Biol. 8, 562–570 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. DeWitt, M. A., Cypranowska, C. A., Cleary, F. B., Belyy, V. & Yildiz, A. The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release. Nat. Struct. Mol. Biol. 22, 73–80 (2015).

    Article  CAS  PubMed  Google Scholar 

  26. Toba, S., Watanabe, T. M., Yamaguchi-Okimoto, L., Toyoshima, Y. Y. & Higuchi, H. Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein. Proc. Natl Acad. Sci. USA 103, 5741–5745 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. DeWitt, M. A., Chang, A. Y., Combs, P. A. & Yildiz, A. Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains. Science 335, 221–225 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Duellberg, C. et al. Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein. Nat. Cell Biol. 16, 804–811 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  30. Elshenawy, M. M. et al. Lis1 activates dynein motility by pairing it with dynactin. Preprint at https://www.biorxiv.org/content/10.1101/685826v1 (2019).

  31. Reck-Peterson, S. L. et al. Single-molecule analysis of dynein processivity and stepping behavior. Cell 126, 335–348 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Can, S., Dewitt, M. A. & Yildiz, A. Bidirectional helical motility of cytoplasmic dynein around microtubules. eLife 3, e03205 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Mitra, A., Ruhnow, F., Nitzsche, B. & Diez, S. Impact-free measurement of microtubule rotations on kinesin and cytoplasmic-dynein coated surfaces. PLoS One 10, e0136920 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Nicholas, M. P. et al. Control of cytoplasmic dynein force production and processivity by its C-terminal domain. Nat. Commun. 6, 6206 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Gennerich, A., Carter, A. P., Reck-Peterson, S. L. & Vale, R. D. Force-induced bidirectional stepping of cytoplasmic dynein. Cell 131, 952–965 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Belyy, V., Hendel, N. L., Chien, A. & Yildiz, A. Cytoplasmic dynein transports cargos via load-sharing between the heads. Nat. Commun. 5, 5544 (2014).

    Article  CAS  PubMed  Google Scholar 

  37. Nishiyama, M., Higuchi, H. & Yanagida, T. Chemomechanical coupling of the forward and backward steps of single kinesin molecules. Nat. Cell Biol. 4, 790–797 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Lu, H. et al. Collective dynamics of elastically coupled myosin V motors. J. Biol. Chem. 287, 27753–27761 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Derr, N. D. et al. Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science 338, 662–665 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Furuta, K. et al. Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors. Proc. Natl Acad. Sci. USA 110, 501–506 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Driller-Colangelo, A. R., Chau, K. W., Morgan, J. M. & Derr, N. D. Cargo rigidity affects the sensitivity of dynein ensembles to individual motor pausing. Cytoskeleton 73, 693–702 (2016).

    Article  CAS  PubMed  Google Scholar 

  42. 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  PubMed  Google Scholar 

  43. Vershinin, M., Carter, B. C., Razafsky, D. S., King, S. J. & Gross, S. P. Multiple-motor based transport and its regulation by Tau. Proc. Natl Acad. Sci. USA 104, 87–92 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Jamison, D. K., Driver, J. W. & Diehl, M. R. Cooperative responses of multiple kinesins to variable and constant loads. J. Biol. Chem. 287, 3357–3365 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Soppina, V., Rai, A. K., Ramaiya, A. J., Barak, P. & Mallik, R. Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes. Proc. Natl Acad. Sci. USA 106, 19381–19386 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Shubeita, G. T. et al. Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets. Cell 135, 1098–1107 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ray, S., Meyhofer, E., Milligan, R. A. & Howard, J. Kinesin follows the microtubule’s protofilament axis. J. Cell Biol. 121, 1083–1093 (1993).

    Article  CAS  PubMed  Google Scholar 

  48. Nicholas, M. P. et al. Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains. Proc. Natl Acad. Sci. USA 112, 6371–6376 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Cleary, F. B. et al. Tension on the linker gates the ATP-dependent release of dynein from microtubules. Nat. Commun. 5, 4587 (2014).

    Article  CAS  PubMed  Google Scholar 

  50. Rai, A. et al. Dynein clusters into lipid microdomains on phagosomes to drive rapid transport toward lysosomes. Cell 164, 722–734 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Dogan, M. Y., Can, S., Cleary, F. B., Purde, V. & Yildiz, A. Kinesin’s front head is gated by the backward orientation of its neck linker. Cell Rep. 10, 1967–1973 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Gibbons, I. R. et al. The affinity of the dynein microtubule-binding domain is modulated by the conformation of its coiled-coil stalk. J. Biol. Chem. 280, 23960–23965 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to the members of the Yildiz laboratory for helpful discussions, A. P. Carter, L. Urnavicius, and S. Lacey (MRC, Cambridge) for providing plasmids and helping with protein expression and purification, and D. Drubin and C. Kaplan for multicolor TIRF microscopy. This work was funded by grants from the NIH (GM094522), and NSF (MCB-1055017, MCB-1617028) to A.Y., a grant from the NIH (GM098859) to S.C.B. and NIH F31 fellowship to L.F.

Author information

Authors and Affiliations

Authors

Contributions

M.M.E., J.C. and A.Y. conceived the study and designed the experiments. M.M.E., J.C. and L.F. prepared the constructs and isolated the proteins. Z.Z. and S.B. synthesized the fluorescent dyes. M.M.E. labeled the proteins with DNA and fluorescent dyes and performed the fluorescence motility experiments. J.C. performed bulk ATPase and MT bridge assays. J.C. and L.O. performed fluorescence tracking assays. M.M.E. performed optical trapping assays. M.M.E., J.C. and A.Y. wrote the manuscript. All authors read and commented on the manuscript.

Corresponding author

Correspondence to Ahmet Yildiz.

Ethics declarations

Competing interests

S.C.B. holds an equity interest in Lumidyne Technologies. All other authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Supplementary Figures 1–8

Reporting Summary

Supplementary Video 1

Processive motility of single DDB and DDR complexes along MTs in 1 mM ATP.

Supplementary Video 2

Helical movement of DDB- and DDR-driven beads along an MT bridge.

Supplementary Video 3

Linking of two DDBs via DNA hybridization.

Supplementary Video 4

Motility of DDB–kinesin and DDR–kinesin colocalizers.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Elshenawy, M.M., Canty, J.T., Oster, L. et al. Cargo adaptors regulate stepping and force generation of mammalian dynein–dynactin. Nat Chem Biol 15, 1093–1101 (2019). https://doi.org/10.1038/s41589-019-0352-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0352-0

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing