Dynein harnesses active fluctuations of microtubules for faster movement

Abstract

The cytoskeleton forms a dynamic network that generates fluctuations larger than thermal agitation of the cytoplasm1. Here, we tested whether dynein, a minus-end-directed microtubule (MT) motor2, can harness energy from these fluctuations using optical trapping in vitro. We show that dynein forms an asymmetric slip bond with MTs, where its detachment rate increases more slowly under hindering forces than assisting forces. This asymmetry enables dynein to generate unidirectional motility towards the minus-end from force fluctuations. Consistent with our model, oscillatory forces exerted by the trap drive dynein stepping without net force and ATP. Dynein is capable of ratcheting towards the minus-end, even when the net force is in the plus-end direction. With ATP, force oscillations increase the velocity and stall force of dynein as it transports cargos and glides MTs. Therefore, dynein is a mechanical ratchet that rectifies cytoskeletal fluctuations to move faster and resists higher forces along MTs.

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Fig. 1: Dynein forms an asymmetric slip bond with an MT.
Fig. 2: Dynein responds asymmetrically to assisting and hindering forces.
Fig. 3: Force oscillations drive minus-end-directed motility of dynein without ATP.
Fig. 4: Force oscillations increase cargo transport and MT gliding velocity of dynein in the presence of ATP.

Data availability

The data represented in Figs. 14 and Extended Data Figs. 2, 3, 4 and 6 are available as source data in the Supplementary Information. All other data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request. Yeast strains used in this study are available from the corresponding author upon request.

Code availability

Software and code used in this study are available from the corresponding author upon request.

References

  1. 1.

    Mizuno, D., Tardin, C., Schmidt, C. F. & Mackintosh, F. C. Nonequilibrium mechanics of active cytoskeletal networks. Science 315, 370–373 (2007).

    ADS  Article  Google Scholar 

  2. 2.

    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  Google Scholar 

  3. 3.

    Fakhri, N. et al. High-resolution mapping of intracellular fluctuations using carbon nanotubes. Science 344, 1031–1035 (2014).

    ADS  Article  Google Scholar 

  4. 4.

    Forth, S., Hsia, K. C., Shimamoto, Y. & Kapoor, T. M. Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks. Cell 157, 420–432 (2014).

    Article  Google Scholar 

  5. 5.

    Buckley, C. D. et al. Cell adhesion. The minimal cadherin–catenin complex binds to actin filaments under force. Science 346, 1254211 (2014).

    Article  Google Scholar 

  6. 6.

    Huang, D. L., Bax, N. A., Buckley, C. D., Weis, W. I. & Dunn, A. R. Vinculin forms a directionally asymmetric catch bond with F-actin. Science 357, 703–706 (2017).

    ADS  Article  Google Scholar 

  7. 7.

    Guo, B. & Guilford, W. H. Mechanics of actomyosin bonds in different nucleotide states are tuned to muscle contraction. Proc. Natl Acad. Sci. USA 103, 9844–9849 (2006).

    ADS  Article  Google Scholar 

  8. 8.

    Bormuth, V., Varga, V., Howard, J. & Schaffer, E. Protein friction limits diffusive and directed movements of kinesin motors on microtubules. Science 325, 870–873 (2009).

    ADS  Article  Google Scholar 

  9. 9.

    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  Google Scholar 

  10. 10.

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

    ADS  Article  Google Scholar 

  11. 11.

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

    ADS  Article  Google Scholar 

  12. 12.

    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).

    ADS  Article  Google Scholar 

  13. 13.

    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).

    ADS  Article  Google Scholar 

  14. 14.

    Qiu, W. et al. Dynein achieves processive motion using both stochastic and coordinated stepping. Nat. Struct. Mol. Biol 19, 193–200 (2012).

    Article  Google Scholar 

  15. 15.

    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  Google Scholar 

  16. 16.

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

    Article  Google Scholar 

  17. 17.

    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  Google Scholar 

  18. 18.

    Khataee, H. & Howard, J. Force generated by two kinesin motors depends on the load direction and intermolecular coupling. Phys. Rev. Lett. 122, 188101 (2019).

    ADS  Article  Google Scholar 

  19. 19.

    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).

    ADS  Article  Google Scholar 

  20. 20.

    Feynman, R. P., Leighton, R. B. & Sands, M. The Feynman Lectures on Physics, Vol. I: The New Millennium Edition: Mainly Mechanics, Radiation, and Heat Vol. 1 (Basic Books, 2011).

  21. 21.

    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  Google Scholar 

  22. 22.

    Gebhardt, J. C., Clemen, A. E., Jaud, J. & Rief, M. Myosin-V is a mechanical ratchet. Proc. Natl Acad. Sci. USA 103, 8680–8685 (2006).

    ADS  Article  Google Scholar 

  23. 23.

    Campas, O. & Sens, P. Chromosome oscillations in mitosis. Phys. Rev. Lett. 97, 128102 (2006).

    ADS  Article  Google Scholar 

  24. 24.

    Pecreaux, J. et al. Spindle oscillations during asymmetric cell division require a threshold number of active cortical force generators. Curr. Biol. 16, 2111–2122 (2006).

    Article  Google Scholar 

  25. 25.

    Yang, G. et al. Architectural dynamics of the meiotic spindle revealed by single-fluorophore imaging. Nat. Cell Biol. 9, 1233–1242 (2007).

    Article  Google Scholar 

  26. 26.

    Gaetz, J. & Kapoor, T. M. Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles. J. Cell Biol. 166, 465–471 (2004).

    Article  Google Scholar 

  27. 27.

    Shingyoji, C., Higuchi, H., Yoshimura, M., Katayama, E. & Yanagida, T. Dynein arms are oscillating force generators. Nature 393, 711–714 (1998).

    ADS  Article  Google Scholar 

  28. 28.

    Lin, J. & Nicastro, D. Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science 360, eaar1968 (2018).

    Article  Google Scholar 

  29. 29.

    Laan, L. et al. Cortical dynein controls microtubule dynamics to generate pulling forces that position microtubule asters. Cell 148, 502–514 (2012).

    Article  Google Scholar 

  30. 30.

    Sanchez, T., Welch, D., Nicastro, D. & Dogic, Z. Cilia-like beating of active microtubule bundles. Science 333, 456–459 (2011).

    ADS  Article  Google Scholar 

  31. 31.

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

    Article  Google Scholar 

  32. 32.

    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  Google Scholar 

  33. 33.

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

    ADS  Article  Google Scholar 

  34. 34.

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

    Article  Google Scholar 

  35. 35.

    Carter, A. P. et al. Structure and functional role of dynein’s microtubule-binding domain. Science 322, 1691–1695 (2008).

    ADS  Article  Google Scholar 

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Acknowledgements

We thank L. Ferro, M. ElShenawy and N. Hendel for their help in the initiation of this project, and the rest of the Yildiz Laboratory members for helpful discussions and technical assistance. This work was funded by grants from the NIH (GM094522) and NSF (MCB-1055017, MCB-1617028) to A.Y. and an NSF Graduate Research Fellowship (DGE 11064000) to V.B.

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Contributions

V.B. built the microscope and initiated the oscillation trap experiments. Y.E. and S.C. prepared the protein and antibody-coated beads. Y.E. performed single-molecule experiments and analysed the data. A.Y. and Y.E. wrote the manuscript. A.Y. supervised the project.

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Correspondence to Ahmet Yildiz.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Force-induced release rate analysis of dynein.

a. When pulled towards the minus-end, dynein exhibits a slip bond behavior with an MT, in which the release rate increases exponentially by assisting force (black dashed curve). In the hindering direction, possible force-detachment kinetics of slip, slip-ideal, ideal and catch bond behavior are shown for comparison (solid curves). b. Cumulative distributions of dynein dwell time on an MT under a given force were fit to a double exponential decay (red curves) to calculate fast (k1) and slow (k2) release rates from MT. n = 504 for 1 pN and 512 for −1 pN. c. k1 of dynein from MT (mean ± s.e.m.; n 270, 243, 479, 407, 385, 354, 487, 461, 380, 260, 208, 325, 137, 325 from left to right). This rate likely represents a weak interaction between dynein and tubulin. Similar to k2 (Fig. 1c), k1 increases with force in both directions. d. Comparison of the single and double exponential fit to example cumulative distributions of dynein dwell time. p values are calculated using F-test.

Extended Data Fig. 2 Measurement of force-detachment kinetics of dynein using a DNA-tethered optical trap.

a. A tail truncated dynein monomer (GFP-Dyn331kD-DHA) was labeled with a 74 bp DNA tether at its C-terminal DHA tag using the HaloTag chemistry. The DNA-labeled motor was attached to a bead via a biotin–streptavidin linkage. b. MT release rates of dynein monomers pulled from the C-terminal AAA ring via a DNA tether (blue, mean ± s.e.m.; from left to right n = 534, 661, 251, 973, 1074, 1742, 3216, 1423, 1003, 984, 792, 501, 310, 240 from three technical replicates). MT release rates of dynein monomers pulled from the N-terminal linker via the GFP-antibody (red, Fig. 1c) are shown for comparison. Source data

Extended Data Fig. 3 Force-induced stepping of dynein in the absence of ATP.

a. Example trajectory of a bead driven by single full-length dynein at 4.5 pN hindering force (blue) in the absence of ATP. Red horizontal lines represent a fit with a step finding algorithm (see Methods). b. Step size distribution of dynein under assisting and hindering forces in the absence of ATP (n = 214, 207, 204, 203, 266, 316, 308, 400, 211, 233, 247, 233, 202, 204, 238, 205, 226, 203, 208, 265 steps from hindering to assisting forces). c. (Top) The average step size of dynein under different forces. Positive steps are in the minus-end direction. Error bars represent s.e.m. The red dashed curve is an interpolation of the data to an exponential function. (Bottom) The ratio of steps taken in the plus-end direction to minus-end-direction. d. The ratio of the experimentally measured velocities (Fig. 2b) to the average step size (c) in the absence of ATP. The errors represent s.e.m. of velocity measurements (Fig. 2b). The blue curve represents a fit to equation 3. The errors of the derived parameters are s.e. of the fit. Source data

Extended Data Fig. 4 Force-induced stepping of dynein in 1 mM ATP.

a. Example trajectory of a bead driven by single full-length dynein at 5.6 pN hindering force (blue). Red horizontal lines represent a fit with a step finding algorithm. b. Step size distribution of dynein under assisting and hindering forces (n = 447, 428, 492, 430, 440, 460, 442, 291, 314, 381, 335, 298, 340 steps from hindering to assisting forces). c. (Top) The average step size of dynein under different forces. Error bars represent s.e.m. The red dashed curve is an interpolation of the data to an exponential function. (Bottom) The ratio of steps taken in the plus-end direction to minus-end-direction. d. The ratio of the experimentally measured velocities (Fig. 2c) to the average step sizes (c) in 1 mM ATP. The errors represent s.e.m. of velocity measurements (Fig. 2c). The blue curve represents a fit to equation 5. The errors of the derived parameters are s.e. of the fit. Source data

Extended Data Fig. 5 Example traces for nucleotide-free oscillations.

a. Example trajectory of a dynein-driven bead oscillated ± 4.9 pN in a square wave pattern without ATP. b. Example trajectory of a dynein-driven bead oscillated with + 6 pN and −1.5 pN in a square wave pattern at 2.5 Hz. Even though dynein was pulled more strongly backward, it moved towards the MT minus-end in the absence of ATP. c. The bead position was subtracted from the trap position to determine the force exerted on the bead during force oscillations. Due to the thermal relaxation of the bead and the rotational freedom of the bead–motor linkage, the bead–trap separation reaches near zero when the bead is moved between forward and backward positions. To determine how force affects velocity, this “dead” period t1 is omitted from total elapsed time and t2 is taken as half period of oscillations. (Left) To switch between assiting and hindering forces, the trap was first moved 250 nm and then with proportional feedback-controlled increments every 10 ms until the desired force was reached. (Right) The trap was first moved 500 nm and then with feedback-controlled increments every 10 ms until the desired force was reached. An increase in this “overshoot” distance decreased t1.

Extended Data Fig. 6 Force oscillations facilitate ratcheting of mammalian dynein–dynactin towards the minus-end of MTs in the absence of ATP.

a. The assembly of the mammalian dynein-dynactin-BicD2 (DDB) complex from human dynein, pig-brain dynactin and the N-terminal coiled-coil of mouse BicD2 (BicD2N, 1–400). BicD2N was fused with GFP at its C terminus for attachment of the complex to anti-GFP-coated beads. b. Example trajectories of DDB-driven beads pulled towards the MT plus- (left) and minus- (right) end under 1.5 pN force. The average velocities (mean ± s.e.m.) were calculated from 44 (left) and 27 (right) trajectories. c. Example trajectory of a DDB-driven bead oscillated ± 2.75 pN in a square wave pattern at 10 Hz. Similar to beads driven by S. cerevisiae dynein, DDB processively drives the bead towards the MT minus-end. d. The velocity of DDB-driven beads increases with ΔF in the absence of net force on the bead (mean ± s.e.m.; from left to the right n = 25, 32, 26 from three technical replicates). In comparison, DDB ratcheted faster towards the MT minus-end than S. cerevisiae dynein. Source data

Extended Data Fig. 7 Force fluctuations increase the power output of dynein towards the minus-end.

a, b. Estimated \(\bar F - V\) relationship under different ΔF in the absence (a) and presence (b) of 1 mM ATP. The model predicts that increasing ΔF leads to faster minus-end directed velocities at \(\bar F = 0\;pN\) and higher average hindering forces to stall the bead movement. The inset in (a) shows that the curves do not intersect at the same point. c, d. Force oscillations increase the power output of dynein in the absence (c) and presence (d) of 1 mM ATP. The power output of dynein at average hindering forces was calculated from the F-V relationship. e. Power output of dynein (black circles, mean ± s.e.m.; n = 42, 56, 58, 47 from left to right) in 1 mM ATP and in the absence of force fluctuations (ΔF = 0 pN). The blue curve corresponds to the 0 pN curve in d.

Supplementary information

Supplementary Information

Force-detachment kinetics of a dynein monomer and the force–velocity relationship of a dynein dimer.

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Ezber, Y., Belyy, V., Can, S. et al. Dynein harnesses active fluctuations of microtubules for faster movement. Nat. Phys. 16, 312–316 (2020). https://doi.org/10.1038/s41567-019-0757-4

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