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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data represented in Figs. 1–4 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.
Software and code used in this study are available from the corresponding author upon request.
Mizuno, D., Tardin, C., Schmidt, C. F. & Mackintosh, F. C. Nonequilibrium mechanics of active cytoskeletal networks. Science 315, 370–373 (2007).
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).
Fakhri, N. et al. High-resolution mapping of intracellular fluctuations using carbon nanotubes. Science 344, 1031–1035 (2014).
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).
Buckley, C. D. et al. Cell adhesion. The minimal cadherin–catenin complex binds to actin filaments under force. Science 346, 1254211 (2014).
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).
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).
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).
Gennerich, A., Carter, A. P., Reck-Peterson, S. L. & Vale, R. D. Force-induced bidirectional stepping of cytoplasmic dynein. Cell 131, 952–965 (2007).
Cleary, F. B. et al. Tension on the linker gates the ATP-dependent release of dynein from microtubules. Nat. Commun. 5, 4587 (2014).
Belyy, V., Hendel, N. L., Chien, A. & Yildiz, A. Cytoplasmic dynein transports cargos via load-sharing between the heads. Nat. Commun. 5, 5544 (2014).
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).
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).
Qiu, W. et al. Dynein achieves processive motion using both stochastic and coordinated stepping. Nat. Struct. Mol. Biol 19, 193–200 (2012).
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).
Reck-Peterson, S. L. et al. Single-molecule analysis of dynein processivity and stepping behavior. Cell 126, 335–348 (2006).
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).
Khataee, H. & Howard, J. Force generated by two kinesin motors depends on the load direction and intermolecular coupling. Phys. Rev. Lett. 122, 188101 (2019).
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).
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).
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).
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).
Campas, O. & Sens, P. Chromosome oscillations in mitosis. Phys. Rev. Lett. 97, 128102 (2006).
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).
Yang, G. et al. Architectural dynamics of the meiotic spindle revealed by single-fluorophore imaging. Nat. Cell Biol. 9, 1233–1242 (2007).
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).
Shingyoji, C., Higuchi, H., Yoshimura, M., Katayama, E. & Yanagida, T. Dynein arms are oscillating force generators. Nature 393, 711–714 (1998).
Lin, J. & Nicastro, D. Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science 360, eaar1968 (2018).
Laan, L. et al. Cortical dynein controls microtubule dynamics to generate pulling forces that position microtubule asters. Cell 148, 502–514 (2012).
Sanchez, T., Welch, D., Nicastro, D. & Dogic, Z. Cilia-like beating of active microtubule bundles. Science 333, 456–459 (2011).
Zhang, K. et al. Cryo-EM reveals how human cytoplasmic dynein is auto-inhibited and activated. Cell 169, 1303–1314 (2017).
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).
Urnavicius, L. et al. Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature 554, 202–206 (2018).
King, S. J. & Schroer, T. A. Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20–24 (2000).
Carter, A. P. et al. Structure and functional role of dynein’s microtubule-binding domain. Science 322, 1691–1695 (2008).
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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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.
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.
Source Data for Fig. 1
Source Data for Fig. 2
Source Data for Fig. 3
Source Data for Fig. 4
Source Data for Extended Data Fig. 2
Source Data for Extended Data Fig. 3
Source Data for Extended Data Fig. 4
Source Data for Extended Data Fig. 6
About this article
Cite this article
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
Nature Communications (2020)
Synthetic biology approaches to dissecting linear motor protein function: towards the design and synthesis of artificial autonomous protein walkers
Biophysical Reviews (2020)