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.
Subscribe to Journal
Get full journal access for 1 year
only $15.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All data that support the conclusions are available from the authors on request.
Code used in this paper is available from the corresponding author upon request.
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).
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).
Carter, A. P., Cho, C., Jin, L. & Vale, R. D. Crystal structure of the dynein motor domain. Science 331, 1159–1165 (2011).
Kon, T. et al. The 2.8 Å crystal structure of the dynein motor domain. Nature 484, 345–350 (2012).
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).
Zhang, K. et al. Cryo-EM reveals how human cytoplasmic dynein is auto-inhibited and activated. Cell 169, 1303–1314 (2017).
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).
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).
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).
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).
Torisawa, T. et al. Autoinhibition and cooperative activation mechanisms of cytoplasmic dynein. Nat. Cell Biol. 16, 1118–1124 (2014).
Moughamian, A. J. & Holzbaur, E. L. Dynactin is required for transport initiation from the distal axon. Neuron 74, 331–343 (2012).
Schlager, M. A. et al. Bicaudal D family adaptor proteins control the velocity of dynein-based movements. Cell Rep. 8, 1248–1256 (2014).
Trokter, M., Mucke, N. & Surrey, T. Reconstitution of the human cytoplasmic dynein complex. Proc. Natl Acad. Sci. USA 109, 20895–20900 (2012).
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).
Urnavicius, L. et al. The structure of the dynactin complex and its interaction with dynein. Science 347, 1441–1446 (2015).
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).
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).
Urnavicius, L. et al. Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature 554, 202–206 (2018).
Splinter, D. et al. BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures. Mol. Biol. Cell 23, 4226–4241 (2012).
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).
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).
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).
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).
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).
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).
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).
Duellberg, C. et al. Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein. Nat. Cell Biol. 16, 804–811 (2014).
King, S. J. & Schroer, T. A. Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20–24 (2000).
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).
Reck-Peterson, S. L. et al. Single-molecule analysis of dynein processivity and stepping behavior. Cell 126, 335–348 (2006).
Can, S., Dewitt, M. A. & Yildiz, A. Bidirectional helical motility of cytoplasmic dynein around microtubules. eLife 3, e03205 (2014).
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).
Nicholas, M. P. et al. Control of cytoplasmic dynein force production and processivity by its C-terminal domain. Nat. Commun. 6, 6206 (2015).
Gennerich, A., Carter, A. P., Reck-Peterson, S. L. & Vale, R. D. Force-induced bidirectional stepping of cytoplasmic dynein. Cell 131, 952–965 (2007).
Belyy, V., Hendel, N. L., Chien, A. & Yildiz, A. Cytoplasmic dynein transports cargos via load-sharing between the heads. Nat. Commun. 5, 5544 (2014).
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).
Lu, H. et al. Collective dynamics of elastically coupled myosin V motors. J. Biol. Chem. 287, 27753–27761 (2012).
Derr, N. D. et al. Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science 338, 662–665 (2012).
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).
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).
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).
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).
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).
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).
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).
Ray, S., Meyhofer, E., Milligan, R. A. & Howard, J. Kinesin follows the microtubule’s protofilament axis. J. Cell Biol. 121, 1083–1093 (1993).
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).
Cleary, F. B. et al. Tension on the linker gates the ATP-dependent release of dynein from microtubules. Nat. Commun. 5, 4587 (2014).
Rai, A. et al. Dynein clusters into lipid microdomains on phagosomes to drive rapid transport toward lysosomes. Cell 164, 722–734 (2016).
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).
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).
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.
S.C.B. holds an equity interest in Lumidyne Technologies. All other authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Tables 1–3 and Supplementary Figures 1–8
Processive motility of single DDB and DDR complexes along MTs in 1 mM ATP.
Helical movement of DDB- and DDR-driven beads along an MT bridge.
Linking of two DDBs via DNA hybridization.
Motility of DDB–kinesin and DDR–kinesin colocalizers.