These authors contributed equally to this work
In vitro reconstitution of a highly processive recombinant human dynein complex
Article first published online: 1 JUL 2014
© 2014 MRC Laboratory of Molecular Biology. Published under the terms of the CC BY 4.0 license
This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
The EMBO Journal
Volume 33, Issue 17, pages 1855–1868, 1 September 2014
How to Cite
The EMBO Journal (2014) 33: 1855–1868
See also: MA Cianfrocco & AE Leschniner (September 2014)
- Issue published online: 1 SEP 2014
- Article first published online: 1 JUL 2014
- Manuscript Accepted: 12 JUN 2014
- Manuscript Revised: 4 JUN 2014
- Manuscript Received: 22 APR 2014
- Medical Research Council, UK. Grant Numbers: MC_UP_A025_1011, U105178790
- Wellcome Trust. Grant Number: WT100387
Cytoplasmic dynein is an approximately 1.4 MDa multi-protein complex that transports many cellular cargoes towards the minus ends of microtubules. Several in vitro studies of mammalian dynein have suggested that individual motors are not robustly processive, raising questions about how dynein-associated cargoes can move over long distances in cells. Here, we report the production of a fully recombinant human dynein complex from a single baculovirus in insect cells. Individual complexes very rarely show directional movement in vitro. However, addition of dynactin together with the N-terminal region of the cargo adaptor BICD2 (BICD2N) gives rise to unidirectional dynein movement over remarkably long distances. Single-molecule fluorescence microscopy provides evidence that BICD2N and dynactin stimulate processivity by regulating individual dynein complexes, rather than by promoting oligomerisation of the motor complex. Negative stain electron microscopy reveals the dynein–dynactin–BICD2N complex to be well ordered, with dynactin positioned approximately along the length of the dynein tail. Collectively, our results provide insight into a novel mechanism for coordinating cargo binding with long-distance motor movement.
Cargo adaptor BICD2 converts dynein from a non-processive to a highly processive motor in presence of dynactin. This might coordinate long-distance movement with cargo availability.
- Production of the 1.4 MDa recombinant mammalian dynein complex using a single baculovirus.
- The dynactin complex and the N-terminus of the cargo adaptor BICD2 (BICD2N) are sufficient to convert dynein into a highly processive motor.
- In the presence of BICD2N, dynactin forms a well-ordered complex with the dynein tail.
- Results suggest a mechanism for coupling cargo availability to the activation of long-distance transport.