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EMBO J. 2014 Sep 1;33(17):1855-68. doi: 10.15252/embj.201488792. Epub 2014 Jul 1.

In vitro reconstitution of a highly processive recombinant human dynein complex.

Author information

1
Division of Structural Studies, MRC-Laboratory of Molecular Biology, Cambridge, UK.
2
Division of Cell Biology, MRC-Laboratory of Molecular Biology, Cambridge, UK.
3
Division of Cell Biology, MRC-Laboratory of Molecular Biology, Cambridge, UK sbullock@mrc-lmb.cam.ac.uk cartera@mrc-lmb.cam.ac.uk.
4
Division of Structural Studies, MRC-Laboratory of Molecular Biology, Cambridge, UK sbullock@mrc-lmb.cam.ac.uk cartera@mrc-lmb.cam.ac.uk.

Abstract

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.

KEYWORDS:

Bicaudal‐D; dynactin; dynein; microtubules; processivity

PMID:
24986880
PMCID:
PMC4158905
DOI:
10.15252/embj.201488792
[Indexed for MEDLINE]
Free PMC Article

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