In vitro reconstitution of a highly processive recombinant human dynein complex

• Published in: The EMBO journal Vol. 33; no. 17; pp. 1855 - 1868
• Main Authors: Schlager, Max A, Hoang, Ha Thi, Urnavicius, Linas, Bullock, Simon L, Carter, Andrew P
• Format: Journal Article
• Language: English
• Published: London Blackwell Publishing Ltd 01.09.2014; Nature Publishing Group UK; BlackWell Publishing Ltd
• Subjects: Animals; Baculoviridae - genetics; Bicaudal-D; Carrier Proteins - metabolism; dynactin; Dynactin Complex; dynein; Dyneins - metabolism; EMBO20; Fluorescence microscopy; Humans; Macromolecular Substances - metabolism; Mammals; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Microtubule-Associated Proteins - metabolism; microtubules; Molecular biology; processivity; Protein Multimerization; Proteins; Sf9 Cells; dynein; Bicaudal‐D; microtubules; dynactin; processivity
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201488792

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. Synopsis 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. Graphical Abstract 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.