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In vitro reconstitution of a highly processive recombinant human dynein complex
Author(s) -
Schlager Max A,
Hoang Ha Thi,
Urnavicius Linas,
Bullock Simon L,
Carter Andrew P
Publication year - 2014
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.201488792
Subject(s) - dynein , dynactin , biology , microtubule , microbiology and biotechnology , processivity , motor protein , molecular motor , biophysics , biochemistry , gene , polymerase
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 BICD 2 ( BICD 2N) gives rise to unidirectional dynein movement over remarkably long distances. Single‐molecule fluorescence microscopy provides evidence that BICD 2N 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– BICD 2N 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.