Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used interferometric scattering (iSCAT) microscopy to track dynein–dynactin–BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive, and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB–kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity of the complexes, in quantitative agreement with the shift of isolated DDB into the diffusive state. The data suggest a DDB activation model in which dynactin p150 enhances dynein processivity not solely by acting as a diffusive tether that maintains microtubule association, but rather by acting as an allosteric activator that promotes a conformation of dynein optimal for processive stepping. In bidirectional cargo transport driven by the opposing activities of kinesin and dynein–dynactin–BicD2, the dynactin p150 subunit promotes retrograde transport and could serve as a target for regulators of transport.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology