Dynactin p150 Promotes Processive Motility of DDB Complexes by Minimizing Diffusional Behavior of Dynein

Abstract

Abstract 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, in quantitative agreement with the increase in diffusivity seen in isolated DDB. The data suggest a DDB activation model in which engagement of dynactin p150 with the microtubule promotes dynein processivity, serves as an allosteric activator of dynein, and enhances processive minus-end motility during intracellular bidirectional transport. TOC Highlight Dynein-dynactin-BicD2 (DDB) is highly processive, but also shows transient pausing and diffusion, which we analyzed using iSCAT microscopy. Blocking dynactin p150 results in more diffusion of isolated DDB and a plus-end shift of kinesin-1 – DDB complexes. Thus, we conclude that p150 is an allosteric activator of dynein in the DDB complex.