Dynactin Functions as Both a Dynamic Tether and Brake during Dynein-Driven Motility

Abstract

Dynactin is a large multi-subunit complex that is an important cofactor for cytoplasmic dynein in cellular processes including organelle trafficking and mitosis. Dynactin increases the processivity of dynein and also targets dynein to specific cellular locations. Although the role of dynactin in dynein-mediated processes is well established, the underlying mechanisms involved are unknown. Here, we use single molecule approaches to investigate the contribution of the dynactin subunit p150Glued to dynein motor function. We first characterized the motility of purified GFP-tagged mammalian dynein isolated from a knock-in mouse brain tissue (Zhang et al., 2013). Consistent with previous reports (Mallik et al., 2005; Ross et al., 2006), single molecules of dynein-GFP switch stochastically from processive to diffusive states of motion. We analyzed this motion by a novel algorithm that we developed to parse trajectories into different states and found that dynein is processive 60% of the time. To examine the activation of dynein by dynactin, we investigated the formation and co-migration of a dynein-p150Glued co-complex using dual-color TIRF microscopy. We provide direct evidence that p150Glued is sufficient to recruit and tether dynein to the microtubule and that this recruitment is concentration-dependent; p150Glued increases the on-rate and decreases the off-rate of dynein from microtubules. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of dynactin is essential for the decreased detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.