Single-Molecule Imaging of Cytoplasmic Dynein in vivo Reveals the Mechanism of Motor Activation and Cargo Capture

Abstract

The minus-end directed motor protein, cytoplasmic dynein is regulated for its activity in living cells by remaining in an inactive conformation until the formation of a tripartite complex comprising dynein, dynactin and a cargo adaptor. Thereupon, dynein processively transports cargo towards the minus ends of microtubules. How this process of dynein activation occurs in living cells is unclear, since this entails the congregation of three kinds of protein complexes inside the crowded environs of cell. Here, we employed high time resolution, single-molecule imaging and tracking techniques to visualize and follow fluorescently-tagged dynein heavy chain in HeLa cells. We observed dynein that bound afresh from the cytoplasm to the microtubule engaged in processive minus end-directed movement only about 30% of the time. Contrary to the existing model of cargo capture at microtubule plus ends by EB1-dynein clusters, we discovered that dynein encountered cargo that were pre-anchored all along the length of the microtubule. Taken together, we propose a search strategy that is facilitated by dynein's frequent microtubule binding-unbinding kinetics: (1) in the event of a futile event when dynein does not encounter pre-anchored cargo on the microtubule, dynein unbinds and diffuses back into the cytoplasm, (2) when dynein encounters cargo upon microtubule binding, it is activated and transports the cargo in a short run towards the minus end. In conclusion, dynein activation and cargo capture are coupled to enable minus-end directed transport in vivo