Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells (1). Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin and a cargo adaptor (2-5). Thereupon, dynein transports cargo towards the minus ends of microtubules. How this process of motor activation occurs is unclear, since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualise and track fluorescently tagged dynein. First, we observed that dynein that bound to the microtubule engaged in minus end-directed movement only ~30% of the time and resided on the microtubule for a short duration. Next, using high-resolution imaging in live and fixed cells, we discovered that dynactin remained persistently attached to microtubules, and endosomal cargo remained in proximity to the microtubules and dynactin. Finally, we employed two-colour imaging to visualise cargo movement effected by single motor binding. Taken together, we discovered a search strategy that is facilitated by dynein's frequent microtubule binding-unbinding kinetics: (1) in a futile event when dynein does not encounter cargo anchored in proximity to the microtubule, dynein unbinds and diffuses into the cytoplasm, (2) when dynein encounters cargo and dynactin upon microtubule binding, it moves cargo in a short run. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on reduction of dimensionality to enable minus end-directed transport in vivo.
Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)