Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells.

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein 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 visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.

Visualization of Single Dynein Molecules in Mammalian Cells.

In vitro single-molecule imaging experiments have provided insight into the stepping behavior, force production, and activation of several molecular motors. However, due to the difficulty in visualizing single molecules of motor proteins in vivo, the physiological function and regulation of motors at the single-molecule level have not been studied widely. Here, we describe how highly inclined and laminated optical sheet (HILO) microscopy can be adapted to visualize single molecules of the motor protein cytoplasmic dynein-1 in mammalian cells with high signal-to-noise ratio and temporal resolution.

Role of Dynactin in the Intracellular Localization and Activation of Cytoplasmic Dynein.

Cytoplasmic dynein, the major minus end-directed motor protein in several cell types, transports a variety of intracellular cargo upon forming a processive tripartite complex with its activator dynactin and cargo adaptors such as Hook3 and BicD2. Our current understanding of dynein regulation stems from a combination of in vivo studies of cargo movement upon perturbation of dynein activity, in vitro single-molecule experiments, and cryo-electron microscopy studies of dynein structure and its interaction with dynactin and cargo adaptors. In this Perspective, we first consolidate data from recent publications to understand how perturbations to the dynein-dynactin interaction and dynactin's in vivo localization alter the behavior of dynein-driven cargo transport in a cell type- and experimental condition-specific manner. In addition, we touch upon results from in vivo and in vitro studies to elucidate how dynein's interaction with dynactin and cargo adaptors activates dynein and enhances its processivity. Finally, we propose questions that need to be addressed in the future with appropriate experimental designs so as to improve our understanding of the spatiotemporal regulation of dynein's function in the context of the distribution and dynamics of dynactin in living cells.