Dynein and dynactin move long-range but are delivered separately to the axon tip.

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.