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

Abstract

The microtubule motor dynein drives retrograde transport from the axon tip back to the cell body and is essential for neuronal survival. Its function requires its cofactor dynactin and regulators LIS1 and NDEL1. However, it is unclear if all dynein components can travel along the axon, how far they move and whether they do so together. Here, we use neuron-inducible (NGN2-OPTi-OX) human-stem-cell lines to endogenously tag dynein components and track them under a near single molecule regime. In the retrograde direction dynein and dynactin can move the entire > 500 um length of the axon in one go. Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with initiation of transport. Intriguingly, in the anterograde direction dynein/LIS1 move faster than dynactin/NDEL1 implying they can travel on different cargos. Therefore, neurons ensure efficient transport by keeping dynein/dynactin on cargos over long distances, but keeping them separate until their function is required.

Human dynein-dynactin is a fast processive motor in living cells

“…While several groups have recently imaged endogenously tagged dynein in mitotic HeLa cells and iNeurons (Fellows et al, 2023; Ide et al, 2023), to our knowledge - this is the first direct visualization of motile endogenously-tagged human dynein-dynactin complexes in proliferating, non-neuronal cells. Two distinct motile populations of dynein-dynactin exist in human interphase cells: one pool that tip-tracks on polymerizing MT plus-ends and a second pool that moves processively at high velocities (∼7X faster than the tip-tracking population) along MTs.…”

“…We propose that the ∼3 μm run lengths measured here are an underestimate of what the dynein-dynactin complex is capable of due to the relatively short and dynamic MT tracks available to it in interphase HeLa cells and because motile puncta could only be visualized while they were in the TIRF field and prior to their photobleaching. Indeed, dynein was recently shown to be incredibly processive in iNeurons where its mean run length was ∼ 35 μm and runs >100 μm were observed (Fellows et al, 2023). It is possible that the tip-tracking pool of dynein-dynactin is regulated in a cell-cycle dependent manner because dynein-dynactin tracking events were less robust in mitotic cells than in interphase cells.…”

“…This suggests that the cargos, which we presume motile dynein-dynactin puncta are bound to, any kinesins associated with the cargos are inactive and/or cannot effectively bind the MT lattice during dynein-dynactin-mediated transport in interphase HeLa cells. Interestingly, directional switching was also uncommon during retrograde dynein-dynactin movements in iNeuron axons (Fellows et al, 2023) and in retrograde trafficking of early endosomes in Ustilago maydis mediated by dynein-dynactin containing the HOOK3 adaptor (Bielska et al, 2014; Schuster et al, 2011).…”

Cargo specificity, regulation, and therapeutic potential of cytoplasmic dynein

“…This finding implies that when traveling distances greater than one micron, dynein-dynactin-cargo adaptor complexes consistently undergo binding and unbinding. While overexpressed GFP-dynein shows a short residence time and short run length inside the cytoplasm of HeLa cells, dynein shows much more robust movement and a longer run length along the axons of human neuronal cells 39 . Endogenously labeled dynein had a much longer run length of up to ~110 μm and was able to move the entire length of the axon, with an average speed of ~1.7 μm/s, as observed via HILO microscopy.…”

Measurements of neurite extension and nucleokinesis in an iPSC-derived model system following microtubule perturbation

“…Then, terminal differentiation into specific neuronal subtypes can be achieved as before, using specific factors. Multiple studies investigating microtubule dynamics and cargo transport using these neurons demonstrated their promise for studying the microtubule network (45)(46)(47), but their physiology remains incompletely characterized.…”