Dynein-mediated microtubule translocation powering neurite outgrowth in chick and Aplysia neurons requires microtubule assembly.

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

Neurite elongation is highly correlated with bulk forward translocation of microtubules.

During the development of the nervous system and regeneration following injury, microtubules (MTs) are required for neurite elongation. Whether this elongation occurs primarily through tubulin assembly at the tip of the axon, the transport of individual MTs, or because MTs translocate forward in bulk is unclear. Using fluorescent speckle microscopy (FSM), differential interference contrast (DIC), and phase contrast microscopy, we tracked the movement of MTs, phase dense material, and docked mitochondria in chick sensory and Aplysia bag cell neurons growing rapidly on physiological substrates. In all cases, we find that MTs and other neuritic components move forward in bulk at a rate that on average matches the velocity of neurite elongation. To better understand whether and why MT assembly is required for bulk translocation, we disrupted it with nocodazole. We found this blocked the forward bulk advance of material along the neurite and was paired with a transient increase in axonal tension. This indicates that disruption of MT dynamics interferes with neurite outgrowth, not by disrupting the net assembly of MTs at the growth cone, but rather because it alters the balance of forces that power the bulk forward translocation of MTs.