Gαi2 promotes microtubule-driven incorporation of neurofilaments to stabilise nascent axons in vivo.

Axon initiation is a critical step in the establishment of neuronal morphology. However, the mechanisms regulating stable axon initiation during embryonic development remain poorly understood. Here, using fixed-tissue super-resolution and high-resolution live-tissue imaging in the developing chick spinal cord, we reveal that stable axon initiation is characterised by microtubule-directed neurofilament accumulation in the emerging axon, and identify the multifunctional G-protein Gαi2 as a regulator of this process. Before axon initiation, microtubule network orientation shifts towards the site of the future axon where coiled Gαi2-associated neurofilaments accumulate asymmetrically before unfurling into the initiating axon behind microtubules. Crucially, Gαi2 is associated with neurofilaments and microtubules at their points of contact. Gαi2 depletion reduces engagement between these cytoskeletal networks and leads to impaired passage of neurofilaments into initiating axons and disrupted axon outgrowth, supporting a role for Gαi2 in regulating microtubule-driven incorporation of neurofilaments in nascent axons. These findings advance mechanistic understanding of polarity establishment and provide insight into cytoskeletal regulation during axon formation.