Neurofilaments form a highly stable stationary cytoskeleton after reaching a critical level in axons.

The ultrastructural view of the axonal cytoskeleton as an extensively cross-linked network of neurofilaments (NFs) and other cytoskeletal polymers contrasts with the dynamic view suggested by axonal transport studies on cytoskeletal elements. Here we reconcile these perspectives by showing that neurons form a large NF network along axons which is unequivocally stationary, metabolically stable, and maintained by NFs and nonfilamentous subunit assemblies undergoing slow transport by intermittent rapid movements and pauses. In mouse primary cortical neurons transfected with EGFP-NFL, formation of this stationary NF network requires a critical level of NFs, which explains its absence in NF-poor developing neurons studied previously. Most NFs at proximal axon regions were in a stationary structure coexisting with a smaller pool of moving EGFP-NFL assemblies that were mainly nonfilamentous. Distally along the same axon, EGFP-labeled NFL was much less abundant, and we detected only short filaments moving bidirectionally by slow transport (rapid movements and pauses) as previously described. In living mice, >25% of radiolabeled newly synthesized NFs remained in optic axons after slowly transported NFs had exited. Retained NF remained fixed over several months in a nonuniform distribution and exhibited exceptionally slow turnover (t(1/2) >2.5 months), implying that, at steady state, >90% of NFs in mature optic axons comprise the stationary cytoskeleton and <10% are undergoing slow transport. These findings reconcile in vitro and in vivo axonal transport observations, showing that slowly transported NFs or subunit oligomers are precursors to a highly stable stationary cytoskeletal network that supports mature axons.