Neurofilament cross-bridging competes with kinesin-dependent association of neurofilaments with microtubules

The phosphorylation of neurofilaments (NFs) has long been considered to regulate their axonal transport rate and in doing so to provide stability to mature axons. Axons contain a centrally situated 'bundle' of closely opposed phospho-NFs that display a high degree of NF-NF associations and phospho-epitopes, surrounded by less phosphorylated 'individual' NFs that are often associated with kinesin and microtubules (MTs). Bundled NFs transport substantially slower than the surrounding individual NFs and might represent a resident population that stabilizes axons and undergoes replacement by individual NFs. To examine this possibility, fractions enriched in bundled NFs and individual NFs were generated from mice and NB2a/d1 cells by sedimentation of cytoskeletons over a sucrose cushion. More kinesin was recovered within individual versus bundled NF fractions. Individual but not bundled NFs aligned with purified MTs under cell-free conditions. The percentage of NFs that aligned with MTs was increased by the addition of kinesin, and inhibited by anti-kinesin antibodies. Bundles dissociated following incubation with EGTA or alkaline phosphatase, generating individual NFs that retained or were depleted of phospho-epitopes, respectively. These dissociated NFs aligned with MTs at a level identical to those originally isolated as individual NFs regardless of phosphorylation state. EGTA-mediated dissociation of bundles was prevented and reversed by excess Ca2+, whereas individual NFs did not associate in the presence of excess Ca2+. These findings confirm that bundling competes with NF-MT association, and provide a mechanism by which C-terminal NF phosphorylation might indirectly contribute to the observed slowing in axonal transport of phospho-NFs