title: Neurofilament cross-bridging competes with kinesin-dependent association of neurofilaments with microtubules.
abstract: 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 Ca(2+), whereas individual NFs did not associate in the presence of excess Ca(2+). 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.
authors.affiliation: Center for Cellular Neurobiology and Neurodegeneration Research, Departments of Biological Sciences and Biochemistry, University of Massachusetts Lowell, Lowell, MA 01854, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Kushkuley
authors.forename: Walter K H
authors.initials: WK
authors.lastname: Chan
authors.forename: Sangmook
authors.initials: S
authors.lastname: Lee
authors.forename: Joel
authors.initials: J
authors.lastname: Eyer
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Leterrier
authors.forename: Franck
authors.initials: F
authors.lastname: Letournel
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Shea
mesh_terms: D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D005260:Female; D007382:Intermediate Filaments / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding
chemical_list: D016900:Neurofilament Proteins; D016547:Kinesins