Phospho-dependent association of neurofilament proteins with kinesin in situ

Recent studies demonstrate co-localization of kinesin with neurofilament (N F) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated wi th NF subunits in situ. Axonal transport of NF subunits in mouse optic nerv e was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, in dicating that NF transport was dependent upon MT dynamics. Kinesin co-preci pitated during immunoprecipitation of NF subunits from optic nerve. The ass ociation of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-pur ify in a microtubule motor preparation from CNS while less phosphorylated f orms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free condi tions diminished the association of NF subunits with kinesin. The nature an d extent of this association was further examined by intravitreal injection of S-35-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits un derwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated wi th kinesin, and (2) that dissociation from kinesin may foster trailing of N F subunits during continued transport. These data suggest that kinesin part icipates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoske leton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport. (C) 2000 Wiley-Liss, Inc.