Dynamic aspects of the neuronal intermediate filament (NIF) network studied by microinjection of biotinylated NIF proteins into living cells

The properties of neuronal intermediate filament (NIF) networks in vivo were studied using embryonic chick dorsal root ganglion neurons in culture. The natural changeover from a primarily vimentin-containing intermediate filament (IF) to a primarily neurofilament-containing IF network was observed to occur gradually and in a non-catastrophic manner as the neurons matured. The dynamics of the NIF system were studied in a more direct manner following the microinjection of purified biotinylated NIF proteins into living cells. Vimentin obtained from bovine lens and neurofilament triplet proteins (NFT) obtained from bovine spinal cord were biotinylated, disassembled, and purified by ion exchange chromatography. Recombination of the purified NFT proteins NF-L, NF-M, and NF-H resulted in the formation of 10 nm IF in vitro. Purified biotinylated vimentin also formed 10 nm IF in vitro. Each of these purified biotinylated proteins was microinjected at concentrations of 0.8-1.0 mg/ml into DRGs obtained from chick embryos at stage E8, when the NIF network is composed primarily of vimentin, and at E14, when the NIF network is composed primarily of the NFT proteins. All four biotinylated proteins were found to become directly incorporated into each type of NIF network within a few hours, with the exception of biotinylated vimentin which became localized in a juxtanuclear cap region in E8 DRGs, prior to its incorporation into the endogenous vimentin network. NF-H was found to become incorporated 1.5 times faster than either vimentin or NF-L. DRGs from newborn mice also incorporated the biotinylated proteins. Microinjection of biotinylated NF-H into DRGs from E17 chicks at concentrations higher than 1 mg/ml were found to result in the accumulation of neurofilamentous structures in the cell body. These accumulations were concentration-dependent and were coincident with a decrease in the measurable neurite width, accumulation of mitochondria in the cell body, as well the recognition of the neurofilamentous accumulations by an antibody specific for the highly phosphorylated form of NF-H. These results emphasize the importance of maintaining the correct stoichiometric ratios among the NFT proteins within cells. These observations also support the view that NIF are dynamic structures in vivo.