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The Journal of Cell Biology, Vol 108, 579-593, Copyright © 1989 by The Rockefeller University Press
ARTICLES |
MJ Monteiro and DW Cleveland
Johns Hopkins University, School of Medicine, Department of Biological Chemistry, Baltimore, Maryland 21205.
We have used transient and stable DNA transfection to force synthesis of the mouse NF-L and NF-M genes in nonneuronal cultured animal cells. When the authentic NF-L gene (containing 1.7 kb of sequences 5' to the transcription initiation site) was transfected into L cells, correctly initiated NF-L mRNA was produced from the transfected gene but not the endogenous NF-L genes. Therefore, the normal restriction of NF-L expression to neurons cannot derive exclusively from absence in nonneuronal cells of neuron-specific transcription factors. When the NF- L coding region was linked to the strong promoter from Moloney Murine Sarcoma virus, we obtained high levels of synthesis of NF-L subunits (accumulating to as much as 9% of cell protein in stable cell lines). Although NF-L and NF-M polypeptides are normally expressed exclusively in postmitotic neurons, NF-L or NF-M polypeptides expressed in fibroblasts were efficiently assembled into intermediate filament arrays, thus demonstrating the competence of both NF-L and NF-M to assemble in vivo in the absence of additional neuron-specific factors. As judged by immunofluorescence localization and by the alteration in the solubility of the endogenous vimentin filaments, filaments containing NF-L appeared to be copolymers with vimentin. Neither the alteration in the properties of the vimentin array nor the accumulation of NF-L to a level that made it the second most abundant cellular protein (after actin) had any observable effect on cell viability or growth rate.
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