Expression of NF-L and NF-M in fibroblasts reveals coassembly of neurofilament and vimentin subunits.
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.
Monteiro
M J
MJ
Johns Hopkins University, School of Medicine, Department of Biological Chemistry, Baltimore, Maryland 21205.
Cleveland
D W
DW
D000818:Animals; D003001:Cloning, Molecular; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D007381:Intermediate Filament Proteins / Q000096:biosynthesis* / Q000235:genetics; D007382:Intermediate Filaments / Q000378:metabolism / Q000648:ultrastructure; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D008870:Microtubules / Q000648:ultrastructure; D008980:Moloney murine sarcoma virus / Q000235:genetics; D016900:Neurofilament Proteins; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D012995:Solubility; D014158:Transcription, Genetic; D014162:Transfection; D014746:Vimentin / Q000378:metabolism*
D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D016900:Neurofilament Proteins; D012333:RNA, Messenger; D014746:Vimentin; C044882:neurofilament protein L