Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts.
The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.
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Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032.
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Liem
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D000818:Animals; D002478:Cells, Cultured; D002872:Chromosome Deletion; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism*; D051379:Mice; D016253:Microscopy, Immunoelectron; D009154:Mutation; D016900:Neurofilament Proteins; D015183:Restriction Mapping; D014162:Transfection; D014746:Vimentin / Q000378:metabolism
D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D014746:Vimentin