Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly.
We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.
Ngai
J
J
Division of Biology, California Institute of Technology, Pasadena 91125.
Coleman
T R
TR
Lazarides
E
E
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002645:Chickens; D003599:Cytoskeleton / Q000648:ultrastructure*; D003907:Dexamethasone / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005796:Genes; D007382:Intermediate Filaments / Q000648:ultrastructure*; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D010957:Plasmids; D014176:Protein Biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014746:Vimentin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D014746:Vimentin; D003907:Dexamethasone