Fate of a headless vimentin protein in stable cell cultures: soluble and cytoskeletal forms.
The non-alpha-helical head domains of cytoskeletal intermediate filaments (IFs) are considered to play an important role in IF assembly and stability. We have investigated the fate of a "headless" mutant vimentin protein in cell types that either lack cytoplasmic IFs or contain preexisting IF networks (keratin and vimentin). Stable clones expressing a transfected headless vimentin cDNA were individually analyzed in order to avoid variabilities introduced by transient transfection and to compare the levels and effects of the mutant vimentin protein more accurately. In cells lacking IFs, the mutant protein existed in a diffuse, soluble form as determined by immunofluorescence and biochemical protein fractionation. In cells possessing vimentin IFs, the headless vimentin was highly dispersed throughout the cytoplasm, including lamellopodia. Expression levels in individual clones were as high as sevenfold greater than the endogenous vimentin component. Although the majority of the headless vimentin was highly soluble, a residual portion of the mutant vimentin colocalized with vimentin filaments and consistently comprised about 25% of the cytoskeletal vimentin network. These results demonstrate that the mutant protein can be stably expressed at relatively high levels without deleterious cellular effects or disruption of endogenous vimentin filaments. The observed specific ratio of mutant to wild-type vimentin (1:3) in the cytoskeleton supports IF in vivo assembly via specific hybrid tetramer formation and, further, that at least three intact head domains are required for competent tetramer formation and IF assembly.
Andreoli
J M
JM
Department of Molecular Biology and Genetics, Wayne State University, Detroit, Michigan 48202.
Trevor
K T
KT
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship; D014162:Transfection; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014746:Vimentin