Colchicine-sensitive and colchicine-insensitive intermediate filament systems distinguished by a new intermediate filament-associated protein, IFAP-70/280 kD.
A monoclonal antibody was produced, using as antigen a BHK-21 cytoskeletal preparation enriched in intermediate filaments (IF) and their associated proteins. This antibody reacted exclusively with a reproducible set of 70-280 kD polypeptides present in minor quantities in this preparation, as detected by immunoblot analysis. Based upon several criteria, this immunologically related group of polypeptides was designated as IFAP-70/280 kD (IF-Associated Protein): (1) it co-isolated with IF in vitro, (2) it co-localized (by both immunofluorescence and immunoelectron microscopy) with IF in situ in all stages of cell spreading, and (3) it segregated in vitro with the 54/55 kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP-70/280 kD to regions of parallel or loosely bundled IF in situ, suggesting a role in regulating the supramolecular organization of IF. When this monoclonal antibody was used for double-label immunofluorescence observations of colchicine-treated BHK cells, it demonstrated the presence of colchicine-sensitive and colchicine-insensitive IF. Anti-IFAP-70/280 kD localized entirely to the drug-induced juxtanuclear IF cap, while a polyclonal antibody directed against the desmin/vimentin structural IF subunits and the previously characterized monoclonal anti-IFAP-300 kD [Yang et al., 1985; J. Cell Biol. 100:620] localized to both the juxtanuclear IF cap and a colchicine-insensitive IF network peripheral to the cap in the same cells. The colchicine-insensitive IF pattern often exhibited similarities to that observed for the actin-based stress fiber system, suggesting that stress fiber association may be an additional factor in IF organization.
Yang
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Department of Anatomy and Cell Biology, College of Medicine, University of Illinois, Chicago 60612.
Lieska
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Goldman
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Goldman
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D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured / Q000187:drug effects / Q000648:ultrastructure; D003078:Colchicine / Q000494:pharmacology*; D006224:Cricetinae; D015151:Immunoblotting; D007382:Intermediate Filaments / Q000737:chemistry / Q000187:drug effects*; D007668:Kidney / Q000737:chemistry / Q000648:ultrastructure; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D003078:Colchicine