Colchicine‐sensitive and colchicine‐insensitive intermediate filament systems distinguished by a new intermediate filament‐associated protein, IFAP‐70/280kD

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 70‐280kD 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‐70/280kD (IF‐Associated Protein): (1) it coisolated 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 54/55kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP‐70/280kD 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‐70/280kD 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‐300kD [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. © 1992 Wiley‐Liss, Inc.