/ 3-1 nternexin , a Protein Ubiquitous Intermediate Filament-associated

In this article we show a Triton-insoluble, intermediate filament-associated protein of ~70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name/3-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. ~-Internexin also shares some degree of homology with a-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of ~-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of ~-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of ~-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70). Cytoskeletal-associated proteins are defined by their ability to co-isolate and interact in situ with one of the filamentous networks indigenous to most eukaryotic cell types. These polypeptides are thought to subserve a number of putative functions with regard to cytoskeletal organization and regulation. They may effect dynamic changes of cytoskeletal architecture in vivo by altering the polymerization state of cytoskeletal subunit components; engendering a redistribution of intracellular filaments; serving as potential crossbridging agents between subcellular organelles (e.g., between neurofilaments and microtubules); or harboring specific catalytic activities (e.g., phosphokinase). Support for some of these hypothetical roles is derived from studies conducted in a number of laboratories. Numerous investigators have observed that high molecular weight microtubule-associated proteins (MAPs) ~ as well as lower molecular weight tau-proteins promote the in vitro assembly of tubulin subunits into polymeric microtubules (4, 26, 32). MAPs have also been impli~Abbreviations used in this paper. " HSP, heat shock protein; IF, intermediate filament; MAP, microtubule-associated protein; SV8, Staphylococcus aureus V8. THE IOURNAL OF CELL BIOLOGY • VOLUME 101 OCTOBER 1985 1323 1331 © The Rockefeller University Press . 0021-9525/85/10/1323/09. $1.00 cated in mediating the interactions between microtubules and neurofilaments (1, 18). Immunoelectron microscopy studies have shown that the peripherally located 200-kD neurofilament-associated subunit appears to serve as an interfilamentous cross-linker in myelinated axons (11, 12, 19). In this report we describe an intermediate filament (IF)associated protein designated ~-internexin which is present in many diverse eukaryotic cell types. We examine the relationship of this polypeptide to a-internexin, an IF-associated protein originally isolated from rat spinal cord and optic nerve, which was found to bind to several IF subunit proteins (22). The presence of B-internexin in all cell types investigated, and the homology of this protein isolated from the different cells were established by two-dimensional gel electrophoresis, peptide mapping, and antibody localization. Its solubility properties and uitrastructural localization render this protein a likely candidate for the class of IF-associated proteins that may play an integral role in the regulation of the IF-reticulum. In this paper we will also discuss the possible relatedness of B-internexin to a protein that co-isolates with microtubules and to the most commonly elicited, constitutively synthesized mammalian heat shock protein of ~70 kD (HSP 68/70). 1323 on A uust 6, 2017 jcb.rress. org D ow nladed fom MATERIALS AND METHODS Cell Culture and Cytoskeletal Extracts: Five culture lines were used to isolate IF-rich fractions and subsequently detect the IF-associated protein dubbed fl-internexin: human glioma cells (U25 I-MG), Chinese hamster o~ary fibroblasts (CHO), rat kangaroo kidney epithelial cells (PIK2), human foreskin fibroblasts (FS-4), and neuroblastoma-glioma hybrids (NGI08-15). All cultures were grown on 100-mm sterile Falcon tissue culture dishes (Falcon Labware, Oxnard, CA) in Dulbecc&s modified Eagle's medium supplemented with 10% fetal calf serum except the neuroblastoma-glioma hybrid line, which was maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum containing 0.1 mM hypoxanthine, l ~M aminopterin, and 16 ~M