3-1Nternexin, a Protein Ubiquitous Intermediate Filament-Associated
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/3-1nternexin, a Ubiquitous Intermediate Filament-associated Protein EUGENE W. NAPOLITANO, JOEL S. PACHTER, STEVENS. M. CHIN, and RONALD K. H. LIEM Department of Pharmacology, New York University School of Medicine, New York, New York 10016 ABSTRACT 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 monoclo- nal 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 cated in mediating the interactions between microtubules and co-isolate and interact in situ with one of the filamentous neurofilaments (1, 18). Immunoelectron microscopy studies networks indigenous to most eukaryotic cell types. These have shown that the peripherally located 200-kD neurofila- polypeptides are thought to subserve a number of putative ment-associated subunit appears to serve as an interfilamen- functions with regard to cytoskeletal organization and regu- tous cross-linker in myelinated axons (11, 12, 19). lation. They may effect dynamic changes of cytoskeletal ar- In this report we describe an intermediate filament (IF)- chitecture in vivo by altering the polymerization state of associated protein designated ~-internexin which is present in cytoskeletal subunit components; engendering a redistribu- many diverse eukaryotic cell types. We examine the relation- tion of intracellular filaments; serving as potential cross- ship of this polypeptide to a-internexin, an IF-associated bridging agents between subcellular organelles (e.g., between protein originally isolated from rat spinal cord and optic neurofilaments and microtubules); or harboring specific cat- nerve, which was found to bind to several IF subunit proteins alytic activities (e.g., phosphokinase). Support for some of (22). The presence of B-internexin in all cell types investigated, these hypothetical roles is derived from studies conducted in and the homology of this protein isolated from the different a number of laboratories. Numerous investigators have ob- cells were established by two-dimensional gel electrophoresis, served that high molecular weight microtubule-associated pro- peptide mapping, and antibody localization. Its solubility teins (MAPs) ~ as well as lower molecular weight tau-proteins properties and uitrastructural localization render this protein promote the in vitro assembly of tubulin subunits into poly- a likely candidate for the class of IF-associated proteins that meric microtubules (4, 26, 32). MAPs have also been impli- may play an integral role in the regulation of the IF-reticulum. In this paper we will also discuss the possible relatedness of ~Abbreviations used in this paper." HSP, heat shock protein; IF, B-internexin to a protein that co-isolates with microtubules intermediate filament; MAP, microtubule-associated protein; SV8, and to the most commonly elicited, constitutively synthesized Staphylococcus aureus V8. mammalian heat shock protein of ~70 kD (HSP 68/70). THE IOURNAL OF CELL BIOLOGY • VOLUME 101 OCTOBER 1985 1323 1331 1323 © The Rockefeller University Press . 0021-9525/85/10/1323/09.$1.00 MATERIALS AND METHODS achieved by sequential addition of components used in the chloramine T method (10). After 20 ul phosphate buffer (0.5 M, pH 7.5) was pipetted into each tube, 20 ~1 Na125I, resuspended in phosphate buffer (0.5 uCi/20 ul), was Cell Culture and Cytoskeletal Extracts: Five culture lines were added. 20 #1 chloramine T ( 1 mg/ml) was applied to each tube, and after the used to isolate IF-rich fractions and subsequently detect the IF-associated absorption of liquid was allowed to proceed for 30 min, 0.5 ml sodium protein dubbed fl-internexin: human glioma cells (U25 I-MG), Chinese hamster metabisulfite (2 mg/ml) was added to terminate the reaction. 0.5 ml NaI (2 o~ary fibroblasts (CHO), rat kangaroo kidney epithelial cells (PIK2), human mg/ml) was placed into each sample tube, and the gel slices were then rinsed foreskin fibroblasts (FS-4), and neuroblastoma-glioma hybrids (NGI08-15). with several changes of 10% methanol. Radiolabeled proteins in these slices All cultures were grown on 100-mm sterile Falcon tissue culture dishes (Falcon were used in peptide mapping studies by the method described above. Labware, Oxnard, CA) in Dulbecc&s modified Eagle's medium supplemented Antiserum Production: Polyclonal antisera directed against gel-pu- with 10% fetal calf serum except the neuroblastoma-glioma hybrid line, which rified a-internexin (22) derived from rat spinal cord were raised in rabbit and was maintained in Dulbecco's modified Eagle's medium with 10% fetal calf guinea pig by use of the following protocol: (a) A subcutaneous injection of serum containing 0.1 mM hypoxanthine, l ~M aminopterin, and 16 ~M 25-50 #g protein, emulsified in Freund's complete adjuvant, was injected thymidine. subcutaneously followed by immediately by two intradermal injections into Cytoskeletal extracts from monolayer cultures were prepared by Dounce the hind paws. (b) 2 wk later, a second inoculation of protein, emulsified in homogenization of freshly harvested cells in an ice-cold (4"C) buffer consisting Freund's incomplete adjuvant, was administered as before. (c) Step b was of l0 mM phosphate (pH 6.8), 0.1 M NaCl, l mM EDTA containing 1% repeated for three more 2-wk intervals. (d) The animals were bled intermittently Triton X-100, 0.5 mg/ml DNase I (Worthington Biochemical Corp., Freehold, during these periods, and the immune sera were tested for specificity by N J), l0 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, and l uM Aprotinin immunoblotting and immunofluorescence assays described below. (Sigma Chemical Co., St. Louis, MO) followed by sedimentation in an Eppen- Mouse monoclonal antibodies and ascites directed against a-internexin were dorf centrifuge at 12,000 g for 10 min at 4*(?. Triton-insoluble pellets were produced as described by Wang et al. (30) employing the procedure of Kohler solubilized in SDS sample buffer (16), diluted in lysis buffer (21), and subjected and Milstein (15) as modified by Dippold et al. (6). The schedule of immuni- to two-dimensional gel electrophoresis and fluorography as described below. zation of female BALB/c mice was similar to that of the guinea pig. Approxi- Labeling of Cell Cultures: Cultures grown in 100-mm Falcon dishes mately 50-100 ug of purified antigen, emulsified in complete Freund's adjuvant, were labeled with [sSS]methionine by washing cells three times with phosphate- was used for the initial immunization. In four subsequent inoculations, purified buffered saline (PBS) and preincubating the cultures with 3 ml methionine-free antigen was mixed with Freund's incomplete adjuvant and injected subcuta- medium (Select-Amine Kit, Gibeo Laboratories Inc., Grand Island, NY) for 1 neously at 2-wk intervals. The last injection of antigen (without adjuvant) was h at 37"C. 100 ~Ci/ml of [sSS]methionine (>1,000 Ci/mmol, New England administered intravenously 3 d before the fusion. Clonal supernatants were Nuclear. Boston, MA) was then added for another hour at 37"C. Cytoskeletal then screened for anti-a-internexin activity using the microspot assay described extracts were then prepared as described above. elsewhere (19). Isolation of MAPs: MAPs were isolated from rat spinal cord by the Affinity Chromatography: Purification of the antisera by affinity method of Vallee (28). The spinal cords were homogenized in ice-cold reassem- chromatography was executed essentially as described in the pamphlet Affinity bly buffer of 0. l M 2-(N-morpholino)ethane sulfonie acid, 0.5 mM MgCl2, and Chromatography, Principles and Methods (Pharmacia, Uppsala, Sweden). In l mM EGTA, pH 6.8, with a Dounce homogenizer, and centrifuged at 40,000 brief, CNBr-activated Sepharose 4B (Sigma Chemical Co.) was swollen for 15 rpm in a Beckman Ti 75 rotor (Beckman Instruments Inc., Palo Alto, CA) for min in l mM HCI and then washed with coupling buffer (0. l M NaHCO3, pH 30 min in the cold. Microtubule proteins were obtained through one cycle of 8.3, 0.5 M NaCl). The gel suspension was immediately transferred (for covalent assembly and disassembly (25, 26). The resulting superuatant containing the coupling) to a solution of purified antigen (~ l mg/ml), dissolved in coupling disassembled microtubules was then removed, and taxol (from the National buffer containing 0.1% SDS, and mixed in an end-over-end rotator overnight Cancer Institute) was added to a final concentration of 20 ~M in the presence at 4°C. Active groups remaining on the gel were then blocked by incubation of I mM GTP. The solution was incubated for 30 min at 37"C and centrifuged with 0.2 M glycine, pH 8.0, overnight at 4"C. Nonspecifically adsorbed protein at 40,000 rpm in a Beckman Ti 75 rotor at room temperature for 30 min. The was eluted from the gel suspension by 0. l M acetate buffer, pH 4, containing MAPs were removed from the resulting pellet by washing the pellet with 0.3 M 0.5 M NaCl and then rinsed extensively with coupling buffer.