Purification of the 300K intermediate filament-associated protein and its in vitro recombination with intermediate filaments

IFAP-300K is a 300,000-mol-wt intermediate filament-associated protein previously identified in the baby hamster kidney fibroblastic cell line (BHK-21) by a monoclonal antibody (Yang H.-Y., N. Lieska, A. E. Goldman, and R. D. Goldman, 1985, J. Cell Biol., 100: 620-631). In the present study, this molecule was purified from the high salt/detergent-insoluble cytoskeletal preparation of these cells. Gel filtration on Sephacryl S-400 in the presence of 7.2 M urea allowed separation of the high molecular weight fraction from the structural intermediate filament (IF) subunits desmin and vimentin, designated 54K and 55K, respectively, and other low molecular weight polypeptides. DE-52 cellulose chromatography of the high molecular weight fraction using a linear NaCl gradient in 8 M urea yielded a pure 300,000-mol-wt species which was confirmed to be IFAP-300K by immunological and peptide mapping criteria. Two-dimensional PAGE of native BHK IF preparations followed by immunoblot analysis demonstrated the inability of the IFAP-300K-immunoreactive material to enter the first dimensional gel except as a 200,000-mol-wt doublet which presumably represented a major proteolytic derivative of IFAP-300K. The molecule's pl of 5.35, as determined by chromatofocusing, and its amino acid composition were extremely similar to those of BHK cell vimentin/desmin despite their non-identity. Ultrastructurally, IFAP-300K preparations in low salt buffers existed as particles composed of one or two elliptical units measuring 16 X 20 nm. In physiological salt buffers, the predominant entities were large, elongated aggregates of the elliptical units, which were able to be decorated by using the immunogold technique with monoclonal anti-IFAP-300K. Compared with the morphology of homopolymer vimentin IF, in vitro recombination studies using column-purified vimentin and IFAP-300K demonstrated the additional presence of aggregates similar in appearance to IFAP-300K at points of contact between IFs. Antibody decoration and immunogold labeling of these recombined preparations using rabbit antidesmin/vimentin and monoclonal anti-IFAP-300K confirmed the identity of the inter-filament, amorphous material as IFAP-300K. The presence of IFAP-300K at many points of intersection and lateral contact between IFs, as well as at apparent inter-filament "bridges," in these recombined specimens was identical to that seen both in situ and in native IF preparations. No such co-sedimentation was found in vitro between actin and IFAP-300K. No effects of IFAP-300K upon the kinetics of IF polymerization were detected by turbidimetric measurements.     

Human epithelial cell intermediate filaments: isolation, purification, and characterization

Intermediate filaments (IF) isolated from human epithelial cells (HeLa) can be disassembled in 8 M urea and reassembled in phosphate-buffered solutions containing greater than 0.1 mg/ml IF protein. Eight proteins were associated with HeLa IF after several disassembly-reassembly cycles as determined by sodium dodecyl sulfate gel electrophoresis (SDS PAGE). A rabbit antiserum directed against HeLa IF contained antibodies to most of these proteins. The immunofluorescence pattern that was seen in HeLa cells with this antiserum is complex. It consisted of a juxtanuclear accumulation of IF protein and a weblike array of cytoplasmic fibers extending to the cell border. Following preadsorption with individual HeLa IF proteins, the immunofluorescence pattern in HeLa cells was altered to suggest the presence of at least two distinct IF networks. The amino acid composition and alpha-helix content (approximately 38%) of HeLa IF proteins was similar to the values obtained for other IF proteins. One-dimensional peptide maps show extensive homology between the major HeLa IF protein of 55,000-mol- wt and a similar 55,000-mol-wt protein obtained from hamster fibroblasts (BHK-21). HeLa 55,000-mol-wt homopolymer IF assembled under conditions similar to those required for BHK-21 55,000-mol-wt homopolymers. Several other proteins present in HeLa IF preparations may be keratin-like structural proteins. The results obtained in these studies indicate that the major HeLa IF protein is the same major IF structural protein found in fibroblasts. Ultrastructural studies of HeLa cells revealed two distinct IF organizational stages including bundles and loose arrays. In addition, in vitro reconstituted HeLa IF also exhibited these two organizational states.     

Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology

Intermediate filaments (IF) isolated from a variety of cultured cells, conventionally described as fibroblasts, are composed predominantely of proteins of molecular weights of 54,000 and/or 55,000. Less than 15% of the protein found in native IF preparations from these cells is composed of three to four polypeptides of molecular weights 60,000- 70,000. We have investigated some biochemical and immunological properties of these proteins isolated from BHK-21 and mouse 3T3 cells. They are capable of forming paracrystals that exhibit a light/dark banding pattern when negatively stained with uranyl acetate. The dark bands are composed of longitudinally aligned approximately 2-nm-diam filaments. The center-to-center spacing between either dark or light bands is 37-40 nm. These dimensions are consistent with the secondary structure of IF polypeptides and suggest that the dark bands represent lateral alignment of alpha-helical coiled-coil domains. Immunoblotting, secondary structure, as well as amino acid composition data indicate that the 60,000-70,000-mol-wt paracrystal polypeptides are similar to keratin. Thus, polypeptides with biochemical and immunological properties of epidermal keratin are present in cells normally considered to be fibroblasts.     

The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.     

Expression of glial filament protein (GFP) in nerve sheaths and non-neural cells re-examined using monoclonal antibodies, with special emphasis on the co-expression of GFP and cytokeratins in epithelial cells of human salivary gland and pleomorphic adenomas

We describe two novel monoclonal antibodies specific for glial filament protein (GFP), i.e., GF12.23 and GF12.24 (both IgG2a]. These cross-react over a broad range of species with epitopes located in the alpha-helical rod domain typical of all intermediate filament (IF) proteins. These monoclonal antibodies were used, in conjunction with other monoclonal GFP antibodies, rabbit antiserum to GFP, and various antibodies to other cytoskeletal proteins, to examine the occurrence of GFP in cells outside of the central nervous system of rodents, cows, and humans. We detected some scattered GFP-containing cells in the neural sheaths in some species but not in others, and we obtained different results when comparing the rabbit antisera with the monoclonal GFP antibodies. In the enteric glia of rats, we observed GFP-positive cells with all of the antibodies used, whereas in human intestine, the various monoclonal antibodies showed no reaction with any intestinal cells. Similarly, no GFP was detected in surface cells of the lens of cows and rats using any of the GFP antibodies, whereas some reaction was seen in murine lens tissue. We were also unable to detect GFP-positive cells in human, bovine, or rat liver with any of the monoclonal antibodies, which is in contrast to the reactivity of the rabbit GFP antisera with some stellate perisinusoidal cells of rat but not bovine or human liver. The possible reasons for the discrepancies between the different species and the different antibody preparations used are discussed. In addition, using double-label immunofluorescence microscopy, we showed that normal human parotid glands contain a certain type of epithelial cell that co-expresses cytokeratins and desmosomal proteins with GFP. The histological distribution of these GFP-positive cells suggests that they represent a subset of the myoepithelial cells present in this tissue. Cells co-expressing cytokeratins and GFP - in some cases, apparently together with vimentin as the third IF protein present - were also identified in tumors derived from this salivary-gland epithelium, i.e., pleomorphic adenomas, in which GFP-positive cells were relatively frequent in the myxoid and chondroid components, thus confirming the work of other investigators. Possible implications for the concept of histogenesis of these tumor cells are discussed, as are possible mechanisms resulting in the co-expression of IF proteins.     

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.     

The detection of smooth muscle desmin-like protein in BHK21/C13 fibroblasts.

Three distinct proteins, actin (42,000 daltons), the principal form of fibroblast 10 nm filament protein (55,000 daltons), and a protein with a molecular weight of 52,000 and a pI of 5.8 were detected in nonionic detergent-insoluble cytoskeletal and 10 nm filament preparations of control BHK21/C13 and line 9 hamster fibroblasts. Cytoskeletal preparations of other hamster fibroblast cell types, such as NIL8 and primary embryo fibroblasts, contained the 55,000-dalton component but lacked the 52,000-dalton protein. A Rous sarcoma virus transformant of the BHK21/C13 line and an adenovirus transformant of line 9, resembled the NIL8 and other fibroblast types in that they contained only the 55,000- and 42,000-dalton polypeptides. The identity of the 52,000-dalton protein in BHK21/C13 cells was studied. This protein co-electrophoreses on both one- and two-dimensional polyacrylamide gels with the predominant muscle form of 10 nm filament protein. Further, one-dimensional peptide maps of the hamster smooth muscle 10 nm filament protein and the hamster fibroblast 52,000-dalton protein are identical to one another and distinct from the peptide maps of both the 42,000- and the 55,000-dalton components of the fibroblast cytoskeletal preparations. We conclude that BHK21/C13 cells contain both the fibroblast and the muscle form of 10 nm filament protein.     

Lamins A, B and C share an epitope with the common domain of intermediate filament proteins

Peripheral lamina were isolated from rat liver nuclei and probed with a monoclonal antibody reactive against a common domain of all intermediate filament (IF) proteins. The antibody reacted against lamins A and C as expected from their known sequence homology with IF proteins. Lamin B was also recognized by the antibody. These results indicate that lamin B, despite important biochemical differences vs lamins A and C, also shares sequence homology with IF proteins.     

Cell-substratum adhesion in embryonic chick central nervous system is mediated by a 170,000-mol-wt neural-specific polypeptide

Embryonal chick neural retina cells release into the culture medium a complex of proteins and glycosaminoglycans, termed adherons, that promote cell to substratum adhesion. A monoclonal antibody (C1H3) blocks adheron-mediated cell to substratum adhesion and specifically binds to a 170,000-mol-wt protein present in retinal adherons (Cole, G.J., and L. Glaser, 1984, J. Biol. Chem., 259:4031-4034). The 170,000-mol-wt protein also can be identified in embryonic chick brain and peripheral nervous tissue. In the neural retina, C1H3 also binds to a second antigen with a molecular weight of 140,000 that is absent in the brain. Embryonic brain, therefore, provides a source for the immunopurification of the 170,000-mol-wt protein. Brain adherons also contain the 170,000-mol-wt protein, and cell to substratum adhesion mediated by these adherons is blocked by the C1H3 monoclonal antibody. The 170,000-mol-wt protein in the brain is therefore functionally identical to that in the retina. To demonstrate that adheron-mediated cell to substratum adhesion is caused by cell binding to the 170,000-mol-wt protein, we showed that (a) protease digestion, but not glycosaminoglycan hydrolase digestion of adherons, blocked their ability to bind cells to substratum; (b) the immunopurified 170,000-mol-wt protein blocks adheron-mediated cell to substratum adhesion; and (c) cells can bind to immunopurified 170,000-mol-wt protein bound to glass surfaces.     

A re-evaluation of cytoplasmic gelsolin localization

Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large-scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co-localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS)