Cellular distribution of a protein related to neuronal microtubule-associated protein MAP-2 in Leydig cells

A monoclonal antibody to neuronal microtubule-associated protein MAP-2 was produced. Immunoblotting of lysates of cultured cells revealed that the antibody, called MA-01, bound to a protein of Mr 210 kDa. Double immunofluorescence microscopy showed that the antibody stained microtubules. No fibrillar structures were observed in cells treated with Colcemid, but the antibody stained vinblastine paracrystals. In cytochalasin B-treated Leydig cells, MA-01 antibody stained star-like structures that codistributed with actin patches and with a star-like arrangement of vimentin. These observations indicate that the protein immunologically related to MAP-2 in Leydig cells could be involved in the interaction of microtubules with intermediate filaments or microfilaments.     

Reorganization of a novel vimentin-associated protein in 3T3-L1 cells during adipose conversion

We have found that the antibody A2, a marker for the capsule of steroidogenic lipid droplets, reacts with an intermediate filament-associated protein, P₂₀₀, in 3T3-L1 preadipocytes. Supporting evidence came from the colocalization pattern of P₂₀₀ with vimentin in double label experiments. The association of P₂₀₀ with vimentin was further confirmed by its copurification with vimentin after high salt extraction and colocalization of these two proteins in high salt-extracted and vinblastine-treated cells. In preadipocytes this protein was distributed on the vimentin filament network. At the early stage of adipose conversion, this protein was found to encircle nascent lipid droplets ranging from 0.1 to 0.2 μm, accompanied with a decreased distribution on the vimentin filament system. This infers a possible translocation of P₂₀₀ from the vimentin filaments to the droplet surface. Meanwhile, the vimentin filaments remained in a normal distribution in the cytoplasm and were apparently not associated with the nascent droplet. The association of vimentin filaments to droplet surfaces became prominent in lipid droplets larger than 0.2 μm, forming a typical vimentin cage. Immunogold staining also confirmed the translocation of P₂₀₀ immunoreactivity from the droplet surface to the vimentin cage. The relocation of P₂₀₀ from the cytoplasmic vimentin filaments to the droplet surface prior to the formation of the vimentin cage, as well as the reorganization of this protein in the vimentin cage, suggests a stabilizing role in the lipid droplet formation and an inducing function of this protein in the formation of the vimentin cage. J. Cell. Biochem. 67:84–91, 1997. © 1997 Wiley-Liss, Inc.     

Epinemin: a new protein associated with vimentin filaments in non-neural cells

In this report I describe a new protein, defined by a monoclonal antibody, which is associated with vimentin filaments in a variety of cultured cells and in skeletal muscle. By immunofluorescence it is absent in smooth muscle, in cells without vimentin, and in neural vimentin containing cells. This protein has a molecular weight of 44,500, a pl of 5, a two-dimensional tryptic peptide fingerprint pattern different from vimentin, is unrelated to actin by Cleveland peptide analysis and by light and electron microscopy, and is not recognized by either a polyclonal antivimentin antibody (Frank, E.D., and L. Warren, 1981, Proc. Natl. Acad. Sci. USA, 78:3020-3024) or a monoclonal antibody against all classes of intermediate filaments (Pruss, R.M., R. Mirsky, M.C. Raff, R. Thorpe, A.J. Dowding, and B.H. Anderton, 1981, Cell, 27:419-428). The protein is resistant to nonionic detergent extraction, is soluble in high salt and can thus be removed from vimentin filaments, but fragments with vimentin in either low salt or anionic detergent and collapses with vimentin in colchicine-treated cells. By light microscopy, the distribution of the protein is indistinguishable from vimentin filaments and appears uniform along them. In contrast, immunoferritin electron microscopy reveals that the molecule is distributed in an intermittent pattern on vimentin filaments. Adopting the terminology of Granger and Lazarides (1980, Cell, 30:263-275), the molecule is called epinemin, meaning "upon filaments."     

A monoclonal antibody that detects vimentin-related proteins in invertebrates

Summary Drosophila melanogaster contains a 46 000 MW cytoplasmic protein which is immunologically related to the intermediate filament protein vimentin of vertebrates. A monoclonal antibody raised against this protein was used to study its cross-reactivity with other vertebrate and invertebrate cells. Indirect immunofluorescence showed filamentous meshworks in all species tested. Protein blotting was used to determine the molecular weights of the proteins responsible for the wide range of cross-reactivity of this antibody. We present evidence that vimentin-like proteins are also present in invertebrates and form a cytoplasmic network in Paramecium. Furthermore, we demonstrate in vertebrates and invertebrates the presence of high molecular weight polypeptides which are immunologically related to vimentin.     

Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.     

Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.     

Intermediate-sized filaments in Drosophila tissue culture cells

In using a monoclonal antibody against a major cytoplasmic protein of 46,000 mol wt, we have characterized an intermediate-sized (10 nm) filamentous cytoskeleton in Drosophila melanogaster tissue culture cells. Indirect immunofluorescence, immunoelectron microscopy, and protein blotting show that this cytoskeleton exhibits features typical of the vertebrate vimentin cytoskeleton, including the diameter and appearance of filaments, sensitivity to 10(-6) M colcemid, and insolubility in buffers containing 1% Triton X-100. The antibody cross- reacts with vimentin and desmin from baby hamster kidney cells and stains a vimentin cytoskeleton in the vertebrate Chinese hamster ovary cell line. We, therefore, conclude that the 46,000-mol wt Drosophila protein is homologous to vertebrate vimentin. Three minor, higher- molecular-weight polypeptides are also detected in the Drosophila cells that react with the antibody. At least two of these are members of a family of proteins with properties resembling those of the 46,000-mol wt intermediate filament protein.     

Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha- internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.     

On the relation between distinct components of the cytoskeleton: an epitope shared by intermediate filaments, microfilaments and cytoplasmic foci.

Monoclonal antibodies were generated against detergent-insoluble cytoskeletal proteins isolated from low-density membrane fractions of rat liver. By immunofluorescence, one of the antibodies stains three distinct structures in cultured rat fibroblast and hepatocyte lines as well as the PtK2 rat-kangaroo kidney epithelial line. These structures are: i) many tangled filaments similar to intermediate filaments (IFs), ii) fewer and variable numbers of straight filaments, and iii) punctate cytoplasmic foci, often most intense around the nucleus. All three of these structures are resistant to extraction by non-ionic detergent. Close examination reveals that the tangled and straight filaments are not stained uniformly, but as a series of bright patches. In cells treated with nocodazole, the antibody reacts strongly with a perinuclear filamentous cage. Very few tangled filaments are detected in these cells, however, the straight filaments and punctate cytoplasmic staining are resistant to nocodazole treatment. Double-label immunofluorescence shows that, even though tangled filament distribution and nocodazole sensitivity are similar to the behavior of vimentin IFs, there is only partial coincidence of staining with either vimentin or cytokeratin IFs. The straight filaments coincide with some actin stress fibers, but the punctate cytoplasmic staining is not related to IFs, actin, or tubulin. Thus, this monoclonal antibody stains a novel group of three seemingly unrelated cytoskeletal structures, including a previously undescribed insoluble nonfilamentous pool. Taken as a whole, two hypotheses are consistent with these data. i) The antigen recognized may be a protein which has a large insoluble cytoplasmic pool and binds both IFs and actin, but only binds to a subset of each class of filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2(+)-calmodulin-dependent protein kinase II phosphorylates various types of non-epithelial intermediate filament proteins

We have investigated the actions of Ca2(+)-calmodulin (CaM)-dependent protein kinase II on various types of non-epithelial intermediate filament proteins, vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament triplet proteins. Most of these filament proteins could serve as substrates. The effects of phosphorylation on the filamentous structure of vimentin were investigated in sedimentation experiments and by using electron microscopy. The amount of unassembled vimentin increased linearly with increased phosphorylation. However, the extent of the effect of phosphorylation on the potential to polymerize was also affected by the MgCl2 concentration, under conditions for reassembly. The actions of Ca2(+)-CaM-dependent protein kinase II on non-epithelial intermediate filaments under physiological conditions are given attention.     

The 2G2 Antibody Recognizes an Acidic 110-kDa Human Mitochondrial Protein

Using fluorescence microscopy, the mouse monoclonal antibody 2G2 was found to label mitochondria in human cells, as assessed by double staining with either Rhodamine 123 or a polyclonal antibody to mitochondrial matrix HSP-60 proteins. No reactivity to the 2G2 antibody was detected in cells from mouse, rat and chicken. Immunoblotting analysis demonstrated that the 2G2 antigen corresponds to a human protein with a relative mobility of 110 kDa and an approximate isoelectric point of 6.5 that co-partitions with HSP-60 proteins during isolation of mitochondria from HeLa cells. Close examination of the 2G2 staining pattern in HeLa and Fanconi's anaemia cells revealed differences in the morphology and organization of mitochondria in these two cell types. In HeLa cells, mitochondria appear as individual tubular compartments of variable length and are closely associated with vimentin filaments, particularly at the periphery of the nucleus. In Fanconi's anaemia cells, mitochondria have a filamentous shape and form an interconnected cytoplasmic reticulum running in parallel with both vimentin filaments and microtubules. After stabilization with aldehyde- or alcohol-based fixation protocols that optimize the preservation of cytoskeletal components, the epitope targeted by the 2G2 antibody may serve as a valuable marker in the investigation of relationships between mitochondria and other cellular structures in human cells.     

Vimentin filaments in spreading,randomly locomoting,and f-met-leu-phe-treated neutrophils

Summary Human neutrophils contain intermediate filaments of the vimentin type. A cytoskeletal preparation, produced by high-salt and Triton X-100 extraction of human neutrophils, reveals a major band at 57000 M _r that comigrates with 3T3 cell vimentin on one-dimensional gels. Two-dimensional gel electrophoresis of whole neutrophils illustrates the presence of vimentin but not desminor keratin-filament subunits. The presence of vimentin in neutrophils is also shown by its specific staining with avian vimentin antiserum by two-dimensional gel immunoautoradiography. Indirect immunofluorescence studies show that vimentin antiserum labels an area on one side of the nucleus in spreading neutrophils. This bright area appears as a loose knot of vimentin filaments; a few filaments may radiate from the knot. In contrast to spreading neutrophils, those undergoing random locomotion contain a fine network of filaments that are located in the cytoplasm between the nucleus and the trailing end of the cell. Similarly, in chemoattractant-treated neutrophils, vimentin filaments are bundled in the uropod. Transmission electron microscopy of human neutrophil monolayers confirms the intracellular distribution of intermediate filaments as shown by immunofluorescence in spreading and randomly locomoting cells.     

Discrimination between the nuclear lamin and intermediate filament (cytokeratin/vimentin) proteins of rat hepatic tumor cells by differential solubility and electrophoretic criteria

1. The major proteins which comprise the high salt/detergent-insoluble cytoskeletal matrix of rat hepatic tumor cells containing abnormal (Mallory body-like) aggregates of intermediate filaments were distinguished on the basis of electrophoretic mobility and differential solubility. 2. Gel electrophoresis of the intermediate filament-enriched cytoskeletal fraction of Mallory body hepatic tumor cells revealed the presence of: (a) intermediate filament proteins typical of cultured liver epithelial cells (cytokeratins A and D, vimentin), (b) some residual actin and, (c) two peptides of Mr = 68,000-72,000. 3. Analysis of the products of filament disassembly/reassembly mixtures indicated that the two Mr = 68,000-72,000 peptide species had the solubility characteristics of nuclear lamins. 4. The presence of nuclear lamin proteins in the high salt/detergent-resistant fraction of cultured liver cells was consistent with the resolution of residual nuclear-like structures in extracted cell monolayers. 5. Thus, while cytokeratin/vimentin-class intermediate filament proteins and nuclear lamins co-isolate from rat liver cells under conditions of high salt/detergent extraction, these two types of cytoskeletal proteins could be distinguished on the basis of their differential solubility and molecular weight.     

The VacA toxin of Helicobacter pylori identifies a new intermediate filament-interacting protein

The VacA toxin produced by Helicobacter pylori acts inside cells and induces the formation of vacuoles arising from late endosomal/lysosomal compartments. Using VacA as bait in a yeast two-hybrid screening of a HeLa cell library, we have identified a novel protein of 54 kDa (VIP54), which interacts specifically with VacA, as indicated by co-immunoprecipitation and binding experiments. VIP54 is expressed in cultured cells and many tissues, with higher expression in the brain, muscle, kidney and liver. Confocal immunofluorescence microscopy with anti-VIP54 affinity- purified antibodies shows a fibrous pattern typical of intermediate filaments. Double label immunofluorescence performed on various cell lines with antibodies specific to different intermediate filament proteins revealed that VIP54 largely co-distributes with vimentin. In contrast to known intermediate filament proteins, VIP54 is predicted to contain approximately 50% of helical segments, but no extended coiled-coil regions. The possible involvement of this novel protein in interactions between intermediate filaments and late endosomal compartments is discussed.     

Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.     

Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm.

The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells.     

Differential sensitivity of vimentin and nuclear lamins from Ehrlich ascites tumor cells toward Ca2+ -activated neutral thiol proteinase

A comparative study of the susceptibility of vimentin and nuclear lamins from cultured Ehrlich ascites tumor (EAT) cells to degradation by Ca2+ -activated neutral thiol proteinase (calpain) has been undertaken. While pure vimentin was degraded very quickly at physiological ionic strength by purified calpain, isolated lamin B was digested comparatively slowly and purified lamins A/C were fairly resistant to proteolytic degradation. Similar digestion patterns were obtained from vimentin and lamin B with intermediary breakdown products close in size to the corresponding alpha-helical rod domains. To exclude the possibility that the low susceptibility of isolated lamins to Ca2+-dependent proteolytic degradation was due to irreversible denaturation during their isolation and purification, Triton cytoskeletons were prepared and their nuclear lamina as well as vimentin filaments were exposed to relatively large quantities of purified calpain. Under these conditions, not only vimentin filaments but also lamins A and B were digested while lamin C remained intact to a high degree. The major breakdown products of vimentin and lamins were identified as polypeptides which were 35 to 45 amino acids longer than the corresponding alpha-helical rod domains. Most of the vimentin-derived material and all high molecular weight polypeptides originating from lamins remained associated with the Triton cytoskeletons as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with immunoblotting. Indirect immunofluorescence and electron microscope analysis of the calpain-digested Triton cytoskeletons revealed that they still contained a laminalike structure around the nuclear chromatin and numerous structurally altered intermediate filaments in the cytoplasmic remnant, although all vimentin had been degraded with the formation of 40/41 kDa polypeptides as major digestion products. In untreated Triton cytoskeletons, the vimentin filaments seemed to be in direct physical contact with the nuclear lamina, whereas in digested Triton cytoskeletons there was a distinct gap between structurally altered filaments and the nuclear surface. This shows that vimentin filaments and the nuclear lamina are differentially susceptible to degradation by calpain under certain ionic conditions and suggests that both filamentous structures are intimately associated with each other.(ABSTRACT TRUNCATED AT 400 WORDS)     

Semiquantitative Postembedding Characterization of Intermediate Filaments in Central Nervous System Lesions Using Immunoelectron Microscopy

Standardized postembedding immunoelectron microscopy was performed to demonstrate glial fibrillary acidic protein (GFAP) and vimentin in individual intermediate filaments to determine the diagnostic value of demonstrating ultrastructural and immunophenotypic characteristics of intermediate filaments in routine brain biopsy specimens. Dual expression of GFAP and vimentin was observed in the astroblastoma and astrocytes of Alexander's disease. The antigen availability for vimentin, however, was too low to allow reliable assessment of the GFAP:vimentin ratio in individual intermediate filaments and/or filament bundles. In meningioma, only vimentin positive intermediate filaments were found. GFAP positive intermediate filaments were present in all other specimens except the oligodendroglial components of the mixed glioma, which were devoid of intermediate filaments. GFAP positivity in the filamentous periphery and electron-dense core of Rosenthal fibers was demonstrated. Technical and tissue processing factors had a significant effect on particle density values obtained for individual specimens. Although the number, distribution, and density of glial intermediate filaments varies in different astroglial entities, correlation of particle density values determined by immunoelectron microscopy with relative GFAP concentrations in different lesions requires utmost caution. Nevertheless, application of the postembedding approach to routinely fixed biopsy specimens indicated an association of different entities with the exclusive presence of GFAP and/or vimentin in individual intermediate filaments, thus emphasizing the diagnostic value of intermediate filament typing for pathological characterization.     

Identification and characterization of a novel mammalian intermediate filament-associated protein

A novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule-associated protein MAP1A (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co-localizes with both vimentin and cytokeratin filaments using double-label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two-dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non-ionic detergent/high-salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament-associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double-label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV-1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament-associated protein for which we have proposed the designation gyronemin (Gr. gyros: around; nemin: filament).     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.