Classes Of Intermediate Filaments Research Articles

Intermediate filaments in the inner ear of normal and experimentally damaged guinea pigs

The hypothesis that proteins known to occur in glial cells in the central nervous system may be present in inner-ear supporting cells was investigated. Immunocytochemical techniques were used to look for the existence of two classes of intermediate filaments, vimentin and glial fibrillary acidic protein (GFAP), in cellular elements of the inner-ear epithelium in normal and experimentally damaged guinea-pig cochleas. Vimentin is present in two types of supporting cells in the normal organ of Corti: Deiters' cells and inner pillar cells. Differences in intensity and distribution of vimentin immunostaining are observed across the three rows of Deiters' cells. GFAP immunoreactivity was not detected in any supporting-cell type in the organ. Cochlear hair cells were not labeled for either GFAP or vimentin. Following hair-cell destruction by exposure to noise or the administration of aminoglycosides, GFAP and vimentin are not present in phalangeal scars replacing lost hair cells.

Effect of aluminum and other multivalent cations on neurofilaments in vitro: An electron microscopic study

Using electron microscopy (EM) of negatively stained samples, we have systematically explored the effect of aluminum and other multivalent cations on neurofilaments (NFs) in vitro. Interactions of these cations were investigated with bovine, rabbit, and rat spinal cord native NFs, and with 10-nm filaments reconstituted from the 68-kDa subunit (NF-L) isolated from bovine spinal cord. Our results indicated that, as has been observed with other classes of intermediate filaments (IFs), all multivalent cations caused significant aggregation of native NFs, suggesting that this phenomenon is a rather general one and not limited to aluminum. In addition, all cations tried caused significant lateral aggregation of filaments reconstituted from NF-L. Aluminum lactate had an identical effect on bovine, rabbit, and rat NFs. Because aluminum causes strong aggregation of NFs in vitro, a similar phenomenon may occur in vivo leading to the observed accumulation of NFs in neuronal perikarya of rabbits after intoxication with aluminum. These in vitro observations support the concept that some human neurological diseases characterized by the accumulation of NFs may be related to abnormal levels of multivalent cations.

Human lung tumours may coexpress different classes of intermediate filaments

Human lung tumours may coexpress different classes of intermediate filaments

Coexistence of cytokeratin, vimentin and neurofilament protein in human choroid plexus

The expression of intermediate filament proteins in human brain ependyma and choroid plexus epithelium has been studied by immunohistochemistry using a panel of monoclonal antibodies directed against all five classes of intermediate filaments. Ependymal cells express GFAP and vimentin filaments, whereas plexus epithelium simultaneously contains neurofilaments, cytokeratins and vimentin, a phenomenon not previously observed in normal cells in vivo. By means of specific antibodies we were able to establish that cytokeratins 8 and 18 but not 19 are present in plexus epithelium.

Phosphorylation and dephosphorylation of neurofilament proteins in retinal ganglion cell neurons in vivo.

The cytoskeleton of most higher eukaryotic cells is composed of three filamentous systems, which serve varying dynamic and structural roles in cellular function. Two of these systems, the 80 A microfilaments and the 230 A microtubules, are composed of subunit proteins that are phylogenetically highly conserved. By contrast, the 100 A intermediate filaments are encoded by a large multigene family, the members of which are differentially expressed in different tissues (Fuchs and Hanukoglu, 1983). On the basis of biochemical and immunological criteria, five major classes of intermediate filaments have been defined (Lazarides, 1980). These include keratin filaments, found in cells of epithelial origin; desmin filaments, predominantly found in smooth, skeletal and cardiac muscle cells; vimentin filaments, present in cells of mesenchymal origin; glial filaments, constituents of certain glial cell types; and neurofilaments, present in many differentiated neurons of vertebrates and invertebrates. This classification emphasizes the tissue specificity of intermediate filaments, although it is now known that subunits from more than one class may coexist in some tissues at certain developmental stages (Osborn et al., 1980; Drager, 1983).

Human lung tumours may coexpress different classes of intermediate filaments.

Ninety four pulmonary neoplasms were examined immunocytochemically with two or three different monoclonal antibodies against the intermediate filament proteins cytokeratin, neurofilament, vimentin, and desmin. In normal tissues these have a different and non-overlapping distribution, and it is generally believed that tumours maintain the same pattern of expression as the tissues from which they arise. In this report, however, the coexpression of at least two (and less commonly three or four) different intermediate filaments was seen in 40% (37 of 94) of the cases of lung cancer. These results, especially if confirmed in other common types of human malignancy, have considerable implications for the use of anti-intermediate filament antibodies in diagnostic pathology.

Idiotypic analysis of a B cell clone with anti-intermediate filament specificity in a patient with Sjögren's syndrome: involvement of five subpopulations producing different immunoglobulin isotypes.

An IgM paraprotein from patient LP with Sjögren's syndrome exhibited an antibody activity to intermediate filaments (IMF) of cells from all vertebrates examined, and appeared to recognize several classes of IMF (i.e., vimentin, desmin, and keratin). A mouse monoclonal anti-idiotype (Id) antibody, K4A, was prepared against the IgMk (LP) and used as a specific probe in two-color immunofluorescence to examine the extent of clonal involvement in the patient's blood and bone marrow mononuclear cells (MNC). Twenty to 30% of MNC in her blood samples were IgMk+ plasmablasts with morphologic similarity to Waldenström's macroglobulinemia cells. IgG+ and IgA+ plasmablasts were demonstrated in lower frequencies (approximately 2%). Almost all of the IgM+ cells and approximately 80% of the IgG+ cells and IgA+ cells in the blood were reactive with the K4A anti-Id antibody. Immunoglobulin (Ig) subclass analysis revealed that the K4A Id was expressed by IgG1+, IgG3+, IgA1+ and IgA2+ plasmablasts. Similar observations were obtained with bone marrow samples, although the proportion of Id+ cells among IgG+ or IgA+ cells was lower in marrow than in blood. IgG and IgA fractions isolated from the patient's serum were also shown to contain anti-IMF activity. Ig biosynthetic analysis of blood MNC revealed that the K4A anti-Id antibody precipitated not only IgM but also IgG and IgA. Because cells simultaneously producing two different Ig isotypes were not detected, these results indicate the presence of five separate subpopulations of the K4A Id+ neoplastic clone. The data thus suggest the occurrence of a neoplastic or pre-neoplastic transformation event before the switching of Ig heavy chain isotypes, and imply a role for the IMF antigen in the exaggerated proliferation and differentiation along five of the nine potential intraclonal pathways.

Cell shape and motility of oligodendrocytes cultured without neurons.

Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), were cultured from newborn rat brain and optic nerve to study how they differentiate in vitro in the absence of neurons. By use of galactocerebroside (GC) as a reference marker, the development of the cell phenotype was studied with video-enhanced differential interference contrast microscopy, immunofluorescence and electron microscopy. After a few days in culture, oligodendrocytes extend 5 to 10 main processes that are very rich in microtubules, but they did not stain with a monoclonal antibody reacting with all known classes of intermediate filaments. The number of processes can vary with the substrate on which the cells are grown; fewer processes form on laminin than on polylysine coated glass. Oligodendrocytes, in a fashion similar to that of neurons appear to keep their body immobile while the long processes grow. However, while neurons display motile activities mostly at the end of the cell processes called growth cones, the oligodendrocytes display motile, actin rich filopodia and lamellipodia along the entire length of all processes. The outgrowth of motile processes from oligodendrocytes sometimes occurs preferentially towards neighboring astrocytes. Oligodendrocyte processes display intense bidirectional movement of cytoplasmic organelles. Movement of surface components also occurs since GC molecules cross-linked by antibodies move from the processes towards the cell body. Thus, oligodendrocytes cultured without neurons develop on schedule a complex phenotype similar to their in vivo counterpart. In addition, their processes are capable of specific motile activities which may function in vivo to find the target axon and to transport myelin membrane components at the site of myelin assembly.

Changes in Some Myelin Protein Markers and in Cytoskeletal Components During Wallerian Degeneration of Mouse Sciatic Nerve

After transection of the mouse sciatic nerve, the sequence of events occurring in the distal degenerating segment was followed by the biochemical changes related to the cytoskeletal components and to the myelin protein markers. The components of the intermediate filaments and of the microtubules undergo early changes. Within 3 days, the neurofilament triplet and the peripherin disappear whereas many peptides bearing the antigenic determinant common to all classes of intermediate filaments accumulate. Several of them persist after 1 month. The tubulin pattern changes from a high level of microheterogeneity--reflecting mostly the axonal contribution--to a lower level displayed by the predominant Schwann cells. A decrease in the amount of the myelin markers is also observed. However, a month after transection, immunoreactive basic protein is still present in the degenerated segment homogenate.

Differentiation of NTERA-2 clonal human embryonal carcinoma cells into neurons involves the induction of all three neurofilament proteins

Monoclonal antibodies were used in indirect immunofluorescence and immunoblot studies to examine the expression of four different classes of intermediate filaments, namely, neurofilaments, glial filaments, cytokeratin, and vimentin, in NTERA-2 cl.D1 (NT2/D1) pluripotent human embryonal carcinoma (EC) cells, and in the neurons derived from these cells by differentiation induced with retinoic acid. In the EC cell cultures, grown in the absence of retinoic acid, cytokeratin was the predominant intermediate filament detected by immunofluorescence; only a few cells expressed vimentin, and none expressed glial filament protein or any of the three neurofilament proteins (NF195, NF170, and NF70). Immunoblot analyses of cytoskeletal extracts of these cells supported these data. Two days after exposure to retinoic acid, all three neurofilament subunits were detected in a few cells with a non-neuronal morphology and, by double indirect immunofluorescence, were observed to colocalize with cytokeratin. The number of neurofilament-positive cells increased with time after initial exposure to retinoic acid, and although 95% of these cells contained cytokeratin initially, less than 5% of the neurofilament-positive cells retained cytokeratin 2 weeks later. By this time, many of the cells expressing all three neurofilaments but no cytokeratin exhibited a neuronal morphology. Vimentin was evident in a large number of cells in the cultures, but it was not detected in the neurofilament-positive cells. Also, many of the neurofilament-negative cells continued to express cytokeratin. No cells expressing glial filament proteins were found. Immunoblot analysis of the differentiated cultures also revealed all three neurofilament subunits, and vimentin and cytokeratin, but no glial filament protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Intermediate filaments in the newborn inner ear of the mouse.

The presence of intermediate filaments in the inner ear of the newborn mouse was analyzed with immunofluorescence techniques using antibodies against the five classes of intermediate filaments: cytokeratins, vimentin, desmin, neurofilaments and glial fibrillary acid protein (GFA). Neurofilaments were found in all nerve fibers from the ganglion cell to the hair cell. In the vestibular ganglion two subpopulations of ganglion cells were identified: a minor part staining intensively with neurofilament and the major part of cells lacking this immunofluorescence. Vimentin occurred in a number of supporting structures in the membranous labyrinth, but not in vestibular or cochlear ganglion cells. Cytokeratins, desmin or GFA were not identified in the inner ear.

Keratin Type Intermediate Filaments in Sweat Gland Myoepithelial Cells

A study was undertaken to clarify the origin of sweat gland myoepithelial cells using monoclonal antibodies EKH1, EKH4, and AN3. EKH1 recognizes all classes of intermediate filaments. EKH4 and AN3 recognize keratin type intermediate filaments. Since within the skin, only epithelial cells of ectodermal origin contain keratin, EKH4 and AN3 could be used as ectodermal markers within the skin. Sweat gland myoepithelial cells were labeled by all three antibodies. In contrast, arrector pili muscle and vascular smooth muscle were recognized only by EKH1, but not by EKH4 and AN3. This study demonstrated that myoepithelial cells of sweat glands contain keratin type intermediate filaments and suggested their ectodermal origin. On the other hand, arrector pili muscle and vascular smooth muscle did not contain keratin type intermediate filaments, despite their ultrastructural similarity to myoepithelial cells. Electron microscopic studies using human fetal and adult skin revealed that myoepithelial cells are developed from basal cells of the coiled tip of fetal gland and not from mesenchymal cells. In order to determine the time of appearance of myoepithelial cells during fetal development, embryonic and newborn mouse skin was also examined. It was found that sweat gland myoepithelial cells first appear around 20 weeks of gestation in humans and after birth in mice.

Alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues.

In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.

Immunoelectron Microscopic Localization of Intermediate Filament Vimentin in Neuroblastoma Cells: Methods

The cytoskeleton of many cell types contains three prominent filamentous elements: intermediate filaments; actin-containing microfilaments and microtubules. Five antigenically distinct classes of intermediate filaments (IFs) have been identified on the basis of their protein subunit composition: keratin; desmin; glial fibrillary acidic protein; neurofilament triplet protein and vimentin. Although substantial progress in identifying and determining the functional roles of these filamentous proteins has been made with immunof1uorescent techniques, relatively little has been accomplished with immunoelectron microscopy. Previously, it has been difficult to simultaneously preserve ultrastructural detail, maintain antigenicity and ensure antibody penetration into dense cytoskeletal structures. The non-ionic detergent, Triton X-100 has been used, prior to or after fixation, to facilitate macromolecular access to deeply localized antigens. This extraction procedure maintains antigenicity as mirrored by immunofluorescence microscopy. The present study presents a reliable method that preserves dense filamentous cellular organelles and permits immunocytochemica1 localization of vimentin in cultured neuroblastoma cells.

INTERMEDIATE FILAMENTS IN CULTURED HUMAN PLEOMORPHIC ADENOMAS

Primary cultures of 9 benign human pleomorphic adenomas were analyzed by immunofluorescence and antibodies against the different intermediate filaments and microfilaments. The cultured cells were also cytogenetically characterized by G- and C-banding techniques. The adenoma cells expressed at least three different classes of intermediate filaments, viz. prekeratin, vimentin and glial fibrillary acidic protein (GFA). No correlation between chromosomal pattern and intermediate filament expression was found. The frequency of cells stained for each of the different antibodies varied considerably among the tumours. Prolonged culturing appeared to induce a reduction of cells positive for prekeratin. This was probably a true loss of antigens and not an effect of overgrowth of stromal cells. It is concluded that the cells studied, on the basis of several criteria, are of neoplastic origin. This in vitro system appears to be well suited for further histogenetic studies on pleomorphic adenomas.

Applications of monoclonal antibodies to neuroscience research.

The preceding discussion documents the diverse ways in which monoclonal antibodies have contributed to neuroscience research. They provide highly specific reagents to membrane-associated proteins, such as pumps, channels, receptors, and cell-adhesion molecules, that are useful for purifying these proteins, studying their structures at high resolution, and mapping their distributions. In many cases, the specific reagents were obtained using only partially purified antigens. Monoclonal antibodies to cytoskeletal proteins, organelles, and protein kinases have revealed that specific molecules are concentrated in anatomically distinct regions of the cell. A protein kinase has been shown to be a major postsynaptic constituent in many synapses. Individual proteins, such as actin, tubulin, and calmodulin appear to have different antigenic epitopes shielded in different parts of the cell. Monoclonal antibodies have provided a diversity of cell-type-specific reagents in both vertebrate and invertebrate nervous systems. They seem likely to be useful in identifying functionally related subpopulations of neurons and describing neural cell lineages. They will also serve to identify molecules that are important in regulating cell migration in the cerebellum, in marking cell position in the retina, and directing axon growth. This review also documents many purposes for which monoclonal antibodies are poorly suited or must be used with caution: A monoclonal antibody to a protein does not always reveal every place where that molecule is located. Pre- or post-translational microheterogeneity can expose different epitopes on the protein, such as may occur on the Na+-channel. Other proteins within the cell may shield antigenic sites on proteins such as calmodulin. Monoclonal antibodies can bind to epitopes on unrelated molecules (Nigg et al 1982, Lane & Koprowski 1982). This is revealed in some cases as multiple bands on immunoblots. Some cross-reactivity, however, may have a functional basis. For example, structural homology is clearly the basis for the antigenic epitopes that are shared among the five classes of intermediate filaments (Pruss et al 1981). The epitope that appears to be shared between the muscarinic and alpha 1-adrenergic receptors may be conserved because the two receptors modulate common effectors. The cross-reactivity between these receptors was only recognized because very specific and sensitive assays exist for each. It is quite possible that these same antibodies also bind sites on many other types of receptors. Mapping the distribution of this epitope may therefore have little relationship to the actual distribution of the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of frog virus 3 with the cytoskeleton.

The eukaryotic cell contains a detergent-insoluble structural framework termed the cytoskeleton (Brown etal. 1976; Lenk etal. 1977; Osborn and Weber 1977; Webster et al. 1978). The principal components of the cytoskeleton are three chemically and morphologically distinct filaments: the microtubules, the intermediate filaments, and the microfilaments. The three filaments have been well characterized both morphologically and biochemically. Microtubules are hollow tubes measuring 22–26 nm in diameter and each microtubule contains protofilaments which in turn are composed of heterodimers of tubulin (Olmsted and Borisy 1973). Intermediate filaments are wavy filaments measuring 7–11 nm in diameter (Lazarides 1981). At present, four classes of intermediate filaments have been identified (Lazaredes 1981). They are keratin filaments of epithelial cells, neurofilaments of neurons, glial filaments of cells of glial origin (e.g., astrocytes), and a class of intermediate filaments common to all cell types. Though morphologically similar, each class of intermediate filaments contains distinct proteins. Microfilaments measure 4–8 nm in diameter (Goldman et al. 1975) and contain subunits of actin (Groeschel-Stewart 1980).KeywordsIntermediate FilamentUninfected CellAssembly SiteVirus ReleaseBaby Hamster Kidney CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Glial fibrillary acidic protein (GFAP) immunoreactivity in peripheral nerve sheath tumors.

A spectrum of 24 benign and malignant nerve sheath tumors and 10 non-neural spindle-cell tumors were studied by light microscopy for the presence of glial fibrillary acidic protein (GFAP) immunoreactivity by the peroxidase-antiperoxidase (PAP) technique. In 8 cases, these results were compared to their electron microscopic appearances. Seventy percent (7 of 10) of benign schwannomas and 50% (4 of 8) of benign neurofibromas demonstrated focal to diffuse GFAP immunoreactivity. None of the malignant nerve sheath tumors nor any of the non-neural spindle-cell neoplasms contained demonstrable GFAP immunoreactivity. Similarly, no GFAP immunoreactivity could be detected in Schwann cells in normal peripheral nerves. The solitary benign schwannoma available for electron microscopic study demonstrated diffuse and abundant cytoplasmic intermediate filaments, and this tumor displayed diffuse and intense GFAP immunoreactivity. Two benign neurofibromas showed a more variable content of intermediate filaments ultrastructurally, and their GFAP immunoreactivity was variable. All five malignant nerve sheath tumors studied by electron microscopy displayed a variable complement of intermediate filaments; however, none of these tumors possessed GFAP immunoreactivity, suggesting that these intermediate filaments are either members of a different class of intermediate filaments or may perhaps represent "altered" GFAP not recognized by these antisera.

Monoclonal Antibodies to Gel‐Excised Glial Filament Protein and Their Reactivities with Other Intermediate Filament Proteins

A series of 14 monoclonal antibodies (MAs) has been obtained from a single rat-mouse fusion using gel-excised bovine glial filament (GF) proteins as immunogens. These MAs were characterized by two separate immunochemical assays and by two different immunohistochemical methods. Nine MAs demonstrated specificity for GF proteins. One MA also recognized an epitope shared by intermediate filaments (IF) of the vimentin class (VF). Using the enzyme-linked immunosorbent assay, four of the MAs recognized 200,000, 150,000, and 51,000 dalton proteins, suggesting that these MAs were specific for GF proteins (the 51,000 dalton protein) and neurofilament (NF) proteins (the two high-molecular-weight proteins). However, in both of the immunohistochemical assay systems, these MAs stained neurons and their processes but not astroglial cells. These observations strongly suggest that the 51,000-dalton protein recognized by these four MAs was not derived from GF proteins but instead represents derivatives of NF protein subunits comigrating in gels with GF proteins. These data provide additional information concerning the unique and shared antigenic determinants of the three classes of IF (NF, GF, and VF) of the CNS. In addition, they draw attention to the fact that proteins of certain IF may undergo degradation and comigrate in gels with the proteins of unrelated IF. This emphasizes the need for the use of independent immunochemical and immunohistochemical assays in the characterization of the specificity of MAs.

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."