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Structure of Vimentin 10-nm Filaments Probed with a Monoclonal Antibody That Recognizes a Common Antigenic Determinant on Vimentin and Tropomyosin
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Antibodies prepared to the constituent proteins of 10-nm filaments have revealed, by immunofluorescence, the organization and distribution of these filaments in many cell types (for review, see Lazarides 1980). These methods have further allowed observations of 10-nm filaments during dynamic cellular events such as myogenesis (Bennett et al. 1979; Lazarides 1981) and mitosis (Aubin et al. 1980; Blose 1979, 1981). In studying the structure of 10-nm filaments at the ultrastructural level, several investigators have demonstrated that monospecific polyclonal antibodies can decorate 10-nm filaments in a uniform manner (Schlaepfer 1977; Cabral et al. 1980; Henderson and Weber 1980; Heuser and Kirschner 1980). Willard and Simon (1981) have demonstrated that conventional antibodies to neurofilament components can decorate neurofilaments in a periodic or helical fashion. From these observations they have then been able to construct a molecular model of the neurofilament. We have employed a similar approach to understand the structure and molecular...
... A monoclonal antibody was produced by Pillemer and Weissman (20) that bound Thy-1 antigen on T lymphocytes and the idiotypic determinant of TEPC-15 myeloma protein. A number of studies reported the production of monoclonal antibodies that bound microfilaments or intermediate filaments as well as other antigens with no known structural homology (21)(22)(23)(24)(25). Reports of monoclonal antibodies displaying cross-reactions between viral proteins and a nuclear antigen (26) or other intracellular components (27) have recently appeared. ...
We report that approximately 1/4 of monoclonal rheumatoid factors produced by hybridomas derived from fusions of spleen cells from MRL/lpr/lpr mice with systemic lupus erythematosus (SLE) and arthritis exhibited multiple reactivities with other autoantigens, including dDNA , histones, and/or cytoskeletal-cytoplasmic elements. The patterns of reactivities of most of these clones differed, indicating that each had a separate B cell ancestor. Studies with eluted antibodies demonstrated that a single species of antibody molecules was responsible for the observed multiple reactivities. Inhibition experiments suggested that an antibody combining site may be large enough to accommodate dissimilar epitopes. These findings may provide further insights into the generation and extent of antibody diversity as well as the etiopathogenesis of systemic autoimmune diseases.
Monoclonal antibodies(McAbs) have been generated againsta preparationof intermediatefilamentproteins(IFP)from adultchickengizzard.Two antibodies,D3 and D76, have been characterizedindetail.They bind specifically to desmin but recognizedifferent epitopes.Inthe adultchicken,both McAbs produced equivalentimmunofluorescent staining patterns,reactinginfrozensectionswithallforms ofmuscle tissue,includingvascularsmooth muscle, butwithno othertissuetypes.Inisolatedskeletalmyofibrilsand inlongitudinalfrozen sectionsofcardiacand skeletalmuscle, desmin was detectedwith both McAbs atthe Z-band and inlongitudinally-oriented filamentbundles between myofibrils.Incontrasttotheseresults intheadult,the intermediatefilaments(IF)ofembryonic cardiacmyocytes inprimarycultures were decorated only with McAb D3, whereas McAb D76 was completely unreactivewith these cells.Similarly,frozen sectionsthrough the heartatearlystagesof embryonic chick development (Hamburger-Hamilton stages17-18)revealedregionsofmyocytes, identifiedby double immunofluorescence with myosin-specificMcAbs, thatwere unstained with McAb D76 even though similarregionswere stainedby McAb D3 .That McAb D76 reactedwith desmin inalladultcardiacmyocytes but not with allembryonic heartcellsindicatesthat embryonic and adultcardiacIFare immunologically distinctand impliesa conversion in IF immunoreactivityduringcardiacdevelopment .
Cytoskeletal proteins are found, as a general rule, in highly organized arrays within the cytoplasm of higher eukaryotic cells. The three filamentous networks that comprise the cytoskeleton are the microfilaments, composed of actin and many accessory proteins; the microtubules, composed of tubulin and several accessory proteins; and the intermediate filaments, composed of vimentin (or one of four other related proteins) and at least two accessory proteins. These networks have been the objects of intense study over the past 10 years in terms of cell motility, structure, and adhesion, and have proven to be most amenable to immunofluorescence and immunoelectron microscopic examination [e.g., R. Goldman et al., 1976)]. Because of the numerous applications of immunological techniques to investigate the structure and function of the cellular cytoskeleton systems, it is easily understood why many researchers in the field have begun utilizing the lymphocyte hybridoma method (Kohler and Milstein, 1975) as a source of antibodies. Studying something as complex as the cytoskeleton requires the use of the purest immunological reagents available, i.e., monoclonal antibodies.
Epithelia-derived tumors (carcinomas) can be distinguished from mesenchymally derived tumors by the presence of intermediate-sized filaments of the cytokeratin type, which usually coincides with the absence of other types of intermediate-sized filaments such as vimentin filaments. In the course of diagnostic examinations of human tumors, using immunofluorescence microscopy, we have come across a case of an unusual carcinoma (primary tumor and lymph node metastasis) positively stained not only with cytokeratin antibodies but also with immunoglobulins present in vimentin antisera. Therefore, this tumor, a cloacogenic carcinoma apparently derived from the rectal-anal transitional region, has been examined in greater detail using both immunofluorescence microscopy and immuno-electron microscopy as well as gel electrophoretic analysis of cytoskeletal polypeptides from total tumor tissue and from microdissected nodules enriched in carcinoma cells. The unusual reaction of the carcinoma cells with immunoglobulins present in seven different (rabbit or guinea pig) antisera raised against vimentin, has been found to be diminished after absorption on purified cytokeratin or total epidermal cytoskeletal material, but not after absorption on purified vimentin. Gel electrophoretic analysis of tumor cytoskeletons showed an unusual complex pattern of cytokeratin polypeptides containing relatively large (Mr 68,000 and Mr 58,000) neutral-to-slightly basic cytokeratins, as are typically found in epidermis and other stratified squamous epithelia, as well as several smaller acidic cytokeratins, including a Mr 40,000 polypeptide found in certain nonstratified epithelia such as colon and small intestine. Total tumor also showed the inclusion of some vimentin which, however, was significantly decreased in analyses of excised carcinoma nodules. Examining antibody binding to polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper, we have found that antisera raised against vimentin contained not only vimentin antibodies but also immunoglobulins which specifically bound to the largest cytokeratin component.We conclude that the unusual reaction of immunoglobulins present in vimentin antisera with cytokeratin filament bundles does not represent specific binding to vimentin in these carcinoma cells, but is due to a component obviously widespread in vimentin antisera which binds specifically to a cytokeratin present in this type of tumor but not in most other carcinomas. It is proposed that use is made in diagnostic examinations of vimentin antisera or affinity-purified vimentin antibodies that have been pre-absorbed on cytokeratin protein, in order to eliminate such disturbing reactions.
Three hybridoma clones producing IgM antibodies against actin were obtained from mice immunized with purified virions of paramyxoviruses. When tested on growing lung fibroblasts, ascites fluids of all clones stained in immunofluorescence cytoplasmic bundles of microfilaments, but also fibrillar networks. On colchicine-treated cells, perinuclear coils were seen in addition to microfilament bundles. In addition, one clone gave a pronounced speckled staining to the nuclei. Absorption of the ascites fluids with purified actin abolished all staining patterns. Using the Western blotting technique the antibodies reacted with both actin and vimentin polypeptides. DNase I abolished the staining of the actin filaments and of the nuclei, but left the vimentin pattern unimpaired. Thus, the monoclonal antibodies evidently reacted with epitopes common to actin and vimentin.
Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.
Systemic lupus erythematosus (SLE) and other autoimmune diseases are characterized by immune responses to intracellular, highly conserved antigens such as DNA and histone. In this study, peripheral blood lymphocytes (PBL) from a patient with histone autoantibodies were used to prepare IgM human-human hybridoma cell lines. Indirect immunofluorescence (IIF) was used to identify monoclonal antibodies that bound to cytoskeletal and other cytoplasmic constituents. These supernatants did not bind double-stranded or single-stranded DNA. However, immunoblotting revealed that 7/20 hybridomas selected for their binding to cytoskeletal components produced antibodies that also bound mammalian and avian histones. When peptide fragments of histone were used in immunoblotting experiments, it was found that the monoclonal antibodies bound to the carboxyl terminus of H1, a region previously shown to bind autoantibodies from sera of patients with SLE and drug-induced lupus (DIL). When the amino acid sequences of histones and cytoskeletal components were compared using the Swiss-Prot protein data bank, it was confirmed that there are eight regions of similarity. While the significance of polyreactive human monoclonal antibodies to cytoskeletal components and histones is not understood at present, it is possible that the human histone antibodies represent polyreactive antibodies that arise through the mechanism of molecular mimicry.
The focus of research on the neurofibrillary pathology (NFP) of Alzheimer disease has been not only to determine the component forming the paired helical filaments but also to determine whether they result from abnormal processes affecting a single protein. Therefore, although these studies have lead to controversy concerning the respective contribution of components of microtubules and neurofilaments, there has been essentially no consideration of whether other cytoskeletal systems might also be involved and of what are the common features for the incorporated components. Particularly relevant to this issue is our finding that several antisera raised to either skeletal or smooth muscle tropomyosin, a microfilament component, intensely recognize NFP. These antibodies continue to recognize NFP after affinity purification to tropomyosin or paired helical filament fractions. We show that the antibodies do not recognize NFP due to cross-reactivity with the previously identified NFP components related to neurofilaments and microtubules, tau, and MAP2, or neurofilament proteins because the antisera did not recognize these proteins on immunoblots or were not adsorbable by the proteins. Ultrastructural analysis of the immunoreaction showed that tropomyosin-related epitopes were clustered rather than uniformly distributed along paired helical and straight filaments. Although the distribution suggests that tropomyosin is an NFP-associated protein, its retention by paired helical and straight filaments after detergent extraction indicates that it is an integral component strongly and specifically associated with the filaments characteristic of NFP. These findings indicate that NFP involves the three primary neuronal cytoskeletal filament systems, microtubules, neurofilaments, as well as microfilaments, and therefore that NFP probably results from the reorganization of these normal filaments that interact to comprise the cytomatrix and may continue this interaction under the pathologic condition of Alzheimer's disease to generate novel, abnormal polymers.
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