[Immunofluorescence study of intermediate filament protein vimentin of CP-activated macrophages].
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Abstract:
Vimentin is an important cytoskeleton protein for the biological function of macrophages. Alterations of vimentin filaments of CP-activated macrophages stained with FITC-labeled anti-vimentin antibody were observed under immunofluorescence microscope. Activated macrophages showed changes with the following characteristics: the intensity of immunofluorescence of vimentin was increased; the filaments of vimentin became thicker than those of normal macrophages when they were treated with colchicine; and the arrangement of vimentin filaments was parallel in direction to the polarization of the activated macrophages.Keywords:
Immunofluorescence
Colchicine
Intermediate Filament Protein
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The cytoskeleton is a biopolymer network composed of intermediate filaments, actin, and microtubules, which is the main mechanical structure of cells. Vimentin is an intermediate filament protein that regulates the mechanical and contractile properties of cells, thereby reflecting their mechanical properties. In recent years, the "nonmechanical function" of vimentin inside and outside of cells has attracted extensive attention. The content of vimentin in atherosclerotic plaques is increased, and the serum secretion of vimentin in patients with coronary heart disease is remarkably increased. In this review, the mechanistic and nonmechanistic roles of vimentin in atherosclerosis progression were summarized on the basis of current studies.
Intermediate Filament Protein
Neurofilament
Biopolymer
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IFAPa-400, a 400-kDa developmentally regulated protein thought to be associated with intermediate filaments, has been purified from chick embryo hearts to investigate its interaction with vimentin and other IF proteins and to identify other cellular components to which this cytoskeletal protein associates. Previous studies suggested that this protein was associated with the vimentin-containing intermediate filament lattice of myoblasts and neuroblasts before their terminal differentiation, providing these cells with a particular intermediate filament cytoskeleton that could satisfy specific mechanical requirements during their intense morphogenetic activities. Although IFAPa-400 partially reassociated with vimentin and desmin in disassembly–reassembly experiments using crude IF preparations from chick embryo hearts, in vitro recombination of purified IFAPa-400 with vimentin and desmin failed to demonstrate any direct association. When purified IFAPa-400 was used as a probe in blot overlay assays, however, specific binding to vimentin and desmin was observed, providing the first evidence of a physical association between IFAPa-400 and intermediate filament proteins. The blot overlay experiments also demonstrated that IFAPa-400 binds to two unidentified polypeptides of 19 and 32 kDa. These results are thus consistent with the hypothesis that a structural lattice requiring a vimentin–IFAPa-400 combination constitutes the intermediate filament system of myogenic and neurogenic cells.Key words: cytoskeleton, intermediate filaments, intermediate filament associated proteins, vimentin, IFAPa-400.
Desmin
Intermediate Filament Protein
Neurofilament
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We studied the distribution of intermediate filament proteins during several stages of chick embryo heart development by indirect immunofluorescence and fluorescence-activated cell surface analysis. Vimentin is the predominant intermediate filament during the early stages of cardiac genesis, while desmin appears essentially with maturation. Desmin is the main subunit protein of intermediate filaments in the mature myocyte.
Desmin
Immunofluorescence
Intermediate Filament Protein
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Certain glia cells, notably astrocytes and tumor cxells derived therefrom, express simultaneously two types of proteins of intermediate‐sized filaments, vimentin and glia filament protein (GFP). We have used an established human glioma (astrocytoma) cell culture line (U 333 CG/343 MG) in which both proteins are seen in partly overlapping fibrillar structures by immunofluorescene microscopy to examine the possible existence of heteropolymer filaments of these two proteins by using reversible oxidative cross‐linking facilitated by the 1,10‐phenanthroline‐cupric ion complex. Dimeric cross‐link products are characterized by one‐dimensional and two‐dimensional gel electrophoresis under non‐reduing and reducing conditions as well as by peptide mapping. The relatively large prportions of heterodimers of vimentin and GFP obtained in cytoskeletal filaments cross‐linked in this way, demonstrate the frequency of heteropolymer filaments in this cell as well as the frequency of face‐to‐face ‘pairs’ of GEP and vimentin in such filaments. Together with our related observations on heteropolymer filaments between vimentin and desmin in some smooth muscle cells [Ouinlan. R. A. and Franke, W. W. (1982) Proc. Natl Acad. Sci. USA, 79, 3452–3456]. we discuss this as evidence for common principles of molecular arrangments of vimentin, GFP and desmin, at least in the cysteine‐containing surface domains. The results are also discussed in relation to cytoskeletal changes during glial differentiation.
Desmin
Neurofilament
Intermediate Filament Protein
Immunofluorescence
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S ummary . The human IgM anti‐Rh D antibody MAD‐2 has previously been shown to react with human and animal tissues and leucocytes. Double labelling immunofluorescence with MAD‐2 and a mouse monoclonal antibody against vimentin, the intermediate filament protein of cells of mesen‐chymal origin, showed coincidental staining which was distinct from that seen with antibodies against other cytoskeletal proteins. Using immunoblotting, both MAD‐2 and anti‐vimentin reacted with a 55 kDa tissue component, and with purified vimentin. These results show that the major tissue and leucocyte protein recognized by MAD‐2 is vimentin.
Intermediate Filament Protein
Immunofluorescence
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Intermediate Filament Protein
Molecular mass
Neurofilament
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Myocardial cells contain a cytoskeleton of intermediate filaments connecting the myofibrils. The present molecular analysis of the myocardial cytoskeleton was designed to identify the intermediate filament proteins and examine their assembly properties. The intermediate filament proteins desmin and vimentin were isolated from adult bovine myocardium by sequential extraction, urea solubilization, and chromatography on hydroxylapatite and DEAE columns. Desmin was obtained virtually pure in one peak and in a mixture of desmin and vimentin in the trailing fractions. Intermediate filaments of different morphologies polymerized in the desmin and the desmin-vimentin fractions. Isolated myocardial desmin occurs as three isozymes and isolated myocardial vimentin as two isozymes, which co-migrate on two-dimensional gels with corresponding isozymes from bovine skeletal and smooth muscle. Polypeptides of 200,000 and 220,000 daltons that fractionate with myocardial desmin and vimentin are also present in cytoskeletons of smooth and skeletal muscle. The results provide direct evidence that myocardial desmin can assemble to form intermediate filaments, suggesting that desmin is the major component of the cytoskeletal filaments in cardiomyocytes.
Desmin
Intermediate Filament Protein
Myofibril
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Vimentin is an important cytoskeleton protein for the biological function of macrophages. Alterations of vimentin filaments of CP-activated macrophages stained with FITC-labeled anti-vimentin antibody were observed under immunofluorescence microscope. Activated macrophages showed changes with the following characteristics: the intensity of immunofluorescence of vimentin was increased; the filaments of vimentin became thicker than those of normal macrophages when they were treated with colchicine; and the arrangement of vimentin filaments was parallel in direction to the polarization of the activated macrophages.
Immunofluorescence
Colchicine
Intermediate Filament Protein
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Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.
Intermediate Filament Protein
Nestin
Neurofilament
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