Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development

Development ◽  
1981 ◽  
Vol 64 (1) ◽  
pp. 45-60
Author(s):  
Rolf Kemler ◽  
Philippe Brûlet ◽  
Marie-Thérèse Schnebelen ◽  
Jean Gaillard ◽  
François Jacob
Keyword(s):  
Monoclonal Antibodies ◽  
Epithelial Cells ◽  
Embryonic Development ◽  
Tissue Distribution ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Adult Tissue ◽  
Reactivity Pattern ◽  
Germ Layers ◽  
Intermediate Filament Proteins

Monoclonal antibodies (mAbs) against a preparation of intermediate filaments from trophoblastoma cells were studied for their reactivity pattern during embryonic development and on adult tissue cells. Up to day 12 of embryonic development, epithelial cells of the three germ layers reacted with these mAbs. Later during development and in adult tissues, positive reactions could be observed only with epithelial cells derived from mesoderm and endoderm. Because of their tissue distribution, the proteins reacting with these mAbs might belong to the keratin family of intermediate filaments or they might represent a new group of intermediate filaments.

scholarly journals Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross- reacting antibodies

1982 ◽  
Vol 95 (2) ◽  
pp. 414-424 ◽  
Author(s):  
AM Gown ◽  
AM Vogel
Keyword(s):  
Monoclonal Antibodies ◽  
Epithelial Cells ◽  
Stratum Corneum ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Human Cells ◽  
Carcinoma Cells ◽  
Intermediate Filament Proteins ◽  
Tissue Sections ◽  
Antikeratin Antibody

Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all, nonsquamous epithelium. Therefore this antistratum corneum antibody and the anti-54-kdalton antibody identify unique epitopes present in the various cytokeratin molecules of epithelial cells. None of the hybridoma antibodies react with neurofilament proteins. The different patterns of reactivity of these antibodies suggest that many of the immunologically distinct intermediate filament proteins contain common antigenic determinants.

scholarly journals Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization.

1982 ◽  
Vol 92 (3) ◽  
pp. 665-673 ◽  
Author(s):  
E B Lane
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Cell Line ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Antigenic Determinant ◽  
Protein Antigens ◽  
Molecular Weight Range ◽  
Intermediate Filament Proteins ◽  
Molecular Weights

The tonofilament-associated protein antigens recognized in epithelial cells by a group of six monoclonal antibodies have been studied by immunofluorescence and gel immunoautoradiography. The monoclonal antibodies were generated against detergent insoluble cytoskeleton extracts from a cultured simple epithelium derived cell line, Ptk1 cells. They show various tissue specificities, and while they all recognize components at the low end of the molecular weight range for intermediate filament proteins, they confirm that single antibody species can react with multiple polypeptides of different molecular weights in the tonofilament complex. The monoclonal antibodies described here demonstrate the presence of a simple epithelium antigenic determinant associated with intermediate filaments that is not detectable in the specialized cells of squamous and keratinizing epithelia but can reappear in such cells after transformation.

Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis

2000 ◽  
Vol 113 (13) ◽  
pp. 2471-2483 ◽  
Author(s):  
I. Hofmann ◽  
C. Mertens ◽  
M. Brettel ◽  
V. Nimmrich ◽  
M. Schnolzer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intermediate Filament ◽  
Solid Phase ◽  
Specific Interaction ◽  
Binding Assays ◽  
Intermediate Filament Proteins ◽  
Amino Terminal ◽  
Vitro Analysis

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

Differential diagnosis of genitourinary tumors using monoclonal antibodies to intermediate filament proteins

Urology ◽  
1989 ◽  
Vol 33 (5) ◽  
pp. 433-439
Author(s):  
Steven A. Lofton ◽  
Allen M. Gown ◽  
Arthur M. Vogel ◽  
John N. Krieger
Keyword(s):  
Differential Diagnosis ◽  
Monoclonal Antibodies ◽  
Intermediate Filament ◽  
Intermediate Filament Proteins

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

1993 ◽  
Vol 122 (6) ◽  
pp. 1323-1335 ◽  
Author(s):  
GY Ching ◽  
RK Liem
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Neuronal Cell ◽  
Intermediate Filament Protein ◽  
Amino Acid Residues ◽  
Intermediate Filament Proteins ◽  
Transfected Cells ◽  
Type Iv

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.

Molecular analysis of intermediate filament cytoskeleton--a putative load-bearing structure

1984 ◽  
Vol 246 (4) ◽  
pp. H566-H572 ◽  
Author(s):  
M. G. Price
Keyword(s):  
Skeletal Muscle ◽  
Molecular Analysis ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Direct Evidence ◽  
Two Dimensional ◽  
Myocardial Cells ◽  
Intermediate Filament Proteins ◽  
Bovine Myocardium ◽  
Bearing Structure

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.

scholarly journals Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells.

1981 ◽  
Vol 91 (1) ◽  
pp. 175-183 ◽  
Author(s):  
F G Falkner ◽  
H Saumweber ◽  
H Biessmann
Keyword(s):  
Drosophila Melanogaster ◽  
Monoclonal Antibodies ◽  
Heat Shock ◽  
Indirect Immunofluorescence ◽  
Intermediate Filament ◽  
Cross Reaction ◽  
Cytoplasmic Protein ◽  
Baby Hamster Kidney ◽  
Intermediate Filament Proteins ◽  
Bhk Cells

Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures.

scholarly journals Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
Author(s):  
G S Blank ◽  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Light Chains ◽  
Binding Region ◽  
Intermediate Filament Proteins ◽  
Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

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