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Biomedical subjects

W Birchmeier

Publications and source records attributed to W Birchmeier.

At least 109 records · Page 6Linked to original sources
PubMed1985

Control of cell locomotion: perturbation with an antibody directed against specific glycoproteins.

A murine monoclonal antibody, SLOW-1, was selected, which inhibits the locomotion of chick embryo fibroblasts (the immunizing cells) in tissue culture. The antibody, an IgM, cross-reacts in locomotion assays with a number of tumor and untransformed cells, and on fixed and permeabilized cells binds 1-5 X 10(5) target sites with an affinity of 10(-8) M. The antigen can be extracted from cells with isotonic buffers containing EGTA, binds to Concanavalin A, and when analyzed on SDS gels by immunoblotting, two major antigenic glycoproteins are detected at 57 kd (isoelectric point, 5.1) and at 44 kd (isoelectric point, 5.4). The antigenic site involves galactosyl or mannosyl residues, or both, within a complex, N-linked carbohydrate tree. The possible contribution of the SLOW-1 antigen to a common control system of locomotion operating over the cell surface is discussed.

Adsorption↗
PubMed1985

Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin.

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.

Animals↗
PubMed1984

Monoclonal antibodies which prevent experimental lung metastases. Interference with the adhesion of tumour cells to laminin.

Cellular adhesion is important during metastasis, as metastatic cells must escape from the primary site into lymph and blood systems, there to adhere specifically to sites in distant organs. We have recently selected monoclonal antibodies which prevent adherence of B16 mouse melanoma cells to tissue culture dishes, and also markedly reduce experimental lung metastasis in mice when injected before or with the tumor cells. Here, we investigated which step in the metastatic process may be affected by the antibodies. The possible inhibitory effect of antibody on tumour cell adherence to vascular endothelial monolayers and to purified components of the underlying extracellular matrix - fibronectin, laminin and collagen type IV - was studied using in vitro assays. We found that the antibodies significantly blocked attachment to laminin, suggesting that specific basement membrane components play an important role in attracting or otherwise modifying the behaviour of metastatic tumour cells.

Animals↗
PubMed1983

Association between coated vesicles and microtubules.

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.

Animals↗
PubMed1983

Cell-cell interaction and polarity of epithelial cells: specific perturbation using a monoclonal antibody.

With functional assays we selected a monoclonal antibody (anti-Arc-1) that perturbs specific cell-cell contacts of MDCK epithelial cells in tissue culture. The cells loosen their tight junctions, become uncoupled, and their polarity is abolished. Several findings show that the corresponding antigen appears to be a cell-surface protein of MDCK epithelial cells but not of other established cell lines: radioactively labeled antibody could be absorbed on the surface of intact cells; immunoscanning electron microscopy showed a homogeneous surface distribution of the antigen; and the antigen was extractable from the cells by EDTA. Absorption on Western blots and gel filtration showed antigenicity at 130 and 40 kd. This study shows that the hybridoma technique, in combination with functional screening of the monoclonal antibodies, allows the identification of new and specific molecules involved in cell-cell adhesion.

Animals↗
PubMed1983

Monoclonal antibodies inhibit the adhesion of mouse B 16 melanoma cells in vitro and block lung metastasis in vivo.

Seven monoclonal antibodies against mouse B 16 melanoma cells (produced in syngeneic C57BL/6 mice) were selected that blocked the adhesion of melanoma cells to tissue culture dishes. These antibodies were found to be directed against antigens on the surface of mouse B 16 melanoma cells but not on normal mouse cells such as 3T3 fibroblasts. Similarly, the antigens could not be detected in normal mouse tissues (e.g., lung, kidney, liver) but were found in lungs colonized by B 16 melanoma cells. Significantly, three of these antibodies virtually abolished lung colonization of highly invasive B 16 sublines injected into the animals' bloodstream. They exerted their effect both when preabsorbed by the melanoma cell in vitro and when delivered to the animals prior to the tumor cells. It is suggested that monoclonal antibodies might be a promising tool for preventing metastasis.

Animals↗
PubMed1983

Monoclonal antibodies that prevent adhesion of B 16 melanoma cells and reduce metastases in mice: crossreaction with human tumor cells.

Monoclonal antibodies raised against B 16 melanoma cells in syngeneic mice were functionally screened for their ability to inhibit cell adhesion in tissue culture. Three of these antibodies (16/43, 16/77, 16/82), when preinjected into C57BL/6 mice, markedly reduced the number of experimental lung metastases produced by B 16 cells, possibly by interference with their adhesion to the lung endothelia. We now report that these monoclonal antibodies block in vitro attachment of the majority of human melanoma cell lines tested and also of carcinoma, neuroblastoma, and glioblastoma cells from both mice and humans but untransformed cell lines such as 3T3 mouse or MRC-5 human fibroblasts are not affected. The antibodies also react with mouse teratocarcinoma stem cells (F9, PCC4) but not with differentiated teratocarcinoma lines (PYS-2, 944). Furthermore, the antiadhesion activity of the antibodies could be quantitatively absorbed by intact human and mouse tumor cells but not by untransformed cells, suggesting that the corresponding antigens may represent tumor-associated cell surface components. Correspondingly, the antigens were found on simian virus 40-transformed 3T3 mouse fibroblasts and are expressed in a temperature-sensitive fashion in chicken fibroblasts transformed with a temperature-sensitive Rous sarcoma virus. On "immunoblots" of NaDodSO4-containing gels the three selected antibodies (16/43, 16/82, 19/1) were absorbed by antigens with molecular weights of 40,000 and 50,000.

Animals↗
PubMed1982

New surface component of fibroblast's focal contacts identified by a monoclonal antibody.

We describe a monoclonal antibody (anti-FC-1) that inhibits the attachment of chicken embryo fibroblasts to artificial substrates. In the immunofluorescence, anti-FC-1 also stained the focal contacts of the fibroblasts, organelles suggested to be involved in cell-substrate adhesion. This antibody was absorbed on the surface of intact fibroblasts, and its action was blocked by cell extracts with nonionic detergents, indicating that the antigen (FC-1) is a surface membrane protein. Triton-extracted FC-1 exhibited a molecular weight of approximately 500,000, and a 60,000 dalton protein was immunoprecipitated from 35S-methionine-labeled cells. We have thus characterized a new protein at the exterior side of fibroblast's focal contacts. To determine to what degree this protein mediates the adhesion of the cells to the substrate, and whether it also interacts with the cytoskeletal elements on the cytoplasmic side, will require further investigation.

Animals↗
PubMed1980

Stress fiber sarcomeres of fibroblasts are contractile.

Living cultured fibroblasts were microinjected with rhodamine-labeled smooth muscle alpha-actinin and visualized by video-intensified fluorescence microscopy. The alpha-actinin incorporated into the stress fibers and exhibited a regularly striped arrangement. The fluorescently labeled stress fibers remained intact despite glycerol or digitonin extraction of the cells; furthermore, these cell models contracted upon addition of MgATP. During this process, sections of alpha-actinin-labeled stress fibers contracted up to 25%; shortening proceeded in the nonfluorescent part of the stress fiber sarcomeres. In glycerol-extracted cell models, adenylylimidodiphosphate, ADP and pyrophosphate inhibited but vanadate, N-ethylmaleimide-modified heavy meromyosin, cytochalasin B, colchicine, phalloidin and DNAase I did not. Cytochalasin B was inhibitory when added to the intact cells before glycerol extraction. These morphological and biochemical findings demonstrate that stress fiber sarcomeres of fibroblasts are contractile elements and support the concept that an actomyosin system may be involved.

Actinin↗
PubMed1980

Phosphorylation and dephosphorylation of spectrin from human erythrocyte ghosts under physiological conditions: autocatalysis rather than reaction with separate kinase and phosphatase.

The mechanism of phosphosylation and dephosphorylation of spectrin from human erythrocyte membranes has been examined under closely physiological conditions. The results support the hypothesis that spectrin is an autophosphorylating and dephosphorylating system. (i) Extraction from ghosts of up to 85% of the kinase (casein kinase) suggested to catalyze the reaction [see Fairbanks, G., Avruch, J., Dino, E. J. & Patel, V. P. (1978) J. Supramol. Struct. 9, 97--112] only slightly reduced spectrin component 2 phosphorylation and did not affect ATP-induced changes in the ghosts' shapes. (ii) A spectrin--actin complex isolated from endocytotic inside-out vesicles under hyperteonic conditions contained virtually no casein kinase activity and still exhibited a largely intact phosphorylation machinery. (iii) Photoaffinity labeling experiments indicated that spectrin component 2 fulfills the necessary prerequisite of the hypothesis--i.e., it contains its own ATP-binding site. (iv) Under various conditions, spectrin phosphorylation and dephospohrylation seem to be tightly coupled. The implications of these findings for the understanding of spectrin function and the maintenance of erythrocyte shape are discussed.

Actins↗
PubMed1980

Corrugated attachment membrane in WI-38 fibroblasts: alternating fibronectin fibers and actin-containing focal contacts.

The distributions of both fibronectin (LETS, CSP) fibers and focal contacts to the substratum, as viewed by fluorescence and reflection contrast microscopy, respectively, have been compared in freshly plated WI-38 human fibroblasts. Most frequently, the actual focal attachment plaques did not contain fibronectin fluorescence and, furthermore, fibronectin spots and fibers often alternated with focal contacts. Overlap, however, was observed between focal contacts and the endings of actin-containing stress fibers [see also Wehland, J., Osborn, M. & Weber, K. (1979) J. Cell Sci. 37, 257-273]. Thus, the fibroblast attachment membrane might best be described as a corrugated sheet that undulates between alternating microfilaments and fibronectin fibers, at the points of closest and farthest distance to the substratum, respectively.

Actins↗
PubMed1979

ATP-induced endocytosis in human erythrocyte ghosts. Characterization of the process and isolation of the endocytosed vesicles.

ATP-induced endocytosis in human erythrocyte ghosts has been studied, and a procedure for the isolation of the endocytotic vesicles is described. Under isotonic conditions and 37 degrees C, optimal endocytosis occurs with concentrations of 4 to 10 mM MgATP. Within 30 min, up to 45% of the membrane is removed from the surface and converted into sealed inside-out vesicles. Local anesthetics, such as chlorpromazine, potentiate ATP-induced endocytosis in ghosts. Forcing cells containing endocytotic vesicles through a hypodermic needle leads to the exclusive fragmentation of the outermost plasma membrane. The endocytosed vesicles can then be separated from these fragments by centrifugation on a gradient of dextran T70. Biochemical analyses indicate that endocytotic vesicles contain full complements of the major membrane proteins (i.e. also spectrin and actin), common phospholipids, fatty acids, and cholesterol. Furthermore, they exhibit a fully intact spectrin component 2 phosphorylation machinery. In contrast, MgATPase activity is largely excluded from these vesicles. The novel inside-out vesicles described have properties different from those of previously analyzed fragments of the erythrocyte membrane. They will permit a detailed study of a native spectrin-actin network now exposed to the outside.

Acetylcholinesterase↗
PubMed1979

In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts.

Graessmann's microinjection technique was chosen to introduce fluorescently labeled muscle actin and other proteins into WI-38 human fibroblasts. The injected cells were examined during culture by fluorescence and reflection contrast microscopy. Within 30 min after injection, rhodamine-labeled actin was incorporated into a distinct network of fluorescent filaments, resembling the stress fibers observed by classical immunofluorescence microscopy. Cytochalasin b prevented the formation of these fibers, but colchicine did not. Neighboring fibers often converged into distinct focal points that appeared to be concentrated near the base of the cell. Examination of these fluorescent fibers and focal points by reflection contrast microscopy confirmed their close location to the substratum. After 6 hr of culture, fluorescent actin and the control proteins were segregated into granules located mainly near the cell nucleus. Thus, the injected actin both enters the intrinsic actin pool and participates in an assembly and disassembly of filamentous structures. Segregation into granules traces the natural turnover of this protein within the cell.

Actins↗
PubMed1977

Structure of cytochrome c oxidase from baker's yeast - a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochrome c binding site.

Cytochrome c oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K). In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtration. Their amino acid composition as well as their amino- and carbosy-terminal amino acid residues have been determined. Sequence determinations of subunits IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids. The binding site of yeast cytochrome oxidase for cytochrome c was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochrome c from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochrome c and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochrome c and from subunit III of cytochrome oxidase.

Amino Acid Sequence↗