Adhesion among neural cells of the chick embryo. I. An immunological assay for molecules involved in cell-cell binding.

Brackenbury, Robert; Thiéry, Jean‐Paul; Rutishauser, Urs; Edelman, Gerald M.

doi:10.1016/s0021-9258(17)39925-8

Cited by 330 publications

(55 citation statements)

References 27 publications

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“…Western blots analysis of the adult rat retina exhibited three major isoforms of NCAM (120, 140, and 180 kD), which are in agreement with previous reports (Lackie et al 1991; Goridis and Brunet 1992; Zuber et al 1992). NCAM, first reported by Brackenbury et al (1977), is an abundant integral membrane glycoprotein that can promote cell-to-cell adhesion through a homophilic binding mechanism (Rutishauser et al 1988; Doherty et al 1990). In the mouse retina, NCAM-180 and NCAM-140 are present during development, whereas in the adult retina only NCAM-180 and NCAM-120 are expressed (Bartsch et al 1989).…”

Section: Discussionmentioning

confidence: 99%

Multistratified Expression of Polysialic Acid and Its Relationship to VAChT-containing Neurons in the Inner Plexiform Layer of Adult Rat Retina

Sawaguchi

Idate

Ide

et al. 1999

J Histochem Cytochem.

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Section: Discussionmentioning

confidence: 99%

Multistratified Expression of Polysialic Acid and Its Relationship to VAChT-containing Neurons in the Inner Plexiform Layer of Adult Rat Retina

Sawaguchi

Idate

Ide

et al. 1999

J Histochem Cytochem.

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“…These assays were based on the inhibition of the aggregation of a particular cell type by monovalent antibody fragments raised against cell surface determinants and on the reversal of this inhibition by neutralization of the antibody with cell surface molecules . The use of such neutralization assays facilitated the identification and purification of molecules from the cell surface of vertebrate neuronal cells (20,26,29), liver cells (15,30), and embryonal carcinoma cells (31,32) ; the involvement of these molecules in homotypic cell-cell adhesion has been amply demonstrated in vitro (reviewed in reference 4).…”

Section: Discussionmentioning

confidence: 99%

“…I-neuronal membrane vesicles (0 .1 ml) were preincubated for 15 min on ice in 0 .4 ml PBS containing a mixture of Fab' fragments from anti-(N-CAM) Ig and anti-brain membrane Ig in different ratios so that the anti-(N-CAM) titer was maintained constant while the anti-brain membrane Fab' was increased in the 1 .2-ml assay volume . The titer of anti-(N-CAM) from each antiserum was measured in an assay involving aggregation of retinal neural cells as described previously (26) . In contrast to the concentration-dependent inhibition of binding found with anti-brain membrane Fab', anti-(N-CAM) Fab' at concentrations as high as 2 mg/ml did not affect the control level of binding or the inhibition by anti-brain membrane Fab' .…”

Section: Specific Antibodies Inhibit Binding and Membrane Antigens Neutralize This Inhibitionmentioning

confidence: 99%

“…High molecular weight fractions (Mr -100,000) from the column neutralized inhibition by anti-brain membrane Fab' of binding of neuronal vesicles to glial cells (Ng-CAM neutralizing activity) . Residual N-CAM neutralizing activity (26) eluted in the lower molecular weight fractions, and was separated from the peak of Ng-CAM neutralizing activity. The 6 Fractionation of a detergent extract of membranes by gel filtration and Ng-CAM neutralizing activity of fractions .…”

Section: Fractionation Of Neutralizing Activity and Isolation Of A Monoclonal Antibody That Inhibits Adhesionmentioning

confidence: 99%

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Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule.

Grumet¹,

Edelman

1984

The Journal of Cell Biology

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155

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By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) w~s,designed to distinguish between homotypic binding (e .g., neuron to neuron) and heterotypic binding (e .g., neuron to glia) . This distinction was essential because a single neuron might simultaneously carry different CAMS separately mediating each of these interactions . The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMS . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125 1-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period . The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125 1 label . Binding increased with increasing concentrations of both glial cells and neuronal vesicles . Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells .The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells . Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition ; after chromatography, neutralizing activity was enriched 50-fold . This fraction was injected into mice to produce monoclonal antibodies ; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an M r =135,000 band in immunoblots of embryonic chick brain membranes . These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells . Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron-glial interaction is heterophilic, i .e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells .

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“…Endothelial cells were isolated from either adult or fetal (48-cm crown to rump) bovine aortas and grown in culture (passage [3][4][5][6][7][8][9][10][11][12] as previously described in Waymouth's complete medium containing 10% heat inactivated FBS (15). For certain experiments endothelial cells were grown on cell culture plates coated with a collagen film that was prepared as described by the manufacturer (Collagen Corporation, Palo Alto, CA).…”

Section: Cells and Cell Culturementioning

confidence: 99%

Identification of a Ca2(+)-dependent cell-cell adhesion molecule in endothelial cells.

Heimark

Degner

Schwartz

1990

The Journal of Cell Biology

103

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Abstract. Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2+-dependent adhesion of endothelial cells. There was no inhibition of CaZ+-independent adhesion of endothelial cells and only a minor effect on Ca2+-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in nonreducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2+-dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca~+-dependent CAM.

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Adhesion among neural cells of the chick embryo. I. An immunological assay for molecules involved in cell-cell binding.

Cited by 330 publications

References 27 publications

Multistratified Expression of Polysialic Acid and Its Relationship to VAChT-containing Neurons in the Inner Plexiform Layer of Adult Rat Retina

Multistratified Expression of Polysialic Acid and Its Relationship to VAChT-containing Neurons in the Inner Plexiform Layer of Adult Rat Retina

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule.

Identification of a Ca2(+)-dependent cell-cell adhesion molecule in endothelial cells.

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