Ultrastructural localization of cell membrane G M1 ganglioside by cholera toxin

Abstract

An immunoelectron microscopic method is described for sensitive high-resolution visualization of tissue-bound cholera toxin. The principle is to incubate cells or tissue sections with toxin and then to localize the bound toxin with toxin-specific peroxidase (donor:H2O2 oxidoreductase; EC 1.11.1.7)-conjugated antibody and enzyme substrate. Thin sections are examined for electron-opaque precipitates in a transmission electron microscope. Because of the specific binding of the toxin to membrane ganglioside GM1, the method can be used for ultrastructural localization of this ganglioside. Semiquantitative data are obtained by titration of the limiting concentration of cholera toxin producing specific precipitates. The specificity of the method was controlled in various ways, including analyses of the correlation between the immunoelectron microscopy results and determinations of ganglioside GM1 in tissues with different ganglioside concentrations, tissues hydrolyzed with Vibrio cholerae sialidase, tissues in which exogenous GM1 was incorporated, and lipid-extracted tissues. The immunoelectron microscopic method demonstrates that membrane GM1 ganglioside is positioned on the external side exclusively. Cell-bound toxin remains in its original location on the plasma membrane surface of cells below 18.degree. C, but appears redistributed laterally and vertically in the membrane of cells incubated at 37.degree. C for 30 min or longer. In the central nervous sytem GM1 is concentrated in the pre- and postsynaptic membranes of the synaptic terminals; a further increase in reactivity of these structures after hydrolysis of the nervous tissue with V. cholerae sialidase suggests that higher gangliosides of the same series are particularly increased in the pre- and post-synaptic junctions.