The two components of botulinum C2 toxin are functionally different proteins; component II is a binding molecule, whereas component I is an ADP-ribosyltransferase, of which substrate is cytoplasmic actin monomers (Ohishi and Tuyama, 1986; Ohishi, 1986; Ohishi et a/., 1990). This indicates that the toxin binds to the cell surface and enters the cytoplasm. These steps, the binding of the two nonlinked components of C2 toxin to the cells and the endocytotic vesicles containing the components, can be visualized either directly by incubating the cells with the two differently fluorescently labeled components (Ohishi, 1992), or indirectly by immunofluorescence labeling of the two proteins with their specific antibodies. This endocytotic incorporation of the protein is not a specific feature of this toxin, but is common to all the proteins that enter cells by receptor-mediated endocytosis. However, the internalization of the toxin may be of an interest to those readers, who would like use this characteristic toxin as a tool to analyze cellular responses, especially those who would like to compare the incorporation processes of the two non-linked components with those of other proteins. In this section, I shall describe the visualization methods for binding and internalization of the two non-linked protein components of C2 toxin in tissue culture cells using their specific antibodies. In addition, I shall describe the immunization method for the two components of C2 toxin, which would be useful for those who wish to work with the toxin as a tool. The method for direct visualization for the cell-bound and the internalized components I and II of the toxin has already been presented elsewhere (Ohishi, 1992).
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