Receptor-mediated endocytosis of diphtheria toxin by cells in culture.
The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture has been examined by using epifluorescence video intensification microscopy. Rhodamine-labeled diphtheria toxin retained significant toxicity on bioassay and in cell culture and was tested for uptake by human WI-38 and mouse 3T3 fibroblasts grown in culture. When added to cells at 37 degrees C, toxin was observed to become concentrated and internalized in discrete vesicles in both cell lines. The appearance of fluorescent clusters could be prevented by addition of excess unlabeled diphtheria toxin to the medium or by addition of ATP (which has been shown to block toxin binding to cells), indicating that the rhodamine-labeled toxin was binding to diphtheria toxin-specific cell surface binding sites. When the simultaneous uptake of rhodamine-labeled diphtheria toxin and fluorescein-labeled alpha 2-macroglobulin was monitored, the two proteins appeared in the same clusters indicating that the toxin undergoes receptor-mediated endocytosis. Despite the difference in susceptibility to diphtheria toxin of cells derived from sensitive (human) and resistant (mouse) tissues, the behavior of the rhodamine-labeled derivative in both cell lines was indistinguishable in terms of toxin required for formation of clusters or inhibition by unlabeled toxin or by ATP. These results demonstrate that diphtheria toxin-specific cell surface binding sites occur on both insensitive and sensitive cells and suggest that toxin is processed similarly by both cell types during its initial cell surface binding and internalization by this pathway. The possible involvement of this uptake system in the mechanism of action of diphtheria toxin in cells is discussed.[1]References
- Receptor-mediated endocytosis of diphtheria toxin by cells in culture. Keen, J.H., Maxfield, F.R., Hardegree, M.C., Habig, W.H. Proc. Natl. Acad. Sci. U.S.A. (1982) [Pubmed]
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