Receptor-mediated endocytosis of diphtheria toxin by cells in culture.

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Abstract
The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture was 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 embryonic lung fibroblastic WI-38 cells and mouse embryonic fibroblastic 3T3 cells grown in culture. When added to cells at 37.degree. 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 2 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. Thus, diphtheria toxin-specific cell surface binding sites evidently occur on both insensitive and sensitive cells. Toxin is apparently 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.