FIG. 2.

Cytotoxic effect of C. botulinum C2 toxin on synchronous HeLa cells. (A) Time course of C2 toxin-induced actin ADP-ribosylation. At 6 h after release from the amethopterin block, C2 toxin (200 ng of C2II and 100 ng of C2I per ml) was added to synchronized HeLa cells. Cells were incubated at 37°C; immediately and every 30 min after toxin addition, cells were lysed and lysate proteins (100 μg) were subjected to an in vitro ADP-ribosylation assay with C2I. The autoradiogram of [³²P]ADP-ribosylated actin is shown. Lane 1, control (without C2 toxin); lanes 2 to 8, incubation for 30 min with C2 toxin, 60 min with C2, 90 min with C2, 120 min with C2, 150 min with C2, 180 min with C2, and 210 min with C2, respectively. (B) C2 toxin-induced morphological changes and F-actin redistribution. Synchronized control cells (8 h after release from the amethopterin block) as well as synchronized cells treated with C2 toxin for 2 h (6 to 8 h after release from the block; 200 ng of C2II and 100 ng of C2I per ml) were fixed, and F-actin was stained with phalloidin-rhodamine.