Figure 4.

The binding of IgG-opsonized targets triggers traveling calcium waves. High-speed fluorescence micrographs of indo-1-labeled cells were acquired by using a 100-nsec shutter speed and a 30-msec duty cycle (frames 1–22, A and B). Experiments were performed by using cells expressing WT FcγRIIA (A) and FcγRIIA (AAA) (B). For both the WT and mutant FcγRIIA, a calcium wave was initiated near the site of target binding. When this initial wave returns to the binding site, two calcium waves are observed propagating along the cell membrane that return to the site of target binding. Thus, no discernable differences exist in the calcium-signaling routes followed during IgG-opsonized target binding for these tranfectants. The locations of the IgG-opsonized erythrocytes (arrows) were confirmed by transmitted light microscopy in frame 23 of both A and B. After completion of the high-speed imaging, the extracellular locations of the targets were confirmed by labeling the opsonizing IgG on the target with a fluorescent anti-IgG second-step antibody. The FITC emission in frame 24 of both A and B demonstrates that the target was accessible to the second-step reagent. (Magnification: ×800.)