Figure 3.
Translocation of fluorescent proteins during IgG-Zym phagocytosis in transgenic PLB-985 neutrophils. (A) Time-lapse confocal microscopy was used to monitor IgG-Zym phagocytosis by PLB-985 neutrophils expressing YFP, p67phox-YFP, YFP-p40PX, or YFP-p47PRR. Similarly, phagocytosis was filmed in X-CGD PLB-985 neutrophils stably expressing p67phox-YFP, YFP-p47PRR, or YFP-p40PX as well as PLB-985 cells stably expressing YFP-p47PRR-P366A or YFP-p47PRR-K383E/K385E, as indicated. Arrows indicate the cup of phagosomes (newly forming phagosomes), asterisks indicate the internalized phagosomes. For YFP-p40PX, the upper phagosome was another internalized phagosome appearing at 120 s. Supplementary Movies are available as Supplementary Data. The frames are labeled in seconds with respect to the time at which closure (sealing) of the phagosome was observed, with time zero being the time of closure. Bar, 5 μm. (B) The relative fluorescence intensity on the phagosomal membrane compared with the cytosol was determined in the indicated cell lines for five phagosomes at indicated stages and is shown in the graph as mean ± SE “Internalized” means ≥150 s after phagosome closure. (C) The time for the first appearance of YFP proteins on phagosome membrane of p67phox-YFP, YFP-p40PX, and YFP-p47PRR was analyzed in total of in 18 positive phagosomes for each fluorescently tagged protein, using films from at least four independent experiments for each cell line. The time at which the phagosome is sealed is defined as time zero. The mean ± SD of the times at which membrane translocation was first observed shown, which were significantly different for p67phox, p40PX, and p47PRR (*p < 0.01, unpaired t test).