A fluorescently tagged C-terminal fragment of p47phox detects NADPH oxidase dynamics during phagocytosis

Abstract

The assembly of cytosolic p47(phox) and p67(phox) with flavocytochrome b(558) at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47(phox) and p67(phox) are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67(phox) translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67(phox) to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47(phox) PRR. This fragment was recruited in a flavocytochrome b(558)-dependent, p67(phox)-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67(phox) SH3b domain and suggest that the p47(phox)/p67(phox) tail-to-tail interaction is disrupted after oxidase assembly such that the p67(phox)-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.

Figure 1.

Interactions of p67^phox with other cytosolic phox components and with p47PRR and its mutants in vitro. (A) Structural motifs and the proposed interactions between p47^phox, p67^phox, and p40^phox are shown schematically, in addition to the YFP-tagged C-terminal fragment of p47^phox, YFP-p47PRR, used in this study. Mutants in the YFP-tagged C-terminal fragment of p47^phox are also indicated, numbered according to their position in full-length p47^phox. (B) COS-7 cells were transfected with the empty pEYFP-C1 vector (YFP), pEYFP-C1 encoding p47PRR (YFP-p47PRR) or p47PRR mutants. Ten micrograms of cell lysate was incubated with glutathione-Sepharose-4B–bound GST-p67^phox, and bound material was electrophoresed and immunoblotted with anti-GFP polyclonal antibody. Blots are representative of three independent experiments. (C) Immunoblot of 10 μg cell lysate, corresponding to samples shown in B. (D) The bar graph shows the relative recovery of GST-p67^phox -bound YFP-p47PRR and mutant derivatives, relative to the amount present in the lysate, based on densitometry of immunoblots of pulldown samples and cell lysates. The recovery of wild-type YFP-p47PRR considered as 1.0. Assays were performed in triplicate, and mean ± SD are shown. *p < 0.01.

Figure 2.

Expression of YFP or YFP-fusion proteins in stably transgenic PLB-985 cells. (A) YFP or YFP-fusion protein expression was measured by flow cytometry. (B) Western blot analysis of YFP or YFP-fusion proteins using anti-GFP polyclonal antibody. (C) PMA-stimulated superoxide production in transgenic PLB-985 neutrophils. Results are expressed as total relative light unit (RLU) value over 30 min, measured at 1-min intervals. Values represent the mean ± SD of triplicate determinations. (D) IgG-Zym–stimulated superoxide production in phagosomes was measured for 60 min in PLB-985 neutrophils expressing empty vector (YFP), or YFP-p47PRR, or YFP-p47PRR-P366A mutant, using SOD-resistant luminol-enhanced chemiluminescence.

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, p67^phox-YFP, YFP-p40PX, or YFP-p47PRR. Similarly, phagocytosis was filmed in X-CGD PLB-985 neutrophils stably expressing p67^phox-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 p67^phox-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 p67^phox, p40PX, and p47PRR (*p < 0.01, unpaired t test).

Figure 4.

Translocation of endogenous p47^phox and p67^phox-YFP during phagocytosis of IgG-Zym in PLB-985 cells stably expressing p67^phox-YFP. Localization of endogenous p47^phox and p67^phox-YFP after immunofluorescent staining with anti-p47^phox and Alexa555-goat anti-mouse IgG 1 in p67^phox-YFP–expressing PLB-985 cells (A), where the asterisk denotes a phagosome with accumulation of both p47^phox and p67^phox-YFP, or X-CGD PLB-985 cells (B). The triangle indicates negative phagosomes on NADPH oxidase assembly. Representative images from three independent experiments. Bar, 5 μm.

Figure 5.

Translocation of YFP-p47PRR during IgG-Zym phagocytosis in p40^phox knockdown PLB-985 cells. (A) Colocalization of p67^phox-YFP and YFP-p40^phox with mCherry-p47PRR on internalized IgG-Zym phagosomes in PLB-985 neutrophils. The green and red fluorescence represent p67^phox or p40^phox, and p47PRR, respectively. (B) The relative fluorescence intensity on the phagosomal membrane compared with the cytosol was determined in the PLB-985 cells coexpressing p67^phox and mCherry-p47PRR for 10 phagosomes at indicated stages and is shown in the graph as mean ± SE. “Internalized” means 222 ± 95 s after phagosome closure (n = 10). The closed arrow on the X-axis indicates the appearance of mCherry-p47PRR on phagosome. (C) Expression of YFP-p47PRR, p40^phox, p67^phox, and actin was determined by immunoblotting. (D) Densitometry analysis of p40^phox and p67^phox expression normalized to actin from three independent experiments. (E) Localization of YFP-p47PRR in p40^phox knockdown PLB-985 cells during IgG-Zym phagocytosis. Bar, 5 μm.

Figure 6.

Accumulation of p67^phox-YFP and YFP-p47PRR with mCherry-p40PX on the phagosome during IgG-Zym phagocytosis in PLB-985 neutrophils. Time-lapse confocal microscopy was used to monitor translocation of coexpressed or p67^phox and mCherry-p40PX (A) YFP-p47PRR and mCherry-p40PX (B) in PLB-985 cells during IgG-Zym phagocytosis. Arrows indicate the cup of phagosomes (newly forming phagosomes), and letters a–d indicate individual internalized phagosomes. 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. Supplementary Movies S11 and S12 are available as Supplementary Data. The relative fluorescence intensity on the phagosomal membrane compared with the cytosol was determined in the PLB-985 cells coexpressing p67^phox-YFP and mCherry-p40PX (C) or YFP-p47PRR and mCherry-p40PX (D). Five phagosomes for each cell line were analyzed at the indicated stages, which are shown in the graph as mean ± SE. “Internalized” means 146 ± 82 and 170 ± 62 s for C and D, respectively, after phagosome closure. The open and closed arrows indicate the appearance of mCherry-p40PX and YFP-p47PRR, respectively, on the phagosome.

Figure 7.

Accumulation of NADPH oxidase probes on the phagosome during IgG-Zym phagocytosis in PLB-985 neutrophils treated with wortmannin or DPI. Translocation of p67^phox-YFP (A) and YFP-p47PRR (B) during IgG-Zym phagocytosis in transgenic PLB-985 neutrophils coexpressing mCherry-p40PX in the presence of 100 nM wortmannin. Letters m and n indicate individual internalized phagosomes. PLB-985 granulocytes expressing p67^phox-YFP or YFP-p47PRR were pretreated with 10 μM DPI at 37°C for 15 min, followed with synchronized phagocytosis as described in Materials and Methods. NADPH oxidase activity was measured in the presence of luminal, SOD, and catalase. Results are expressed as total relative light unit (RLU) values more than 45 min, measured at 1-min intervals. (C) Values represent the mean ± SD of triplicate determinations. Live image was monitored using confocal microscopy as described in Figure 3A. (D) Arrows indicate presealing (newly forming phagosomes) and letters o and p indicates internalized phagosome. Time-lapse confocal microscopy was used as described in Figure 3A. Bar, 5 μm.

Figure 8.

Activation of the NADPH oxidase in Fc OxyBURST phagosomes. (A) Superoxide production was monitored in individual phagosomes after the addition of Fc OxyBURST to PLB-985 neutrophils expressing mCherry-p40PX. The green and red fluorescence represent DCF (a probe for H₂O₂) and mCherry-p40PX, respectively. 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. (B) The relative intensity of DCF fluorescence in five phagosomes from three independent experiments was analyzed with Image J software. The intensity of DCF was considered as 1.0 at the time at which mCherry-p40PX first appeared on internalized phagosomes and is shown over time (s) after the appearance of the mCherry-p40PX probe. (C) Phagosome superoxide production induced by Fc OxyBURST was measured for 46 min by chemiluminescence in the presence of 20 μM luminol, with or without SOD and catalase. The inset shows the intracellular phagosome superoxide production measured for 46 min in PLB-985 cells expressing mCherry-p40PX, using nonsynchronized (as described in Figure 2D) or synchronized phagocytosis method, as indicated.