Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes

Abstract

Flavocytochrome b(558), the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91(phox) (NOX2) and a smaller subunit, p22(phox). Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91(phox) and p22(phox), we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22(phox) and gp91(phox) subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.

FIGURE 1

Fluorescently tagged p22^phox and gp91^phox form functional heterodimers. A, Schematic of constructs encoding fluorescently tagged p22^phox and gp91^phox. B, 22YFP or CFP91 were transiently expressed in CHO-WT cell lines. Cell lysates prepared 48 h posttransfection were analyzed by Western blotting using polyclonal anti-p22^phox and monoclonal anti-gp91^phox (CL5) Abs. Cell lysates from CHO-22 and CHO-91 cells were used as controls. C, Cell lysates from CHO-91 cells transiently expressing YFP or 22YFP, as well as CHO-WT cells expressing CFP91 or coexpressing CFP91 and 22YFP, were analyzed by Western blotting at 48 h posttransfection for maturation of untagged and CFP-tagged gp91^phox (mAb CL5). YFP and 22YFP protein expression were validated with an Ab to YFP. Data shown are representative of at least three independent experiments. D, NADPH oxidase activity was measured using isoluminol chemiluminescence in CHO cells transiently and/or stably expressing p47^phox and p67^phox with untagged or fluorescently tagged flavocytochrome subunits as indicated. Integrated relative light units are shown. From left to right: CHO-91-67-47 cells transfected with 22YFP (n = 5), untagged p22^phox (n = 3), or YFP (n = 2); CHO-22 cells transfected with p47^phox, p67^phox, CFP91 (n = 2), or CFP (n = 1); CHO-47-67 cells transfected with 22YFP and CFP91 (n = 5) or YFP and CFP (n = 2). Mean ± SD of data for the number of experiments listed above.

FIGURE 2

The subunits of flavocytochrome b, gp91^phox, and p22^phox localize to the ER when expressed individually in CHO cells. A, CHO cells stably expressing untagged p22^phox (top panel) or gp91^phox (bottom panel) were fixed, permeabilized, and stained for p22^phox (mAb 44.1) or gp91^phox (mAb 54.1). 22YFP or YFP91 transiently expressed in CHO-WT cells was imaged 48 h posttransfection in (B) living cells and (C) cells fixed, permeabilized, and costained for calnexin. Merged images show 22YFP and calnexin (top panel) and YFP91 and calnexin (bottom panel), and they are single plane z-stack slices acquired from the bottom of the cell.

FIGURE 3

Heterodimers of p22^phox and gp91^phox traffic to the plasma membrane, while the subunit in “excess” localizes to the ER. CHO-YFP91 cells were transiently transfected with 0.5, 1.0, 2.0, or 4.0 μg of the 22CFP vector. A, YFP91 stably expressed in CHO-YFP91 cells was imaged in living cells. A single-plane z-stack slice acquired from the bottom of the cell is shown. B, Cell lysates were analyzed for gp91^phox, p22^phox, and β-actin protein expression 48 h posttransfection (n = 2 experiments in which protein expression was evaluated). C, Confocal microscopy of YFP91 (left column), 22CFP (middle column), and merged images (right column) following transient expression of 1.0 μg of 22CFP (top panels) or 4.0 μg of 22CFP (bottom panels) 72 h posttransfection of CHO-YFP91 cells. YFP91 localized to the ER in cells that lacked expression of 22CFP (asterisks), to the ER and plasma membrane in some cells expressing 22CFP (arrowheads), and to the plasma membrane but not the ER in other CHO-YFP91 cells that coexpressed 22CFP (arrows). 22CFP localized to the plasma membrane in most CHO-YFP91 cells (arrows), although it was present in both the plasma membrane and ER in some cells overexpressing 22CFP (wide arrows). Single-plane z-stack slices acquired from the bottom of the cell are shown. D, YFP91 and 22CFP colocalize in CHO-YFP91 cells transiently transfected with 0.5 μg of 22CFP. Single-plane z-stack slices acquired from the bottom and middle of the cell are shown.

FIGURE 4

Characterization of model cell line, CHO-91-22YFP, to investigate trafficking of flavocytochrome b. A, Immunoblots of cell lysates from CHO-91 and CHO-91-22YFP cells probed with mAb 54.1 and mAb NS2 to detect gp91^phox maturation and 22YFP protein expression, respectively. B, Cell surface gp91^phox detected by 7D5 (mAb) using flow cytometry in CHO-91 cells (gray) and CHO-91-22YFP cells (black line). CHO-91-22YFP cells were fixed, permeabilized, and stained to detect calnexin (C) gp91^phox (D), or gp91^phox and calnexin (E). Arrowheads denote areas of colocalization. Single-plane z-stack slices acquired from the bottom and/or middle of the cell are shown.

FIGURE 5

Flavocytochrome b localizes to the Rab-11-positive endocytic recycling compartment in CHO cells. A, CHO-91-22YFP cells were fixed, permeabilized, and stained for Rab11. Insets within the merged image (bottom panel) show colocalization of 22YFP and Rab11 in a perinuclear compartment. B, CHO-91-22YFP cells transiently expressing Rab11CFP were imaged in living cells 24 h posttransfection. C, CHO-91-22YFP cells were incubated with Alexa Fluor 647-conjugated transferrin (TfnAF647, 20 μg/ml) for 5 min at 37°C followed by a 25-min chase at 37°C to label the endocytic recycling compartment. Living cells were imaged, and colocalization of 22YFP and Tfn was evaluated in the merged image (n = 2 experiments where 22YFP and Tfn colocalization was evaluated). Single-plane z-stack slices taken from the bottom and middle of the cell are shown.

FIGURE 6

p22^phox C-terminal aa 149–195 are not required for trafficking of the heterodimer to the plasma membrane or to the perinuclear recycling compartment. CHO-WT or CHO-91 cells were transfected with vectors for 22YFP, 172YFP, 149YFP, or 131YFP and evaluated 24 h posttransfection (n = 2 experiments where YFP-tagged p22^phox C-terminal deletion proteins were evaluated). A, Top panel, Flow cytometry analysis of YFP shows similar transfection efficiency of all YFP-tagged p22^phox constructs when transiently expressed in CHO-91 cells. Bottom panel, Analysis of gp91^phox cell surface expression (mAb 7D5) by flow cytometry reveals increased gp91^phox surface expression with coexpression of 22YFP, 172YFP, and 149YFP, but not 131YFP (bottom panel, black lines). CHO-91 cells were used as the control (gray). B, Cell lysates were evaluated for gp91^phox (mAb 54.1), YFP (anti-GFP), and β-actin protein expression. C, Left column, Live imaging of YFP-tagged p22^phox derivatives transiently expressed in CHO-WT cells. Right columns, CHO-WT cells transiently expressing YFP-tagged p22^phox derivatives were fixed, permeabilized, and stained for calnexin. D, Left column, Live imaging of YFP-tagged p22^phox derivatives transiently expressed in CHO-91 cells. Arrows show that 22YFP, 172YFP, and 149YFP associate with the endocytic recycling compartment, but 131YFP did not. Z-stack slices collected from the middle of the cell are shown. Right columns, CHO-91 cells transiently expressing YFP-tagged p22^phox derivatives were fixed, permeabilized, and stained for gp91^phox (mAb 54.1). 22YFP, 172YFP, 149YFP, but not 131YFP, colocalized with gp91^phox at the plasma membrane (arrowheads).

FIGURE 7

In macrophages, flavocytochrome b associates with Rab11-positive recycling endosomes. A, RAW-WT cells transiently expressing Rab11GFP were fixed, permeabilized, and stained for gp91^phox (mAb 54.1) 48 h posttransfection. Colocalization of gp91^phox and Rab11GFP is shown in vesicles near the plasma membrane (top panel, arrowheads) and in the endocytic recycling compartment (bottom panel, insets). Localization of gp91^phox to the endocytic recycling compartment was not dependent on expression of Rab11GFP (top panel, arrows). Single-plane z-stack slices taken from the bottom and middle of the cell are shown. B, RAW-WT transfected with Rab11GFP were incubated with Alexa Fluor 647-conjugated transferrin (TfnAF647, 5 μg/ml) for 5 min at 37°C followed by a 25-min chase at 37°C to label the endocytic recycling compartment. Cells were fixed, permeabilized, and stained for gp91^phox. Colocalization of gp91^phox and Rab11GFP (top panel) and gp91^phox and Tfn (bottom panel) was observed at the perinuclear compartment (arrowheads) and near the plasma membrane (arrows).

FIGURE 8

In macrophages, flavocytochrome b is present in plasma membrane and some Rab5-positive sorting endosomes, but not in Rab7-positive late endosomes. A, RAW-PH-GFP cells were fixed, permeabilized, and stained for endogenous gp91^phox (mAb 54.1). Colocalization of gp91^phox and the plasma membrane marker, PH-GFP, was seen at the cell surface in RAW-PH-GFP cells (insets). A single-plane z-stack slice taken from the middle of the cell is shown (n = 2 experiments where colocalization of gp91^phox and PH-GFP was evaluated). RAW-WT cells transfected with Rab5GFP (B) or Rab7GFP (C) were fixed, permeabilized, and stained for gp91^phox (mAb 54.1) 48 h posttransfection. B, gp91^phox and Rab5GFP showed some colocalization in vesicles near the plasma membrane (top panel, arrowheads) and near the nucleus (bottom panel, arrows). C, Rab7GFP accumulated on large vesicles dispersed throughout the cell that did not colocalize with gp91^phox near the plasma membrane (top panel) or near the nucleus (bottom panel, arrowheads).

FIGURE 9

Unassembled fluorescently tagged flavocytochrome b subunits localize to the ER in RAW 264.7 macrophages, while assembled subunits localize to endosomes and the plasma membrane. Protein expression and subcellular targeting of 22YFP and YFP91 were analyzed in RAW-22YFP and RAW-YFP91 cells. 22YFP and YFP91 protein expression in RAW-22YFP and RAW-YFP91 cells was examined using flow cytometry to detect YFP (A) and using Western blotting to detect p22^phox (mAb NS2) or gp91^phox (mAb 54.1) (B). RAW-WT cells were used as controls for analysis by flow cytometry (filled gray) and Western blot (labeled RAW). C, Subcellular distribution of 22YFP in living RAW-22YFP cells. D, RAW-YFP91 cells were fixed, permeabilized, and stained for calnexin (n = 2 experiments where YFP91 and calnexin colocalization was evaluated). E, Live RAW-22YFP cells transfected with CFP91 were examined 24 h posttransfection. Single z-stack slices from the bottom and/or middle of the cells are shown as indicated.

FIGURE 10

22YFP expressed in p22^phox-deficient BMDM localizes to the plasma membrane and endocytic recycling compartment. p22^phox-deficient (p22^def) BM cells were transduced with MSCV-22YFP and differentiated into macrophages for 6 –7 days. A, Lysates of WT, p22^def, and 22YFP-expressing p22^def BMDM were probed to detect p22^phox (mAb NS2), gp91^phox (mAb 54.1), and β-actin (mAb). Top bar graph shows the relative density of p22^phox protein, normalized to β-actin. Bottom bar graph shows the relative density of gp91^phox protein normalized to β-actin. Data shown are representative of three independent experiments. B, 22YFP expressed in p22^phox-deficient BMDM was imaged in living cells. 22YFP localizes to vesicles near the plasma membrane (insets, arrows) and vesicles that cluster near the nucleus (arrowheads). C, 22YFP-expressing p22^phox-deficient BMDM were incubated with Alexa Fluor 647-conjugated transferrin (TfnAF647, 5 μg/ml) for 5 min at 37°C followed by a 25-min chase at 37°C to label the endocytic recycling compartment. 22YFP and Tfn colocalized in vesicles near the plasma membrane (top panel, insets) and near the nucleus (bottom panel, insets).

FIGURE 11

Flavocytochrome b colocalizes with Rab11GFP in or near phagocytic cups and nascent phagosomes. A, Colocalization of 22YFP and gp91^phox, detected by immunofluorescence, in p22^phox-deficient BMDM following 2 min (top panels) and 15 min (bottom panels) of synchronized phagocytosis. A series of z-stack planes (0.16 μm) were collected. Five single planes (total of 0.8 μm) were merged using the z-stack projection tool in ImageJ. Arrowheads denote phagocytic cups, and asterisks indicate nascent phagosomes. Arrows indicate discrete foci of increased 22YFP and gp91^phox colocalization near phagocytic cups and nascent phagosomes. Representative images from two independent experiments in which at least 10 cells were evaluated at each time point. B, RAW 264.7 cells transiently expressing Rab11GFP were fixed, permeabilized, and stained to detect gp91^phox (red) 15 min after synchronized phagocytosis of opsonized zymosan. Arrowheads indicate colocalization of gp91^phox and Rab11GFP near a forming phagocytic cup (merged image). Arrows show colocalization on or near nascent phagosomes. Wide arrows indicate gp91^phox in the plasma membrane with subjacent to Rab11GFP, and asterisks mark a phagocytic cup that is gp91^phox-positive, but Rab11GFP-deficient.