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PNAS 103 (48): 18226-18231
Copyright © 2006 by the National Academy of Sciences.
Immunoglobulin G signaling activates lysosome/phagosome docking
Vishal Trivedi*,
Shao C. Zhang*,
Adam B. Castoreno*,
Walter Stockinger*,
Eugenie C. Shieh*,
Jatin M. Vyas ,
Eva-Maria Frickel , and
Axel Nohturfft*,
*Department of Molecular and Cellular Biology, Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138; Division of Infectious Disease, Department of Medicine, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114; and Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142
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Fig. 1.. IgG opsonization promotes phagosome maturation in vivo. (A) On day 0, RAW 264.7 cells were set up on coverslips in a 24-well plate. On day 1, the medium was replaced with 0.3 ml of culture supernatant containing lentivirus encoding CD63 fused to cherry fluorescent protein. On day 2, cells were refed fresh medium and grown for another 2 days. Cultures were switched to suspensions of medium B plus 1-µm amine latex beads coupled to BSA or to mouse IgG as indicated. Plates were centrifuged at 1,000 x g for 2 min and incubated at 15°C for 1 h, washed three times with PBS to remove unattached beads, and incubated again for 1 h at 15°C. The plates were then moved to 37°C for the indicated time. Cells were fixed, stained with filipin (15), and analyzed by microscopy. (B) RAW cells were set up as above and on day 1 received medium C plus 0.1 mg/ml fixable tetramethylrhodamine-conjugated dextran (10 kDa; Invitrogen). After incubation at 37°C overnight, the cells were switched to medium A and exposed to 1-µm amine beads or to amine beads conjugated to mouse IgG. The protocol for bead uptake and chase was as in A. Arrowheads point at selected phagosomes.
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Fig. 2.. Lysosome/phagosome interactions in vitro require macrophage cytosol. (A) Lysosome/phagosome targeting was measured with a cell-free scintillation proximity assay. Complete reactions contained 3H-cholesteryl oleoyl ether-labeled lysosomes, J774 cell phagosomes containing scintillant latex beads plus or minus cytosol from J774 cells and rat liver, and an ATP-regenerating system. After 1 h of incubation, proximity scintillation was measured and is expressed in cpm. (B) Reactions were as in A except that macrophage cytosol concentrations were varied in the absence and presence of 6 mg/ml rat liver cytosol.
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Fig. 3.. IgG-opsonized beads activate a cytosolic factor required for promoting lysosome/phagosome interactions. Cell-free assays were performed as in Fig. 2A. (A) Low-salt cytosol was from J774 cells grown for 1 h in the absence or presence IgG-coated beads in medium B. (B) Low-salt cytosol was from J774 cells grown in medium B for 1 h in the absence of beads, with 0.25 mg per well of 6-µm amine beads (NH2), amine beads coupled to BSA, or amine beads coupled to mouse IgG (data were calculated by comparing the effects of low-salt cytosol from bead-treated versus untreated controls). (C) Low-salt cytosol was from J774 cells exposed to the indicated concentrations of 6-µm BSA beads (open circles) or IgG beads (filled circles) in medium B for 1 h. (D) Low-salt cytosol was from J774 cells exposed to 0.2 mg/ml 6-µm IgG beads in medium B for the indicated time. Background scintillation counts in reactions performed without macrophage cytosol were 148 ± 15 cpm (A), 176 ± 11 cpm (B), 446 cpm (C), and 195 ± 41 cpm (D).
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Fig. 4.. Characterization of IgG-induced signaling pathway. (A) CHO-K1 cells were transfected with 0.7 µg each of plasmids expressing human Fc RIIa (pCMV-FcRIIa-IRES-Neo; FcR) and a yellow fluorescent protein (YFP)/mouse PKC- fusion protein (pEX-EF1-YFP-PKC-; PKC). Total DNA amounts were adjusted to 2.1 µg per well by addition of pIRESneo2. Cells were cultured plus or minus human IgG beads in medium A for 1 h at 37°C, harvested, and fractionated. Low-salt cytosol was used for in vitro reactions as in Fig. 2A. Data indicate percentage increase of scintillation in reactions containing low-salt cytosol from IgG-treated versus control cells. (B) CHO-K1 cells were transfected for 48 h with 70 ng each of plasmids expressing FcRIIa, YFP-PKC-, and Lamp1-RFP. Cultures were exposed to 1-µm BSA beads or IgG beads at 15°C as in Fig. 1 and chased at 37°C for 15 min. Cells were fixed, stained with filipin, and analyzed by microscopy. For each condition, two representative sets of images are shown. (C) Lysosome/phagosome targeting was measured as in Fig. 2A. Reactions were set up without (gray bars) or with (black bars) low-salt cytosol from J774 cells cultured for 1 h in the absence or presence of 0.2 mg/ml 6-µm IgG beads, 1 µM Gö6976 (Gö), and 10 µM bisindolylmaleimide (Bis). (D) Reactions were set up as in C. Low-salt cytosol was from J774 cells that had been cultured plus or minus 1 µM Gö6976 and 10 µM bisindolylmaleimide for 1 h and then received solvent or 1.62 pM PMA for 1 h. (E) Reactions were set up with low-salt cytosol from J774 cells grown under control conditions. The indicated drugs were added in vitro at the concentrations used above. (F) RAW cells were loaded with fluorescent dextran and exposed to 1-µm IgG beads at 15°C as in Fig. 1B and chased at 37°C for 15 min. Where indicated, 10 µM Gö6976 was present during the final hour at 15°C and during the 37°C chase. For each condition, two representative sets of images are shown. Arrowheads point at selected phagosomes.
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Fig. 5.. IgG signaling activates the tethering/docking phase of lysosome/phagosome targeting. (A) Cell-free scintillation proximity assays were performed as in Fig. 2A. Low-salt cytosol was obtained from J774 cells cultured for 1 h plus or minus IgG beads. In vitro reactions were set up in the absence or presence of 1 µM latrunculin A (LA) and 20 µM cytochalasin D (CD) as indicated and incubated for 1 h at 37°C. Proximity scintillation was determined in a scintillation counter (black bars). Reactions then were supplemented with 0.1 M sodium carbonate (pH 11) and vortexed for 10 s, and scintillation was immediately measured again (gray bars). (B) Cell-free reactions were set up with low-salt cytosol from J774 cells cultured for 1 h in the absence or presence of 1.6 nM PMA as indicated. Latrunculin A and cytochalasin D were added in vitro as in A. Scintillation was measured before and after carbonate treatment as above. Background scintillation counts in the absence of low-salt cytosol were 264 ± 34 cpm (A) and 166 ± 21 cpm (B).
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