SNAP-23 regulates phagosome formation and maturation in macrophages

Abstract

Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus-tagged SNAP-23 was established. These cells showed enhanced Fc receptor-mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H(+)-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.

FIGURE 1:

Stable overexpression of mVenus-SNAP-23 in J774 macrophages. (A) Total lysates from J774 macrophages stably expressing mVenus (mV), mVenus-SNAP-23 (mV-S23), and mVenus-SNAP-23ΔC8 (mV-S23ΔC8) were analyzed by Western blotting using the indicated antibodies. (B) Confocal live-cell imaging of J774 cells expressing mV-S23 or mV-S23ΔC8 during the ingestion of Texas Red–conjugated zymosan A (TR-zymosan) particles previously opsonized with rabbit anti-zymosan IgGs. The asterisks denote the phagosomes containing Texas Red–conjugated zymosan A particles. Scale bar: 10 μm. (C) Three-dimensional SIM image of J774/mV-S23 cells during the ingestion of IgG-opsonized latex beads (3.0 μm in diameter). Beads outside the cells were stained with an anti-IgG Alexa Fluor 594 antibody. The asterisks denote the bead-containing phagosomes. (D) J774/mV, J774/mV-S23, and J774/mV-S23ΔC8 cells were incubated with IgG-opsonized FITC-zymosan particles, and the efficiency of association and FcR-mediated phagocytosis were then measured as described in Materials and Methods. Arbitrary fluorescence units were normalized to the maximum value obtained for mV cells within the same experiment, defined as 100%. Data presented are the mean ± SE of three independent experiments.

FIGURE 2:

mVenus-SNAP-23 enhances not only phagosome maturation processes but also uptake activity. (A) J774/mV, J774/mV-S23, and J774/mV-S23ΔC8 cells were incubated with nonopsonized FITC-conjugated microbeads to quantify a phagocytosis efficiency for synthetic particles, as described in Materials and Methods. Arbitrary fluorescence units were normalized to the maximum value obtained for mV cells within the same experiment, defined as 100%. Data presented are the mean ± SE of six independent experiments. (B) The J774 cells expressing mVenus-tagged proteins were incubated with luminol-bound microbeads. The efficiency of ROS production within the phagosomes of the cells was determined by measuring chemiluminescence from cells that had ingested the beads. Chemiluminescence was measured on a GloMax 20/20n luminometer every 1 min for up to 15 min. Relative light units were normalized to the maximum value obtained for mV cells within the same experiment, defined as 100%. The amount of signal from mV and mV-S23 cells incubated with beads in the presence of cytochalasin B (cyto B; final: 10 μM) was not significant. Data presented are the mean ± SE of three independent experiments. (C) Left, the cells were stained with LysoTracker Red DND-99 (final: 50 nM) for 20 min. After being washed in HBSS, each cell line's fluorescence intensity was measured, as described in Materials and Methods. Arbitrary fluorescence units were normalized to the value obtained for mV cells within the same experiment, defined as 100%. Data presented are the mean ± SE of five independent experiments. Right, these LysoTracker-treated cells were incubated first with IgG-opsonized latex beads (3.0 μm in diameter) for 5 min at 30°C to allow phagosome formation, and the cells were incubated at 30°C in the presence of cytochalasin B (final: 20 μM) to halt the initiation of phagocytosis. At the indicated time points, the cells were cooled on ice, and then LysoTracker-positive phagosome and nonlabeled phagosome were analyzed under a microscope, as described in Materials and Methods. The results are expressed as the percentage of LysoTracker-positive phagosome (more than 30 individual phagosomes from at least 30 different cells for each experiment) at the indicated time points. Data presented are the mean ± SE of five independent experiments. *, p < 0.02, compared with control mV cells using Student's paired t test, one-tailed. (D) The cells were incubated with IgG-opsonized Texas Red–zymosan particles, and the efficiency of FcR-mediated phagocytosis was then measured, as described in Materials and Methods. Arbitrary fluorescence units were normalized to the maximum value obtained for mV cells within the same experiment, defined as 100%. Data presented are the mean ± SE of three to six independent experiments. *, p < 0.005, compared with control mV cells using Student's paired t test, one-tailed.

FIGURE 3:

mVenus-SNAP-23 recruits the NOX2 complex and H⁺-ATPase to phagosomes. J774/mV and J774/mV-S23 cells were incubated with IgG-opsonized latex beads for a 10-min pulse at 37°C (10/0). After a 120-min chase incubation (10/120), the phagosome fraction was isolated from the cells by sucrose density-gradient centrifugation. The total cell extract (9 and 3 μg) and the isolated phagosome fraction (3 μg) were analyzed by SDS–PAGE and subsequently by Western blotting with the indicated antibodies (left). LAMP-1 is a lysosomal marker protein. The intensity of the signals on the Western blot (left) was quantified by densitometry using ImageJ Version 1.44 (National Institutes of Health, Bethesda, MD), with the values for each protein expressed as a ratio of phagosome (10/0 or 10/120) to extract containing 3 μg of protein (right).

FIGURE 4:

SNAP-23 interacts with syntaxin 11 and/or endocytic SNARE proteins more effectively than its C-terminal truncated form. J774 cells stably expressing mV, mV-S23, or mV-S23ΔC8 were lysed, and the lysates were immunoprecipitated (IP) with anti-EGFP antibodies. The immunocomplexes were subjected to SDS–PAGE, which was followed by Western blot analysis using the indicated antibodies.

FIGURE 5:

Knockdown of SNAP-23 expression inhibits FcR-mediated phagocytosis. (A) J774 cells were transfected with SNAP-23 siRNAs (#1 and #2) or a control nonspecific siRNA. Total cell lysates from siRNA-transfected cells were analyzed by Western blotting using the indicated antibodies (see also Figure S4). The bands of the Western blotting experiment were quantified using ImageJ. The value for each protein was expressed as a ratio of SNAP-23 siRNA#1 or siRNA#2 cells to control siRNA cells and then normalized to the GAPDH internal control, defined as 100%. Data presented are the mean ± SE of three independent experiments. *, p < 0.01, **, p < 0.02, compared with GAPDH in each cells using Student's paired t test, one-tailed. (B) J774 cells transfected with siRNAs were fixed and then double-stained with antibodies against SNAP-23 (green) and GM130 (red), a Golgi marker protein. SNAP-23 expression was efficiently reduced in almost all cells. Scale bar: 10 μm. (C) J774 cells transfected with siRNAs were analyzed by the luminol bead assay described in Materials and Methods. Relative light units were normalized to the maximal value obtained for control siRNA cells within the same experiment, defined as 100%. Transfection with the SNAP-23 siRNA reduced the efficiency of phagosomal ROS production. Data presented are the mean ± SE of three independent experiments. (D) siRNA-transfected cells were incubated with IgG-opsonized Texas Red–zymosan particles and the efficiencies of association (left) and phagocytosis (right) were measured, as described in Figure 1D. Arbitrary fluorescence units of each cell line were normalized to the maximal value obtained for control siRNA cells within the same experiment, defined as 100%. Data presented are the mean ± SE of three independent experiments. *, p < 0.005, compared with control siRNA cells using Student's paired t test, one-tailed.

FIGURE 6:

Knockdown of SNAP-23 expression delays phagosome maturation. (A) J774 cells stably expressing FcγRIIA fused to TagRFP at its C-terminus (J774/RIIa-TagRFP) were transfected with control siRNA or SNAP-23 siRNA#1. These cells were incubated first with IgG-opsonized EGFP-bound beads for 5 min to allow phagosome formation (phagocytosis) and then in the presence of cytochalasin B at a final concentration of 20 μM (chase incubation). At the indicated time points, the cells were cooled on ice and subsequently treated with sodium citrate buffer (pH 4.0) to quench the EGFP signal of beads outside the cell, as described in Materials and Methods. Microscopic analysis of the phagosome enclosed with RIIa-TagRFP is presented as follows. (B) Left, arrows denote phagosomes enclosed with RIIa-TagRFP (bottom, red fluorescence) and containing an EGFP-bound bead. These phagosomes were classified into four types depending on the EGFP signal intensity (middle). Scale bar: 10 μm. Right, each type of phagosome was quantified over time. The results are expressed as a percent of the total number of the four phagosome types at the indicated time points. Data presented are the mean ± SE of seven independent experiments. The fraction of type D phagosomes in SNAP-23 siRNA cells was significantly reduced at 30 min (Figure S6) compared with control siRNA cells. (C) At 2 d after transfection with siRNAs, J774 cells were incubated with RB-dextran (final: 50 μg/ml) for 12 h. The medium was replaced with fresh dextran-free growth medium, and the cells were chased for 5 h. These cells were incubated first with IgG-opsonized beads for 5 min at 30°C to allow phagosome formation. After the beads outside the cells were stained with Alexa Fluor 488–conjugated secondary antibodies on ice, the cells were incubated at 30°C in the presence of cytochalasin B (final: 20 μM). At the indicated time points, the cells were cooled on ice and then RB-dextran–positive phagosome and nonlabeled phagosome were analyzed under a microscope, as described in Materials and Methods. The results are expressed as the percentage of RB-dextran–positive phagosomes (more than 30 individual phagosomes from at least 30 different cells for each experiment) at the indicated time points. Data presented are the mean ± SE of four independent experiments. *, p < 0.005, **, p < 0.001, compared with control siRNA cells using Student's paired t test, one-tailed. (D) Rescue effects of SNAP-23 expression. The cells transfected with control siRNA or SNAP-23 siRNA#2, which targets to 5′ UTR of SNAP-23 mRNA, were incubated with RB-dextran for 8 h, which was followed by replacing and chasing in dextran-free growth medium for an additional 5 h prior to overnight transfection with plasmids of mVenus-tagged proteins. The cells were analyzed as described above and in Materials and Methods. The results are expressed as the percentage of RB-dextran–positive phagosomes at 15-min chase time. Data presented are the mean ± SE of four independent experiments. Student's paired t test, one-tailed.

FIGURE 7:

The FRET signal of SNAP-23 probe (tG-S1-tR-S2) on the phagosome membrane is increased by the overexpression of VAMP7. (A) Schematic representation of various SNAP-23 constructs (see Materials and Methods). Approximation of the N-termini of the two SNARE motifs (SN1(1-75) and SN2(148-211)) is expected when the complete SNARE complex is formed with other SNARE proteins for membrane fusion. For restriction of the flexibility of the fluorescent proteins at the connection, the C-terminal 11 residues were truncated from TagGFP2, a truncated form of TagGFP2 (Shimozono et al., 2006), and fused to SNAP-23. The inset shows the predicted conformation of the SNAP-23–based FRET construct upon forming fusogenic SNARE complexes. The asterisk indicates a potential lipid-modified cysteine residue. (B) Transiently expressed tG-S1-tR-S2 and tG-S1-S2-tR were predominately localized at the plasma membrane of J774 cells. Spectroscopic data were obtained using the N-SIM system. The asterisks denote the phagosomes containing IgG-opsonized latex beads (3.0 μm in diameter). Scale bar: 10 μm. (C–E) J774 cells were transiently cotransfected with SNAP-23 FRET probes and the Myc-tagged SNARE proteins as indicated. The spectroscopic function of a laser-scanning microscope was used to excite the plasma membrane of living cells at 458 nm, and the resulting emission spectra were analyzed (see Figure S9). Emission at 580 nm was divided by that at 505 nm (TagRFP/TagGFP) and normalized to the value obtained for cells cotransfected with the Myc-vector in the same experiment. This was set to 1.00. Data presented are the mean ± SE of three to six independent experiments. Student's paired t test, two-tailed. (F) J774 cells cotransfected with SNAP-23 FRET probes and the indicated Myc-tagged constructs were incubated with IgG-opsonized zymosan particles at 37°C for 20 min. Extra particles were removed in a washing step, and the FRET efficiency on the phagosome membrane of living cells was then analyzed as described above. Student's paired t test, two-tailed.