SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

Abstract

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted "flipped" cell-cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type-specific exocytic processes such as platelet secretion.

Keywords: CTL; Munc18-2; SM protein; SNARE; Syntaxin 11.

Fig. 1.

Flipped STX11–TMD drives complete fusion, whereas flipped STX11–GPI mainly promotes lipid mixing. (A) Cell fusion assay design monitoring lipid mixing and content mixing. CHO cells (GM1⁻) that expressed flipped VAMP2, -3, -4, -7, or -8 were labeled in the cytoplasm with RFP fused to nuclear export signal (RFP-nes). MEF 3T3 cells (GM1⁺) that express flipped t-SNAREs, including STX11 with the PDGF receptor transmembrane domain or a GPI anchor, were labeled in the nucleus with CFP-nls. The t cells were harvested with an EDTA buffer and overlaid on the v cells. Cells were fixed after 12 h at 37 °C. GM1 was stained with FITC-cholera toxin β-subunit (green). Complete fusion resulted in cells contained red cytoplasm, cyan nuclei, and green cell surface staining. In cells that underwent lipid mixing, GM1 transferred from t cells to the contacting CHO v cells in the absence of the mixing of the cytoplasmic markers. In the no-fusion cells, all of the markers remained within the original cells. (B) The domain structure of flipped SNAREs or GPI-anchored SNAREs. The preprolactin signal sequence (SS, dark green) was fused to the N terminus of VAMPs, SNAP23, and the STX11. The cysteine-rich domain of STX11 (CCxCC residues 277–286) was replaced by either the transmembrane domain (TMD) of the platelet-derived growth factor (PDGF) or by the GPI-anchoring sequence of decay accelerating factor. A Myc tag (light green) was engineered between the N terminus of the STX11 full-length (residues 1–276, STX11, or to the STX11–H3 domain (amino acids 199–276, STX11^H3) and the signal sequence. Cysteine residues of STX11 and SNAP23 were mutated for serines and the putative N-glycosylation of SNAP23 sites were mutated from asparagine to alanine. (C) Representative confocal images of a cell fusion experiment showing complete fusion events (Top row, asterisk), lipid mixing events (Middle rows, arrows), and no fusion (Lower row, arrowheads). (Scale bar, 5 μm.) (D) CHO v cells expressing flipped VAMPs and RFP-nes were detached and overlaid onto MEF 3T3 cells expressing flipped STX11 constructs, flipped SNAP-23, and CFP-nls. The cells were fixed after 12 h at 37 °C. GM1 was stained with FITC-cholera toxin β-subunit (green). Graph shows the percentage of v and t cells in contact that underwent complete fusion (solid bars) or lipid mixing (dashed bars). (E) Time course of complete fusion and lipid mixing mediated by flipped STX11–TMD, –GPI, or STX11–GPI in presence of cytoplasmic domain of VAMP8 (cdV8). CHO v cells were mixed with MEF 3T3 stable t cells. At different time points, the percentage of v and t cells in contact that underwent complete fusion (solid lines) or lipid mixing (dashed lines) was determined using the assay described in D. Images in 50 random fields were used for calculation of each time point. Values are mean ± SE of three independent experiments. *P < 0.1; **P < 0.01.

Fig. S1.

Flipped SNAP25 and SNAP23, but not SNAP29, promote cell surface expression of flipped STX11 constructs. (A) MEF 3T3 cells were transiently cotransfected with either STX11–PDGF, Syx11^H3–PDGF, STX11–GPI, or STX11^H3–GPI and with flipped SNAP25, -23, -29, or vector alone. Cells were fixed and incubated in the presence (permeab.) or absence (nonpermeab) of 0.1% Triton X-100. Cells were stained with anti–c-myc antibody and images were taken using confocal microscopy. (B) Cell surface quantification of different flipped STX11 constructs. Total fluorescent intensity per cell in nonpermeabilized cells stained with anti–c-myc antibody as shown in A were quantified in 75 cells for each condition. Results are mean ± SD of three independent experiments.

Fig. S2.

Characterization of the antibodies used in assays. (A) Anti-STX11 antibody recognizes both endogenous STX11 as well as ectopically expressed EYFP–STX11 construct in human CTL (CD8⁺) and HeLa cells. HeLa cells and CTLs were transiently transfected using an empty vector (mock-transf.) or EYFP–STX11 and lysates of these cells were analyzed by Western blot (W.B.) using both anti-GFP (Upper) and anti-STX11 (Lower) antibodies. (B) Anti-STX11 antibody recognizes recombinant STX11 expressed and purified from bacteria. Different amounts of recombinant STX11 were loaded in a gel and analyzed by Western blot using anti-STX11 antibody. (C) Anti-STX11 antibody specifically recognizes human STX11. Human CTLs were treated with either STX11, STX3, or nontargeting (NT) siRNAs for 48 h and cell lysates were analyzed by Western blot using the rabbit anti-STX11, anti-STX3, or mouse anti-tubulin. Data show that anti-STX11 antibody specifically recognizes STX11 but not STX3, which is the syntaxin with the highest amino acid sequence homology within the epitope recognized by the antibody. (D) Human CTLs do not express SNAP25. The expression of SNAP25 was analyzed in lysates of CTLs (CD8⁺), wild-type HeLa cells (HeLa-wt), or HeLa cells transiently transfected with SNAP25 construct by Western blot using a mouse monoclonal anti-SNAP25 antibody (clone 71.2). (E) CTLs cells do not express VAMP2. The expression of VAMP2 was analyzed in lysates of CTLs (CD8⁺), wild-type HeLa cells (HeLa-wt), or HeLa cells transiently transfected with VAMP2 construct by Western blot using a mouse monoclonal anti-VAMP2 antibody (clone 61.9). (F) Expression of endogenous VAMP2 in human CTLs, YTS (human NK cell line), and HeLa cells is below the detection level of the affinity-purified anti-VAMP2 polyclonal antibody. Equivalent amounts of cell lysates of the indicated cells were analyzed by Western blot for the expression of VAMP2 using a polyclonal antibody anti-VAMP2. Blots in the figures are representatives of three independents experiments. (G) Specificity of Munc18-2 antibodies. Human CTLs were treated with either Munc18-2 or nontargeting (NT) siRNAs for 48 h and cell lysates were analyzed by Western blot using the goat anti–Munc18-2, rabbit anti–Munc18-2, or mouse anti-tubulin. Data show a reduction of the only band detected with the goat anti–Munc18-2 antibody (Upper, arrowhead) and of one of the three bands detected by the rabbit anti–Munc18-2 antibody (Lower, arrowhead).

Fig. 2.

Munc18-2 promotes the transition from incomplete to complete fusion. (A) Cell fusion experiments were performed as described in Fig. 1 using STX11–GPI (orange bars) or STX11^H3–GPI (cyan bars) in the absence (control) or presence of either 5.0 μM Munc18-2^WT and 5.0 μM cdV8, 5.0 μM Munc18-2^WT, 5.0 μM Munc18-2^R65Q, or 5.0 μM Munc18-2^E132A. Graph shows the percentage of v and t cells in contact that underwent complete fusion (solid bars) or lipid mixing (dashed bars). Values are mean ± SE of three independent experiments using different preps of Munc18-2 proteins. (B) Dose-dependent effect of Munc18-2^WT of cell fusion outcomes mediated by STX11–GPI. Cell fusion experiment was performed as Fig. 3C in the presence of increasing concentration (0, 2.5, 5.0, and 10 μM) of Munc18-2^WT. (C) Time course of complete fusion and lipid mixing mediated by flipped STX11–GPI in the absence (blue lines) or presence of either 5.0 μM Munc18-2^WT alone (red lines) or with cytoplasmic domain of VAMP8 (cdV8, gray lines). CHO v cells were mixed with MEF 3T3 stable t cells. At different time points, the percentage of v and t cells in contact that underwent complete fusion (solid lines) or lipid mixing (dashed lines) was determined using the assay described in B. Images in 50 random fields were used for calculation of each time point. Values are mean ± SD of three independent experiments. *P < 0.1; **P < 0.01.

Fig. 3.

Endogenous STX11 interacts with SNAP23 and VAMP8 in activated CTLs. (A) Normal human donor CTLs purified from peripheral blood were activated using beads coated with anti-CD3 and -CD28 antibodies for 4 h. Endogenous STX11 and STX5 were immunoprecipitated from cell lysates using either rabbit anti-STX11 or rabbit anti-STX5 antibody and the indicated coimmunoprecipitated SNAREs or Munc18-2 were analyzed by Western blotting. (B) Bands in the Western blot that corresponded to the fraction of VAMP4/7/8 that coprecipitated with either STX11 or STX5 were quantified by densitometry and normalized to the total amount of STX11 or STX5, respectively, and immunoprecipitated in the same lane. Results represent mean ± SD of three independent experiments. (C) Endogenous VAMP8 was immunoprecipitated from activated CTL lysates as in A, and the indicated coimmunoprecipitated SNAREs or Munc18-2 was analyzed by Western blotting. Note that a goat anti–Munc18-2 antibody was used for Western blot in A, D, and E, giving a single band, whereas a rabbit anti–Munc18-2 antibody was used in C, giving a doublet. (D and E) HeLa cells were transiently transfected with HA–STX11. Immunoprecipitation from cell lysates was performed using either anti-HA (D) or IgG control antibody (E), and coimmunoprecipitated VAMP8, SNAP23, and Munc18-2 were analyzed by Western blotting. Blots are representative of three experiments.

Fig. 4.

VAMP8 or VAMP2 bind to a recombinant STX11/GST–SNAP23 complex. (A) Equivalent amounts of recombinant GST (GST alone) or GST–SNAP23 were bound to glutathione-Sepharose beads, and increasing concentrations (0.5, 1.0, or 1.5 μg) of recombinant VAMP2, -3, -4, -7, and -8 were added to the beads in the absence or presence of 1.0 μg recombinant STX11 (amino acids 1–276) and incubated for 1 h at room temperature. Bound fractions were analyzed by SDS/PAGE and Coomassie blue staining. Proteins-alone lane shows representative images of the total load of each protein used in these experiments. (B) Plot showing quantification of the amount of VAMP protein bound to the beads normalized to the amount of STX11 retained in each experiment. Data represent mean ± SD of three independent experiments. (C) Recombinant GST–STX11 was bound to glutathione-Sepharose beads, and increasing concentrations (0.5, 1.0, or 1.5 μg) of recombinant SNAP23 or VAMP2, -3, -4, -7, and -8 were added to the beads. Bound fractions were analyzed by SDS/PAGE and Coomassie blue staining. Proteins-alone lane is identical to the one shown in A because both experiments were performed in parallel with the same batch of proteins. (D) Recombinant His–SUMO–STX11 was incubated with SNAP23 and either VAMP8 or VAMP7 for 1 h at room temperature, and the formation of high molecular weight complexes was analyzed by SDS/PAGE without boiling the samples. Arrowhead shows the band corresponding to His–SUMO–STX11, which largely disappeared upon SNARE complex formation with SNAP23 and VAMP8 (asterisk), but not with VAMP7. Proteins-alone lane shows the load of each protein used to generate the SNARE complexes. M.W., molecular weight markers (shown to the Right of the gel).

Fig. 5.

Munc18-2 binds to STX11 alone and to STX11/SNAP23/VAMP8 SNARE complex. (A) Coimmunoprecipitation experiments using lysates generated from normal control human CTLs activated with beads coated with anti-CD3 and CD28 antibodies. Endogenous Munc18-2 was immunoprecipitated using an anti–Munc18-2 antibody and the amount of the indicated SNAREs that coimmunoprecipitated was analyzed by Western blotting. (B) Pull-down experiments in which equivalent amounts of recombinant GST protein alone (GST alone), GST–STX11(amino acids 1–276), or GST–STX11–H3 (amino acids 158–276) were bound to GS beads and increasing concentrations (0, 1.0, 2.5, or 5.0 μM) of recombinant Munc18-2^WT was added to the beads. Bound fractions were analyzed by SDS/PAGE and Coomassie blue staining. Proteins-alone lanes show representative images of the proteins used during the experiment. (C) Pull-down experiments in which equivalent amounts of recombinant GST–STX11 or GST–STX11–H3 were bound to GS beads in the presence of SNAP23 and soluble VAMP8. Beads were extensively washed to remove any unbound soluble protein and then they were incubated with increasing concentrations (0, 1.0, 2.5, or 5.0 μM) of recombinant Munc18-2^WT. Bound fractions were analyzed by SDS/PAGE and Coomassie blue staining. Gels are representative of two independent experiments.

Fig. S3.

Munc18-2 promotes transition from incomplete to complete fusion by acting on partially assembled SNARE complex. (A) Schematic representation of order of addition experiments using the cell fusion assay as described in Fig. 1A in which Munc18-2^WT was added at different times as indicated. Prefusion condition, Munc18-2^WT was added at the time the v- ant t- cells were mix (Pre); Postfusion, Munc18-2^WT was added after 10 h of the v ant t cells were mix (post); Munc18-2^WT (partially assembled) or buffer (control) were added at the time of mixing v ant t cells, incubated for 6 h and cdV8 was added for an additional 6 h. (B) Graph showing the percentage of cells that underwent complete fusion (black bars) and lipid mixing (striped bars) in the cell fusion experiments described above in A.

Fig. 6.

Munc18-2 induces STX11/SNAP23/VAMP8 complex formation. (A) Pull-down experiments in which equivalent amount of recombinant GST–SNAP23 was bound to GS beads and incubated for a short period (0, 1, 5, and 10 min) with either 5.0 μM cdV8 and 5.0 μM Munc18-2 in the absence of His–SUMO–STX11 (Left), or with of 5.0 μM cdV8 and 5.0 μM His–SUMO–STX11 in the absence (Middle) or in the presence of 5.0 μM Munc18-2 (Right). Bound fractions were analyzed by SDS/PAGE and Coomassie blue staining. Proteins-alone lane shows representative images of the proteins used during the experiment. (B) Plot shows the quantification of the amount of cdV8 protein bound to the beads normalized by the amount of STX11 retained on each condition in the absence (black) or in the presence (red) of Munc18-2. Gel and plots are mean ± SD of three independent experiments.

Fig. S4.

Munc18-2 induces STX11/SNAP23/VAMP8 complex formation. (A) Schematic representation showing the assessed conditions for the trans-SNARE complex formation assay. Cells expressing flipped STX11–GPI, with or without flipped SNAP23 (Lower), were incubated with VAMP8 protein reconstituted onto artificial liposomes in the presence or absence of recombinant Munc18-2 protein for the indicated period 0, 15, 30, or 60 min. After this time, cells were washed with cold PBS and treated with 1 unit/mL of PI-PLC for 15 min at 37 °C to cleave off STX11 from its GPI-anchor motif. Supernatants were collected and STX11 was IP using anti-STX11 antibody. (B) Bound fractions for each IP described in A were analyzed by Western blot using anti-STX11 and anti-VAMP8 antibodies. Asterisks mark an unspecified band obtained with anti-STX11 antibody. (C) Plot shows the quantification of the amount of co-IPed VAMP8 protein normalized by the amount of STX11 retained on each condition in the absence (black) or in the presence (red) of Munc18-2. Gel and plots are mean ± SD of three independent experiments.

Fig. 7.

Munc18-2 and the STX11/SNAP23/VAMP8 SNARE complex is required for CTL-mediated cytotoxicity. (A) Cytotoxicity assay to measure CTL-mediated cell killing of human CTLs electroporated with either nontargeting (NT), Munc18-2, STX11, STX3, or VAMP8 siRNAs and cultured for 48 h. Equivalent numbers of CTLs (effectors) from each condition were incubated with anti-CD3 antibody in the presence or absence of P815 target cells (targets) at the indicated cell ratios. The killing assay was run for 4 h at 37 °C and the amount of lactate dehydrogenase (LDH) released into the supernatant was quantified using a Cytotox-96 assay. Knockdown efficiency for each siRNAs is shown in Fig. S5 A and B. Results are the mean ± SD of three independent measurements for each condition. *P < 0.01. (B) Human CTLs transfected with EYFP–STX11 using the Neon electroporation system were incubated in the presence of anti-CD3 antibody and P815 cells at a 1:1 ratio for 15 min at 37 °C and seeded onto polylysine-coated coverslips. Cells were fixed, permeabilized, and stained using first goat anti–Munc18-2 and followed by anti-perforin–Alexa 647 antibodies. (Scale bar, 3 μm.) Arrows show perforin granules close to the IS. Arrowheads show accumulation of EYFP–STX11 and Munc18-2 at the IS membrane. (Scale bar, 3 μm.)

Fig. S5.

Munc18-2 and the STX11/SNAP23/VAMP8 SNARE complex is required for CTL-mediated cytotoxicity. (A) Human CTLs were electroporated with either nontargeting (NT), Munc18-2, STX11, STX3, or VAMP8 siRNAs and cultured for 48 h. Cell lysates were analyzed by Western blot for the indicated proteins. (B) Plot showing the quantification of the knockdown efficiency of each protein compared with NT siRNA-treated cells as shown in A. Blots are representative of three independent experiments.