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Munc18-2 and Syntaxin 3 Control Distinct Essential Steps in Mast Cell Degranulation Cristiana Brochetta, Ryo Suzuki, Francesca Vita, Maria Rosa Soranzo, Julien Claver, Lydia Celia Madjene, Tarik This information is current as Attout, Joana Vitte, Nadine Varin-Blank, Giuliano Zabucchi, of October 2, 2021. Juan Rivera and Ulrich Blank J Immunol 2014; 192:41-51; Prepublished online 9 December 2013; doi: 10.4049/jimmunol.1301277 http://www.jimmunol.org/content/192/1/41 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Munc18-2 and Syntaxin 3 Control Distinct Essential Steps in Mast Cell Degranulation

Cristiana Brochetta,*,†,1,2 Ryo Suzuki,‡,1 Francesca Vita,x Maria Rosa Soranzo,x Julien Claver,*,† Lydia Celia Madjene,*,† Tarik Attout,*,† Joana Vitte,*,†,3 Nadine Varin-Blank,{,‖ Giuliano Zabucchi,x Juan Rivera,‡ and Ulrich Blank*,†

Mast cell degranulation requires N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) and mammalian uncoordinated18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. In this study, we found that small interfering RNA–mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE-binding partner syntaxin 3 (STX3) also markedly inhibited degranulation, whereas combined knockdown produced an additive inhibitory effect.

Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion, demonstrating unique roles of Downloaded from each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation, clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and

STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast http://www.jimmunol.org/ cells. The Journal of Immunology, 2014, 192: 41–51.

ast cells (MC) are key effectors in immunity, but are also with release of inflammatory mediators stored in abundant cyto- notoriously known to cause allergies (1). Activation plasmic secretory granules (SGs). This is followed by de novo M through IgE receptors (FcεRI) triggers degranulation synthesis and secretion of cytokines/chemokines (2). In contrast to neuronal/neuroendocrine cell exocytosis, in which individual SGs fuse at the plasma membrane (PM), MC degranulation involves † *INSERM Unite´ Mixte de Recherche-699, 75018 Paris, France; Universite´ Paris granule–granule and SG-PM fusion by compound/multigranular Diderot, Sorbonne Paris Cite, Laboratoire d’Excellence INFLAMEX, 75018 Paris, by guest on October 2, 2021 France; ‡Laboratory of Molecular Immunogenetics, National Institute of Arthritis and exocytosis (3). Despite these differences, MC and other regulated- Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD secretion competent cells share a common molecular machinery of 20892; xDepartment of Life Sciences, Physiology, and Pathology, University of Trieste, 34127 Trieste, Italy; {INSERM U978, 93000 Bobigny, France; and ‖Universite´ membrane fusion (2, 4, 5). It includes SNARE fusion (6, 7) and Paris 13, Sorbonne Paris Cite´, “Adaptateurs de Signalisation en He´matologie,” Laboratoire fusion regulatory proteins such as Munc18 (8–10), Munc13 (11, 12), d’Excellence “Inflamex,” Unite´ de Formation et de Recherche Sante´-Me´decine-Biologie Rab (13–15), complexin (16), SCAMP (17), and Synaptotagmin- Humaine, 93000 Bobigny, France 1 family members (18, 19). SNAREs promote fusion by forming a C.B. and R.S. contributed equally to this work. tetrameric complex through their SNARE motif (20). A functional 2 Current address: Centre Me´dicale Universitaire Department of Cellular Physiology complex in MC comprises the vesicular SNARE VAMP8 and the and Metabolism, School of Medicine, University of Geneva, Geneva, Switzerland. target SNAREs syntaxin (STX)4 and SNAP-23 (6, 7, 21, 22). In 3Current address: Aix-Marseille University, Assistance Publique Hopitaux de Marseille, Laboratoire d’Immunologie, Marseille, France. agreement with the heterogeneity of the SG compartment and the Received for publication May 14, 2013. Accepted for publication October 30, 2013. possibility of granule–granule fusion, other SNAREs may also play a role (9, 23–25). During degranulation, SNAP-23 redistributes from This work was supported by Investissements d’Avenir Programme ANR-11-IDEX- 0005-02, Sorbonne Paris Cite, Laboratoire d’Excellence INFLAMEX. The work of the PM into nascent degranulation channels, which may be a par- J.R. and R.S. is supported by the intramural research program of the National Insti- ticularity of compound exocytosis (6). tute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health. This work was also supported by Cooperation in Science and Technology Action Mammalian homologs of Caenorhabditis elegans uncoordinated- BM1007 (Mast cell and basophils–targets for innovative therapies) of the European 18 (Munc18) proteins are essential regulators of exocytosis, as Community. The research project of U.B. and C.B. has also been supported by evidenced after genetic deletion of the neuronal isoform Munc18-1 a Marie Curie Early Stage Research Training Fellowship of the European Commun- ity’s Sixth Framework Programme under Contract 504926 and by the Fondation de (20) and the more ubiquitously expressed isoforms Munc18-2 and Recherche Me´dicale. Munc18-3 (10, 26–28). Their action involves different SNARE- Address correspondence and reprint requests to Dr. Ulrich Blank, INSERM U699, binding modes (20, 29, 30). Whereas binding to the closed con- Faculte´ de Me´decine X. Bichat, 16 Rue Henri Huchard, 75780 Paris Cedex 18, formation of STX was thought to block SNARE assembly (31), new France. E-mail address: [email protected] evidences rather support that this complex serves as an organizer of The online version of this article contains supplemental material. productive membrane fusion, allowing privileged interactions with Abbreviations used in this article: BMMC, bone marrow–derived mast cell; FHL-5, other fusion effectors and the promotion of a conformational switch familial hemophagocytic lymphohistiocytosis type 5; GP, gold particle; IF, immunofluorescence; MC, mast cell; PM, plasma membrane; RPMC, rat peritoneal MC; RT, to an open form (20) able to bind tetrameric SNARE complexes at room temperature; SCF, stem cell factor; SG, secretory granule; shRNA, short hairpin its C terminus, thereby activating fusion (30, 32, 33). Although this RNA; siRNA, small interfering RNA; SNARE, N-ethylmaleimide–sensitive factor attachment protein receptor; STX, syntaxin; WGA, wheat germ agglutinin; WT, defines a minimal fusion entity (32), additional evidence indicates wild-type. that in living cells Munc18 proteins participate in steps promoting www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301277 42 MAST CELL DEGRANULATION CONTROL BY Munc18-2 AND SYNTAXIN 3 vesicle translocation, tethering, and docking that are required to nucleofection program. BMMC were cultured in IL-3– and SCF-containing support fusion (29). medium for 2 d until experiments. MC express the ubiquitous isoforms Munc18-2 and Munc18-3 Degranulation measurements and determination of CCL-2 (8). Multiple studies support a role for Munc18-2 in degranulation production (8–10, 34, 35). Furthermore, familial hemophagocytic lympho- b histiocytosis type 5 (FHL-5) patients carrying Munc18-2 mutations Release of granule content was determined by measuring -hexosaminidase release (7). CCL2 release was quantified using a Duoset cytokine ELISA kit causing misfolding show impaired lytic granule exocytosis in NK (R&D Systems). Degranulation of BMMC was evaluated by a CD63 expression and cytotoxic T cells (36, 37). In contrast to other immune effector assay. Briefly, BMMC were deprived of SCF overnight, followed by sensitiza- cells (34, 38, 39), a role for PM-localized Munc18-3 in MC has not tion with DNP-specific IgE in Tyrode’s buffer (7) for 3 h at 37˚C. IgE-sensitized 3 6 been demonstrated, and the implication of the neuronal-specific BMMC (2 10 cells) or unsensitized BMMC were stimulated with DNP-HSA (Ag) or PMA/ionomycin (20 ng/ml 1 mM) for 30 min at 37˚C. Cells were in- Munc18-1 is controversial (35, 40). cubated with anti-CD63 Abs (2 mg/ml; MBL International) or isotype control The cytoskeleton also plays an important role in vesicular traf- (2 mg/ml) at 4˚C, followed by incubation with Alexa Fluor 488–conjugated ficking. MC activation is accompanied by cortical actin disassembly anti-rat IgG before analysis by flow cytometry using a FACSCalibur (BD and membrane ruffling that favors access of SGs to the PM (41–43). Biosciences, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR). Similarly, the actin regulatory proteins coronin 1a/b have recently Immunoblotting and immunoprecipitation been shown to differentially effect on degranulation and cytokine 3 7 production (44). FcεRI stimulation also triggers microtubule for- IgE-sensitized or unsensitized RBL cells (1 10 ) were resuspended in medium and challenged with Ag (DNP-HSA, 100 ng/ml) or PMA/ mation through a Fyn/Gab2/RhoA signaling pathway necessary for ionomycin (20 nM/1 mM) for the indicated times. Stimulation was arrested SG translocation (43, 45). using ice-cold PBS. Lysates were prepared in 50 mM HEPES (pH 7.3) In this study, we investigated the role of Munc18-2 and STX3 containing 10 mM CHAPS, 0.1% SDS, 150 mM NaCl, 5 mM KCl, 1 mM Downloaded from in degranulation. We show that they have essential but comple- MgCl2, 1 mM sodium orthovanadate, and protease inhibitors, as described (6, 22), and used for Munc18-2–tubulin coimmunoprecipitation studies. mentary roles and define a new stimulatory Munc18-2 microtubule- Munc18-2–STX3 interactions were explored using BMMC (48 h). After dependent axis enabling SG translocation and fusion. washing, IgE-sensitized cells were resuspended in medium and challenged with DNP-HSA (10 ng/ml). Stimulation was arrested using ice-cold PBS Materials and Methods containing 0.25 mM N-ethylmaleimide for 15 min, followed by a wash in

PBS. Lysates were prepared in 50 mM HEPES (pH 7.2) containing 1% http://www.jimmunol.org/ Reagents and Abs Triton X-100, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM sodium Rabbit Abs to STX3, Munc18-2, rabbit isotype control anti-GST, mouse orthovanadate, and protease inhibitors before immunoprecipitation. Im- isotype control IgG (15.1), and mouse monoclonal anti–DNP-IgE have been munoblotting was carried out as previously described (22, 47). a described (8, 22, 46). Anti– -tubulin, DNP human serum albumin (DNP30–40- Confocal microscopy HSA, Ag), ionomycin, and PMA were purchased from Sigma-Aldrich. Alexa Fluor 555 wheat germ agglutinin (WGA) was from Invitrogen. Murine IL-3 siRNA-transfected RBL cells were seeded in 24-well plates on coverslips and stem cell factor (SCF) were from PeproTech (Paris, France). Nocodazole for 16 h at 37˚C in a humidiﬁed atmosphere with 5% CO2. After PMA/ and Paclitaxel were purchased from Merck-Calbiochem (Nottingham, U.K.). ionomycin stimulation, cells were washed and ﬁxed for 20 min on ice in The Myc–Munc18-2 plasmid was a gift from V.M. Olkkonen (BIOMEDICUM 10 mM PIPES (pH 6.8), 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, and Helsinki 2U).

5 mM glucose containing 4% paraformaldehyde (immunofluorescence by guest on October 2, 2021 [IF] buffer), followed by two washes in IF buffer. Fixed cells were per- Cell lines, primary MC cultures, and preparation of peritoneal meabilized in IF buffer containing 0.025% saponin for 20 min at room MC temperature, followed by blocking in IF buffer containing 0.012% saponin and 7% horse serum (Invitrogen) for 30 min at room temperature (RT). The rat RBL-2H3 MC line and COS7 cells were maintained, as described (8, Staining with primary Abs was performed in IF buffer containing 0.012% 22, 46). Bone marrow was isolated from femurs of C57BL/6 mice (8–12 saponin and 5% horse serum for 2 h at RT, followed by incubation with wk old), and bone marrow–derived MC (BMMC) were derived, as de- secondary Abs for 60 min at RT. Cells were mounted in Prolong-Gold scribed (22). Cell cultures were used after 4–8 wk after routine checking antifading reagent (Molecular Probes) and were analyzed using confocal for purity by evaluating expression of c-kit and FcεRI (6, 22). Wistar male laser-scanning microscope LSM 510 (Zeiss, Oberkochen, Germany). or female rats (200–400 g) were purchased from the University of Trieste. Rats were euthanized by CO2 inhalation. Rat peritoneal MC (RPMC) were Real-time imaging of CD63-GFP–expressing granules obtained and stimulated using compound 48/80, as described (22). All animal studies have been approved by the local institutional animal care For real-time imaging of secretory granules, CD63-GFP–expressing granules and use committees. were tracked. CD63-GFP–transduced BMMC were incubated with Alexa Fluor 555 WGA (5 mg/ml) and placed on poly-d-lysine–coated glass-bottom Gene modifications in RBL-2H3 cells and BMMC dishes for 10 min at 37˚C. Cells were washed and subsequently stimulated with PMA/ionomycin (20 nM/1 mM). A Zeiss LSM-510 META confocal The following small interfering RNAs (siRNAs) (sense) were used (Euro- microscope with 488-nm and 543-nm wavelength light from an argon laser gentec): Munc18-2-siRNA1 (59-GGCUCAUCGUGUACAUUG-39); Munc18- and HeNe was used for SG-PM colocalization measurements. The fluores- 2-siRNA2 (59-CGCUCACAGUUGCUCAUAA-39); STX3-siRNA1 (59-GG- cence of GFP and Alexa Fluor 555 was detected through a band pass filter CUCAACAUCGACAAGAU-39), sSTX3-siRNA2 (59-GGAGCUCCAUGA- (505–530 nm) and a long pass filter (.560 nm), respectively. Fluorescence CAUGUUU-39); and universal scramble siRNA (59-GCCCCGUCUACAU- images were collected every 2 s, and the acquired data were processed by ACAUGU-39). RBL cells were transfected with annealed siRNAs (0.05 mM Adobe Photoshop. Colocalization coefficient analysis was made using each) in two successive electroporations 24 h apart, adjusting cell density each Medical Image Processing Analysis and Visualization (National Institutes of time to 1 3 107/ml in electropermeabilization buffer (120 mmol/L KCl, 10 Health) software. Data of colocalization coefficient were normalized to the mmol/L NaCl, 1 mmol/L KH PO , 10 mmol/L glucose, 20 mmol/L HEPES 2 4 starting point (t = 0). Increase of value indicates SG-PM membrane fusion. [pH 7.0]) at 250 V and 2100 mF. Cells were used for functional analysis the Imaris software (Bitplane AG, Zurich, Switzerland) was used for tracking of next day. For gene silencing of BMMC, short hairpin RNA (shRNA) targeting CD63-GFP–expressed SG and for analyzing granule velocities by the spot- of Munc18-2 or STX3 was performed using lentiviral vectors. Viruses were detection function using the following parameters: autoregressive motion, produced by transfecting vectors with Munc18-2 or STX3 shRNAs into the gapclose 2, 2 mm maximum distance. Average velocities of each granule packaging cells 293LTV using Lipofectamine 2000 (Invitrogen), as described were analyzed before (1–20 frames) and after (21–50 frames) stimulation. (47). For infection, 10-d-old bone marrow cells were resuspended in the concentrated viral supernatants for 2 d at 37˚C with 5% CO2. Postinfection, Electron microscopy cells were grown in IL-3– and SCF-containing medium for 2 d before initiating selection of transduced cells with 8 mg/ml blasticidinS (Invitrogen) for 2 wk. Double immunogold labeling of RPMC ultrathin sections was as described For transient expression of CD63-GFP, Nucleofection (Amaxa) was per- before (22, 34). Briefly, RMPC were fixed in 1.5% glutaraldehyde (Serva) formed, according to the manufacturer’s instructions, using mouse macrophage diluted in 0.1 M cacodylate buffer (pH 7.4) stored for 20 min at room The Journal of Immunology 43

temperature, postfixed in 1% OsO4 for 60 min at 4˚C, dehydrated in eth- two-tailed Student’s t tests or by one-way ANOVA, as appropriate. Two- anol, and finally embedded in Dow Epoxy Resin (DER 332; Unione Chi- way ANOVA was used to examine the overall effects of phenotype and mica Europea, Milano, Italy). For double immunogold labeling, ultrathin time on the changes in granule-plasma membrane colocalization. Differences sections, cut by an ultramicrotome (Ultracut UCT; Leica, Wien, Austria), were considered significant if the p value was #0.05 with the confidence were mounted on nickel grids etched for 1 min with 1% periodic acid and intervals of 95%. rinsed in distilled water three times (2 min each time). The grids with the section sides facing downward were incubated in 20 mM Tris-HCl (pH 8.2), containing 2% BSA, 1% goat serum, 0.05% Tween 20, and 0.1% Triton X- Results 100 containing rabbit anti–Munc18-2 (50 mg/ml) at 4˚C overnight. After Munc18-2 and STX3 have independent roles and complement washing in 20 mM Tris-HCl (pH 8.2) containing 225 mM NaCl, 20 mM in MC degranulation NaN3, 0.05% Tween 20, 0.5% BSA, 0.1% Triton X-100, and goat serum 0.5% grids were incubated for 1 h at room temperature with 10 nm gold- We explored the role of Munc18-2 in MC secretory responses by conjugated goat anti-rabbit (British Biocell International, Cardiff, U.K.) siRNA-mediated silencing in RBL-2H3 MC. Munc18-2 expression diluted 1:50 in Tris-HCl-BSA-Triton. After rinsing thoroughly, grids were was downregulated in RBL MC using siRNA. Consistent with the turned over with the section sides facing upward, according to Bendayan m extent of specific knockdown (Fig. 1A), IgE-induced release of (48), and exposed to rabbit anti-STX3 (50 g/ml). The procedure followed b thereafter was as described above, with the exception that 20 nm gold- the granule-stored enzyme -hexosaminidase was diminished (Fig. conjugated protein A-G (British Biocell International) diluted 1:50 in 1B). RNA silencing of the Munc18-2–interacting partner and SG- Tris-HCl-BSA-Triton was used as revealing system. Sections were analyzed localized SNARE protein STX3 (Fig. 1C) also inhibited IgE- by a Philips EM208 transmission electron microscope mounted to a 4008 3 induced degranulation with two independent siRNAs (Fig. 1D and 2672 pixel Morada camera (Olympus). Control experiments using irrelevant IgG performed in parallel showed no significant immunogold labeling. data not shown). Interestingly, combined silencing of STX3 and Munc18-2 resulted in an additive inhibitory effect (Fig. 1D), dem- Statistical analysis onstrating that they can contribute to degranulation independently of Downloaded from Results were analyzed using GraphPad Prism (GraphPad Software). Sta- each other. Degranulation was also inhibited after stimulation with tistical analysis of differences between groups was determined by unpaired, PMA/ionomycin (Fig. 1D), which bypasses early FcεRI-induced http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Additive effect of Munc18-2 and STX3 on MC degranulation. (A) Munc18-2 expression in RBL cells treated with indicated Munc18-2– specific siRNAs. The means of relative expression levels compared with scrambled siRNA are indicated below lanes (n = 6). (B) Effect of Munc18-2 silencing on b-hexosaminidase release after stimulation with IgE/Ag. Results are means 6 SEM of six experiments. **p , 0.01. (C) Degree of silencing of Munc18-2, STX3, or both. One representative experiment (n = 3) is shown. (D) Effect of silencing of Munc18-2, STX3, or both on b-hexosaminidase release after stimulation with IgE/Ag or PMA/ionomycin. Results are means 6 SEM of three experiments. *p , 0.05, **p , 0.01. (E) Effect of silencing Munc18-2, STX3, or both on IgE/Ag-induced CCL2 chemokine secretion. Released CCL2 at 4 h in cells treated with scrambled siRNA was arbitrarily set to 100%. Results are means 6 SEM of three experiments. 44 MAST CELL DEGRANULATION CONTROL BY Munc18-2 AND SYNTAXIN 3 signaling, supporting the view that both effectors function in the pared with PMA/ionomycin-stimulated cells (Supplemental Fig. late steps of stimulus-secretion coupling. This is also supported by 2). Munc18-2 silencing did not affect microtubule formation (Fig. 2, data showing that more upstream responses such as calcium mo- lower right panel); however, SG movement to the periphery was bilization were not affected (data not shown). No effect of Munc18-2 markedly altered with most SG remaining in the cytosol, although silencing on STX3 and Munc18-3 expression was observed (Fig. 1C they remain attached to microtubules. This indicated that the in- and data not shown). This excludes a chaperon function of hibitory effect on degranulation in the absence of Munc18-2 was Munc18-2 for STX3, as observed for other Munc18-binding proteins due to the inhibition of SG translocation and subsequent incorpo- (36, 49). We also analyzed the effect on de novo CCL2 chemokine ration into the PM. secretion. Neither silencing of Munc18-2 or of STX3, nor a combined To further confirm and extend these findings, we used dynamic knockdown of both, affected IgE-induced CCL2 release when com- image analysis of primary bone marrow–derived MC (BMMC) pared with cells transfected with scrambled siRNA (Fig. 1E). As after silencing the expression of Munc18-2 or its binding partner STX3 has not been previously implicated in MC degranulation (7), STX3. Cells were transduced with lentiviruses encoding shRNAs we evaluated the respective contribution of STX3 and STX4 in the corresponding to Munc18-2 (shM18-2), STX3 (shSTX3), or scram- degranulation response. Surprisingly, siRNA experiments (Supple- bled controls (shScr). Fig. 3A shows that partial, but specific si- mental Fig. 1) showed that the effect of STX3 silencing was more lencing of Munc18-2 or STX3 was achieved when compared with pronounced than that of STX4. No additive effect was observed when wild-type (WT) or scrambled control shRNA. Like in RBL cells, both STXs were downregulated. silencing of STX3 had no effect on Munc18-2 expression or vice versa. Viral transduction also did not affect expression levels of Silencing of Munc18-2 expression restricts SG translocation FcεRI or the general phenotype or growth properties of these cells and fusion (Fig. 3B and data not shown). As observed before in RBL cells, Downloaded from To explore the mechanism of action of Munc18-2, in MC we in- silencing of Munc18-2 or STX3 markedly diminished cell surface vestigated the consequences of silencing its expression on the expression of CD63 (a surrogate marker of MC degranulation at location of SGs in resting and stimulated RBL MC. SGs were the PM) after IgE/Ag or PMA/ionomycin-induced degranulation labeled with an Ab to STX3 (a SG resident protein), and their when compared with WT or control shRNA-transduced cells (Fig. distribution was compared with microtubules, based on findings 3C–E). Release of granule-stored b-hexosaminidase was similarly that SGs are mobilized along microtubules (8, 43, 45). Fig. 2 shows inhibited (Supplemental Fig. 3). To follow SG movement and SG http://www.jimmunol.org/ that in resting, siRNA controls, SGs (in green) are randomly dis- fusion events with the PM in real time, we additionally transfected tributed along microtubules (in red) and often concentrate around cells with CD63-GFP, which incorporates into MC SGs (43). The the microtubule-organizing center or in proximity to the PM (Fig. PM was labeled by loading cells with Alexa Fluor 555–conjugated 2, upper left panel). This distribution was not altered in Munc18- WGA, and cells were then stimulated using PMA/ionomycin. The 2–silenced cells (Fig. 2, lower left panel). PMA/ionomycin stim- dynamics of SG movement and their tracking using real-time CD63- ulation of control siRNA-treated cells induced the formation of GFP imaging analysis are shown in Supplemental Movies 1A–D. microtubular tracks reaching out to the plasma membrane into Representative images of the monitored fusion events during time filopodia-like extensions (Fig. 2, upper right panels). At the same (yt and xt) as compared with the xy image at the beginning and by guest on October 2, 2021 time, SGs aligned along these tracks and substantial STX3 end of the stimulation period are depicted in Fig. 4A. In WT (Fig. immunolabeling was observed at the periphery, indicating that 4A [WT], Supplemental Video 1A) or control shRNA-treated SGs had translocated and fused with the PM. Similar data were BMMC (Fig. 4A [shScr], Supplemental Video 1B), SGs became also obtained when cells were stimulated with IgE/Ag, albeit STX highly mobile and moved toward the PM (open arrowhead in Fig. membrane translocation was somewhat less prominent as com- 4A [WT, shScr]). Velocities of these SGs were increased after

FIGURE 2. Munc18-2 knockdown in RBL cells inhibits SG translocation to the plasma membrane. RBL cells treated with scrambled control (upper panel) or Munc18-2 (lower panel) were grown on coverslips and stimulated with PMA/ionomycin for 20 min (right panels) or not (left panel). Cells were ﬁxed, permeabilized, and stained with anti-STX3 (green) and anti–a-tubulin (red). Cells were visualized by confocal microscopy. Scale bar, 10 mm. Images shown represent single optical sections of indicated colors or the merge of two colors, as indicated. Shown is one of ﬁve representative experiments. The Journal of Immunology 45

FIGURE 3. Impairment of granule fusion and exocytosis upon knockdown of Munc18-2 andSTX3inBMMC.(A)Expressionof Munc18-2 or STX3 in WT and BMMC treated with Munc18-2 (shM18-2), STX3 (shSTX3)- speciﬁc shRNA, or scrambled shRNA (shScr). Relative expression compared with WT BMMC is indicated below lanes. Numbers are the mean B of all experiments relative to WT levels. ( ) Downloaded from Flow cytometric analysis of FcεRI expression on Munc18-2 or Synatxin3-targeted shRNA- transduced BMMC. (C) Flow cytometric analysis of surface expression of CD63 as a surrogate degranulation marker after stimulation with IgE/Ag (blue line) or PMA/ionomycin

(green line) (D, E). Quantitative analysis of http://www.jimmunol.org/ CD63 expression in IgE/Ag (D)- or PMA/ionomycin-stimulated BMMC. Data are means 6 SE of at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on October 2, 2021

stimulation (Fig. 4B [WT, shScr]). Furthermore, SGs and PM of CD63-GFP–positive granules, fewer SGs reached the PM (Fig. fusion were observed in WT or control shRNA-treated BMMC 4A, 4B). In contrast, STX3 silencing showed that, although CD63- (arrows in Fig. 4A [WT, shScr]). Following specific silencing of GFP–positive SGs were observed in close proximity to the PM, either Munc18-2 or STX3, SGs showed distinct features and dif- fusion events were diminished. ferential behavior when compared with control-treated or WT BMMC. When Munc18-2 was silenced (Fig. 4A [shM18-2], Sup- Microtubule-dependent regulation of Munc18-2 function plemental Video 1C), SGs lost their direction and moved randomly during stimulation in the cytosol (open arrowhead). In addition, SGs in Munc18-2– During analysis of SG dynamics, we noticed that in some instances silenced BMMC showed strongly reduced velocities compared with SGs remained immobile in areas that appeared to be restricted or WT or shScr controls. Conversely, silencing of STX3 (Fig. 4A relatively sparse in microtubular networks, whereas they readily [shSTX3], Supplemental Video 1D) showed that SGs remained moved and fused in other areas (Supplemental Video 2). This argued highly mobile, yet upon reaching the PM they rarely fused (indi- for the need of cytoskeletal reorganization to coordinate SG mobility cated by the arrowhead in Fig. 4A [shSTX3]). To quantify the oc- and fusion. In light of the deficiency in translocation, we explored curring fusion events, we measured colocalization of SG and PM whether Munc18-2 could be part of a complex that couples the SG markers as a function of time by correlation coefficient analysis fusion machinery with microtubules. As shown in Fig. 5A (upper (Fig. 4C). An increase in value indicates improved fusion of CD63- panel), myc-tagged Munc18-2 transfected into COS7 cells was GFP–positive granules with the PM. The number of fusion events in specifically coimmunoprecipitated with a-tubulin. Similarly, en- WT and control shRNA-transduced cells rapidly rose to a maximum dogenous Munc18-2 was found to associate with a-tubulin in RBL before starting to decline at the end of the monitoring period. In MC (Fig. 5A [lower panel], 5B). This association was decreased in Munc18-2–silenced cells, a small rise in fusion events was ob- cells treated with the microtubule-dissolving drug nocodazole (Fig. served; however, this rise was delayed and was minor relative to 5B). We next examined whether the Munc18-2–binding partner control cells. Importantly, due to the decreased and random mobility STX3 and the noninteracting protein STX4 cognate SNARE fusion 46 MAST CELL DEGRANULATION CONTROL BY Munc18-2 AND SYNTAXIN 3 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Silencing of Munc18-2 and STX3 in BMMC alters SG translocation and fusion, respectively. (A) Real-time imaging of secretory granule movements. CD63-GFP (green)–transduced BMMC were loaded with Alexa Fluor 555–conjugated WGA (red). The yt or xt images show the time course of cross sections indicated in the xy image (as shown in white cross section for 0 s [0s] images). WT and control shRNA (shScr)-transduced BMMC show numerous granule membrane and plasma membrane fusions (indicated by the arrows). In Munc18-2–silenced cells (shM18-2), few CD63-GFP–positive granules moved toward the membrane, and observed fusion events were rare. In STX3-silenced cells (shSTX3), CD63-GFP–positive granules moved to the PM, but fusion of granules with the PM was rare. The proximity of SG membrane with PM is indicated with an arrowhead. (B) Velocities of granules were analyzed. In Munc18-2–silenced cells (shM18-2), but not in STX3-silenced cells, velocities of granules were significantly decreased. Data shown are mean 6 SE of at least three independent experiments. Statistical significance was **p , 0.01, ***p , 0.001. (C) Analysis of SG membrane and PM colocalization based on correlation coefficient analysis at the indicated time points. Data shown were normalized to the starting point (t = 0). Silencing of Munc18-2 (shM18-2) or STX3 (shSTX3) revealed a diminished colocalization at each time point when compared with the WT or control (dhScr) shRNA-transduced BMMC. Data shown are mean 6 SE of at least four independent experiments. Statistical significance relative to WT was *p , 0.05. proteins also coimmunoprecipitated with tubulin. However, al- able to coimmunoprecipitate tubulin supporting a Munc18-2–de- though we confirmed previous data that Munc18-2 interacts with pendent but STX3-independent interaction (Fig. 5C). We further STX3,butnotwithSTX4orSNAP23,neitheroftheseAbswas analyzed whether the Munc18-2–tubulin interaction was modulated The Journal of Immunology 47 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Munc18-2 interacts with tubulin in a stimulation-dependent manner. (A) Lysates (lys) from COS7 cells transfected with myc-tagged Munc18-2 (+) or empty vector (2)(upper panel) or RBL MC were immunoprecipitated with anti–a-tubulin Ab or normal mouse IgG (NMG), as indicated before immunoblotting with anti-myc (upper panel) or anti–Munc18-2 (lower panel). (B) RBL cell lysates were immunoprecipitated with anti–Munc18-2 or irrelevant normal rabbit IgG (NRG) before immunoblot analysis with indicated Abs. Relative expression levels as compared with sham-treated cells arbitrarily set to 1 are indicated below lanes. Results are representative of at least three independent experiments. (C) RBL cell lysates (left lane) were immunoprecipitated with irrelevant normal rabbit IgG (NRG), anti–Munc18-2, anti-STX3, anti-STX4, or anti-SNAP23 before immunoblot analysis with indicated Abs. Results are representative of at least three independent experiments. (D) RBL were stimulated with IgE/Ag or PMA/ionomycin for indicated times. Lysates were prepared and immunoprecipitated with anti–Munc18-2 before immunoblotting with anti–a-tubulin and anti–Munc18-2 for equal loading. Relative expression levels as compared to unstimulated cells arbitrarily set to 1 are indicated below lanes. *p , 0.05, ***p , 0.001. upon stimulation. Our kinetic analysis showed a time-dependent The latter finding could relate to the fact that MC granules and decrease of the interaction in both IgE/Ag and PMA/ionomycin- those of other secretory cells have been found to contain exosomes stimulated cells (Fig. 5D). (25, 50). A sizable proportion of Munc18-2 (20%) was also found in zones connecting SGs each to the other. These connections Ultrastructural analysis of Munc18-2 and STX3 localization appeared to be constituted of filamentous tangles that appear at- and interaction tached to membranous extensions of the SGs (Fig. 6A, 6C, thin We next used immunogold labeling and electron microscopy to black arrows). PM localization, although occasionally seen, was visualize at the ultrastructural level the distribution of Munc18-2 rare. Similar to Munc18-2, the majority of STX3 was found on the (10 nm gold particles [GP]) and STX3 (20 nm GP) in RPMC. SG surface (22.5%) (Fig. 6A, 6C, white arrows) and within SGs The latter contain a highly differentiated granular compartment and (68.1%) (Fig. 6A, 6E, white arrowheads). STX3 was also clus- upon stimulation release granular content by a process involving tered at sites with filamentous connections (9.4%) often together both SG-SG fusion and SG-PM fusion. Representative images of with Munc18-2 (Fig. 6A, thin white arrows). Treatment with mi- the GP distribution in resting and stimulated cells are depicted in crotubule-depolymerizing or -stabilizing drugs induced the dis- Fig. 6. Quantitative analysis of GP distribution in resting cells appearance of Munc18-2 and STX3 labeling at the level of the shows that Munc18-2 is majorly (80%) associated with SGs (Table filamentous connection in nocodazol- and taxol-treated RPMC, I) both on the surface (Fig. 6A, 6C, 6E, black arrows), but to respectively (Supplemental Fig. 4, Table I). Quantitation of immu- a large part also inside the matrix (Fig. 6A, 6E, black arrowheads). nogold labeling also showed that the amount of the granule matrix 48 MAST CELL DEGRANULATION CONTROL BY Munc18-2 AND SYNTAXIN 3 Downloaded from http://www.jimmunol.org/

FIGURE 6. Ultrastructural localization of Munc18-2 and STX3 in resting and stimulated RPMC. Ultrathin sections of unstimulated RPMC (A, C, E) and 48/ 80-stimulated RPMC (B, D, F) were double-immunogold labeled with anti–Munc18-2 (10 nm GP) and anti-STX3 (20 nm GP). Cells were visualized by electron microscopy. Bars are in nm, as indicated. In unstimulated cells, Munc18-2 and STX3 are present either on the granular surface or within granules, as indicated by arrows, as explained in the text. Munc18-2 are often accumulating in tangles at ﬁlamentous connections between SG that sometimes also contain STX3 (C, E). In stimulated cells, such tangles can be seen to concentrate at fusion sites either between SG (D)oratthePM(F), where they are enriched in both Munc18-2 and STX3 GP, as shown by the arrows. by guest on October 2, 2021

GP in these cells did not change, although on the average an in- Nonetheless, STX3 function is apparently intimately linked to crease of granule surface labeling was noted (Supplemental Fig. 4, Munc18-2, as indicated by the observed coclustering at fusion Table I). After stimulation, numerous fusion events with the ex- sites in degranulating cells. Our findings also demonstrate that pansion of the granular matrix can be observed (Fig. 6B). Inter- Munc18-2 is coupled to cell signaling, which modulates its in- estingly, large clusters of STX3 and Munc18-2 can frequently be teraction with the microtubule cytoskeleton. seen often coinciding with possible sites of fusion between gran- Munc18 proteins are essential fusion effectors in regulated ules (Fig. 6B [white arrowheads], 6D [white arrowheads at higher exocytosis; however, their precise function remains incompletely magnification]), but also between granules and the PM (Fig. 6F). understood (20). In addition to their direct fusion-promoting role (20, 30), Munc18 proteins were proposed to regulate additional Discussion steps such as vesicle translocation, docking, postdocking, and In this study, we report that the SNARE regulator Munc18-2 and its modulation of cytoskeletal functions (29, 51, 52). In this line, STX3-binding partner have distinct, but complementary roles in Munc18-1–deficient chromaffin cells revealed a largely altered MC degranulation. Munc18-2 supports exocytosis by facilitating distribution of dense core vesicles, attributed to a docking defect translocation of SGs, whereas STX3 plays a direct role in fusion. (53). The defect was overcome by overexpressing STX1, which

Table I. Subcellular distribution of Munc18-2 and STX3 in RPMC and effect of nocodazol and taxol treatment

Munc18-2 GP STX3 GP

Treatment SG Surface SG Matrix Filamentous Connections SG Surface SG Matrix Filamentous Connections No treatment (25) 13.8 6 0.9a 66.4 6 5.0 19.9 6 5.0 22.5 6 2.7 68.1 6 3.0 9.4 6 2.1 Nocodazole (17) 26.9 6 3.9b 67.9 6 3.7 3.3 6 1.6 38.8 6 6.4 54.9 6 5.1 4.4 6 2.2 p , 0.001 p , 0.02 Taxol (16) 29.4 6 2.5 66.0 6 4.5 4.5 6 1.4 31.4 6 5.1 67.8 6 5.1 0.8 6 0.6 p = 0.001 p , 0.02 aValues are the mean (6SEM) of the percentage of GP in each subcellular compartment, taking the total number of GP in each picture as 100%. The numbers of pictures scored are shown in parentheses. Total GP scored were at least 200 and 1500 for STX3 and Munc18-2, respectively. bNumbers in bold indicate statistical significant differences as compared with untreated cells. Statistical analysis was carried out using GraphPAD Prism5 program. The p value was calculated using Student t test for unpaired data, two tailed. The Journal of Immunology 49 prevents formation of nonproductive SNARE (STX1-STX1-SNAP- (FEZ1) (64), providing a link to axonal-dependent vesicular transport. 25) complexes (53), suggesting that Munc18-1 could serve as an or- We therefore asked whether Munc18-2 could interact with micro- ganizer of productive SNARE assembly. This view is also validated by tubules. Using coimmunoprecipitation experiments showed that reconstitution experiments (33). Munc18-2 associated in a specific manner with a-tubulin. This Early studies on the ubiquitously expressed Munc18-2 showed interaction decreased in the presence of nocodazole. Impor- that it controls exocytosis in various epithelial cells (54, 55), am- tantly, the interaction was downmodulated after stimulation, ylase release from pancreatic acinar cells (56), or insulin release suggesting a dynamic coupling during cell signaling. Likely, from pancreatic b cells (57). Munc18-2 was also implicated in SG Munc18-2 is not directly responsible for this interaction, as release by immune cells, including MC and neutrophils (8–10, 34). SGs remain attached to microtubules after Munc18-2 silencing The discovery of Munc18-2 mutations in immune deficiency FHL- (Fig. 2). Likewise, attachment seems to take place indepen- 5 patients leading to misfolding was shown to affect STX11 ex- dently of Munc18-2 binding to STX3, as tubulin was not found pression and to lead to a defect in lytic granule exocytosis in NK to coimmunoprecipitate with anti-STX3 and specific knockdown of and cytotoxic T cells (CTL) (36, 37). Although these results in- STX3 did not affect the interaction (data not shown). However, to dicate that Munc18-2, similarly to Munc18-1, plays an important date, we have been unable to detect interactions of Munc18-2 with role in regulated secretion, the functions of these two SNARE other microtubule-associated proteins like kinesin or the FEZ1 ho- accessory proteins may not be completely identical. Thus, in MC molog FEZ2 (data not shown). Based on our results that SG trans- and other cell types (34, 57), the granular localization of Munc18-2 location is clearly affected, it is possible that Munc18-2 could pro- and STX3 is strikingly different. In epithelial cells, Munc18-2 was mote dynamic exchanges between the fusion and the cytoskeletal associated with polarized secretory events, as it was found to transport machinery, perhaps by enabling multiple cycles of docking. regulate, together with STX3, apical secretion in both intestinal This could probably take place independent of its binding to STX3, as Downloaded from and kidney epithelial cells (54, 55, 58). Interestingly, a recent also suggested by the additive effect of downregulation of both STX3 study performed with pancreatic acinar cells suggested a prepon- and Munc18-2. These data are also in agreement with the described derant role of Munc18-2 in sequential fusion (59). Other differ- docking defects in Munc18-1 null chromaffin cells (53), but suggest ences are the specific binding of calcium EF-hand protein Cab45 that docking could be a dynamic process involved in granule trans- to Munc18-2 in the absence of calcium, whereas Munc18-1 binds location. In agreement, FHL-5 patients carrying mutations in only in its presence (60). It has been proposed that this interaction, Munc18-2 retain persistent membrane-trafficking defects in epi- http://www.jimmunol.org/ which occurs in the absence of STX3, might regulate the associ- thelial cells even after hematopoietic stem cell transplantation with ation of Munc18-2 with STX partners during exocytosis (57). inappropriate accumulation of granules in the cytoplasm similar to Concerning MC, our data show that downregulation of either what is seen in MC (65). Munc18-2 or STX3 expression markedly inhibited MC degranulation, The importance of microtubules in organizing the cellular whereas CCL2 chemokine secretion was not affected, arguing that distribution of the core fusion machinery is also supported by both effectors function only in release from storage organelles. These ultrastructural studies. Munc18-2 GP are found mainly in as- experiments implicate STX3, in addition to STX4 (7, 61), in regu- sociation with secretory granules, confirming our previous data lating degranulation. STX11 has also been a potential candidate for (8), but also labeled granule-connecting filamentous structures. by guest on October 2, 2021 regulating MC degranulation. However, although STX11 decreases This distribution, in particular those present in the filamentous in Munc18-2–deficient MC (35) in regulating degranulation, STX11 connections, is decreased in the presence of microtubule-targeting has also been a potential candidate for regulating MC degranulation. drugs, supporting the above hypothesis that it could depend on However, although STX11 decreases in Munc18-2–deficient MC, no dynamic interactions of Munc18-2 with the microtubule net- effect on degranulation was seen in MC from STX11-deficient mice work. This is in agreement with early transmission electron (35, 40, 62). Interestingly, STX3 downregulation had a more pro- microscopy observations in RPMC, which revealed an impor- nounced effect on degranulation than downregulation of STX4. tant cytoskeletal meshwork around SGs and microtubules con- Given the SG localization of the former, this finding supports the necting the SG surface to the subplasmalemmal network or hypothesis that sequential granule–granule fusion is an important to the adjacent granule surface (66, 67). On some occasions, contributor in MC degranulation. Nonetheless, STX3 was also found however, large clusters of both molecules accumulated at sites in clusters at the membrane, suggesting that during this process it is of granule–granule or granule–PM fusion, suggesting that at the able to relocate to this compartment in a manner similar to SNAP23, fusion site productive interactions are maintained to support which in a reverse manner relocates from the plasma membrane to fusion. Taken together, these data support the important role of the SG during degranulation (6). No additive effect on degranulation was dynamic interactions of the secretory machinery with the cyto- observed, when both STX3 and STX4 were simultaneously down- skeleton, as already shown in the case of Rab3D, that could play regulated, suggesting that they function independently. By contrast, a role in actin coating of secretory granules during exocytosis (68, inhibitory effects on degranulation were enhanced when Munc18-2 69) or Rab27 isoforms that could regulate the transition of mi- andSTX3expressionwerejointlyreduced, suggesting unique, but crotubule to actin-based motility of granules (70). complementary functions for each protein in this process. Collectively, our findings establish Munc18-2 as an important Importantly, confocal imaging and live cell imaging studies of regulator of MC-prestored mediator release that functions by the degranulation process support that Munc18-2 controls granule enhancing SG translocation. Concerning STX3, our findings sup- translocation contrasting with STX3, which, as expected, directly port a major role in SG-SG and SG-PM fusion. We also define a impacts on fusion. As degranulation involves microtubule-dependent new Munc18-2–microtubule–dependent regulatory axis that co- SG transport (43, 45, 63), this suggested that Munc18-2 might play ordinately organizes productive interactions between promoters of a role in connecting the fusion apparatus with the microtubule cyto- SG translocation and fusion with the cytoskeleton. skeleton. In support, previous studies have shown that Munc18- 2–labeled SG align along microtubules (8). Similarly, a sizable Acknowledgments proportion of Munc18-2 can be found at cytoskeletal granular con- We thank Samira Benadda, IFR-02, for help with confocal imaging. nections.Furthermore,neuronalMunc18-1wasshowntobindtothe We thank V.M. Olkkonen for providing the Myc–Munc18-2 plasmid Kinesin-1 adaptor protein, fasciculation and elongation protein zeta-1 construct. 50 MAST CELL DEGRANULATION CONTROL BY Munc18-2 AND SYNTAXIN 3

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