Rab5 Is a Novel Regulator of Mast Cell Secretory Granules: Impact on Size, Cargo, and Exocytosis This information is current as of February 12, 2015.

Nurit P. Azouz, Neta Zur, Adi Efergan, Norihiko Ohbayashi, Mitsunori Fukuda, Dina Amihai, Ilan Hammel, Marc E. Rothenberg and Ronit Sagi-Eisenberg J Immunol 2014; 192:4043-4053; Prepublished online 2 April 2014; doi: 10.4049/jimmunol.1302196 http://www.jimmunol.org/content/192/9/4043

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Supplementary Material

The Journal of Immunology

Rab5 Is a Novel Regulator of Mast Cell Secretory Granules: Impact on Size, Cargo, and Exocytosis Nurit P. Azouz,* Neta Zur,* Adi Efergan,* Norihiko Ohbayashi,† Mitsunori Fukuda,† Dina Amihai,‡ Ilan Hammel,‡ Marc E. Rothenberg,x and Ronit Sagi-Eisenberg*

M

ast cells, hematopoietic cells of the immune system, are best known for their involvement in maladaptive acquired immune responses, such as in allergy and asthma (1). However, recent data clearly document their important protective roles in innate host defenses against parasites, bacteria, and even arthropod and reptile toxins (2–5). Indeed, by responding to a wide spectrum of stimuli and releasing multiple biologically active substances, mast cells can initiate innate responses, which in turn contribute to the resolution of infection and to resistance to envenomation. The primary response involves exocytosis and release of preformed mediators, including vasoactive amines such as histamine and serotonin that are prestored in cytoplasmic secretory granules (SGs) (6, 7). This initial event is followed by the de novo synthesis and release of a large array of biologically potent substances, including arachidonic acid metabolites, multiple cytokines, and chemokines (8, 9). Consistent with the diversity of the inflammatory processes that mast cells are involved in, they *Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; †Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan; ‡Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; and xDivision of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, OH 45229 Received for publication August 19, 2013. Accepted for publication February 25, 2014. This work was supported by a grant from the Israel Science Foundation, founded by the Israel Academy for Sciences (1139/12 to R.S.-E.), and partial support was provided by a travel grant from the Constantiner Institute (to N.P.A.). Address correspondence and reprint requests to Dr. Ronit Sagi-Eisenberg, Tel Aviv University, Sackler Faculty of Medicine, Department of Cell and Developmental Biology, Room 312, Tel Aviv 69978, Israel. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: BMMC, bone marrow–derived cultured mast cell; CA, constitutively active; CN, constitutively negative; HSA, human serum albumin; mRFP, monomeric red fluorescence protein; NPY, neuropeptide Y; RBL, rat basophilic leukemia; SG, secretory granule; shRNA, short hairpin RNA; Tfn, transferrin; TPA, 12-O-tetradecanoylphorbol-13-acetate; wt, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302196

respond and are activated by a wide spectrum of external triggers including the immune, allergen/Ag-IgE–mediated trigger, cytokines, bacterial products, neuropeptides, and venom components (10–14). Strikingly, although extensive efforts dedicated to decipher the stimulus–secretion coupling mechanisms have provided significant insights into the signaling networks that bring about exocytosis in mast cells, the mechanisms underlying the genesis of their inflammatory mediators storing SGs remain poorly understood. Rab GTPases are master regulators of vesicular trafficking that mediate vesicle transport, tethering, and fusion within the secretory and endocytic pathways (15, 16). Therefore, unveiling the Rab networks associated with a cellular process provides invaluable tools for decoding the intermediate pathways and mechanisms involved in such process regulation or execution. Along this line of thought, we recently screened 44 Rabs for their phenotypic and functional features using the mucosal mast cell line rat basophilic leukemia (RBL)-2H3 (herein referred to as RBL) (17). The latter provide a good model system to study mast cell exocytosis because they recapitulate mast cell responses to external triggers by releasing the inflammatory contents of their SGs by exocytosis (18, 19). Moreover, studies using electron microscopy, high-resolution fluorescence microscopy, and atomic force microscopy (19–23) have confirmed similar degranulation mechanisms in bone marrow–derived cultured mast cells (BMMCs) and the RBL cells. Because RBL cells allow genetic manipulations, they provide a useful model for functional genomics analyses of exocytosis. Indeed, we applied a coexpression approach cotransfecting GFP-fused wild-type (wt) or Rab mutants and neuropeptide Y fused to monomeric red fluorescent protein (NPY-mRFP) as a reporter for exocytosis (17). Extending this experimental approach, in the present study we analyzed the role of Rab5 and, to our knowledge, we demonstrate for the first time that Rab5 is an important regulator of mast cell SG biogenesis.

Materials and Methods Materials Calcium ionophore A23187, p-nitrophenyl-N-acetyl-b-D-glucosaminide, and DNP-human serum albumin (has) were purchased from Sigma-Aldrich

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Secretion of inflammatory mediators prestored in mast cells secretory granules (SGs) enhances immune responses such as in allergy and host defense. However, the mechanisms underlying the biogenesis of the SGs remain largely unresolved. By combining high-resolution live cell imaging and quantitative morphometric analyses, we show that the small GTPase Rab5 controls the SG size and cargo composition by a VAMP8-dependent fusion mechanism. Knockdown of the endogenous Rab5, or expression of constitutively negative mutants, significantly reduces the size of SGs and increases their number. Conversely, expression of constitutively active Rab5 mutants induces few, but giant, SGs. Both the small and giant SGs maintain their exocytosis competence. Finally, we show that Rab5-mediated fusion between Golgi-derived SGs and early endosomes precedes the maturation of the SGs, as reflected by the recruitment of Rab27B, and allows the incorporation of cargo, such as CD63, that traffics through endosomes. Collectively, our results assign Rab5 a key role in mediating mast cell SG fusion during biogenesis, thereby controlling the amount and composition of the SGs content and maintaining the communication between new and pre-existing SGs. The Journal of Immunology, 2014, 192: 4043–4053.

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(St. Louis, MO). 12-O-tetradecanoyl-13-acetate (TPA) was from Calbiochem (San Diego, CA), Transferrin (Tfn) Alexa Fluor 633 conjugate was from Molecular Probes (Carlsbad, CA).

Abs used in this study HiLyte Plus 647–conjugated goat anti mouse and goat anti-rabbit IgG were from Anaspec (Fremont, CA); Cy2-conjugated donkey anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA); polyclonal anti-serotonin and anti-DNP monoclonal IgE and mAbs against the FLAG tag were from Sigma-Aldrich; monoclonal anti-serotonin was from Novus Biologicals (Littleton, CO); mAbs directed against the T7 tag were from Novogen (North Ryde, NSW, Australia); mAbs against the HA tag were from Covance (Princeton, NJ); polyclonal anti-cathepsin D was from Santa Cruz Biotechnology (Richmond, CA); and monoclonal PE anti-mouse CD117 (c-Kit) was from BioLegend (San Diego, CA).

Plasmids used in this study

Cell culture RBL cells were maintained in adherent cultures in DMEM supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37˚C as previously described. BMMCs were isolated from 6- to 10-wk-old BALB/c mice in complete medium consisting of RPMI 1640 supplemented with 10% FBS (Invitrogen, Carlsbad, CA), glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), pyruvate (1 mM), nonessential amino acids, HEPES (10 mM), and 2-ME (50 mM). BMMCs were subsequently cultured for 2 wk in the presence of both IL-3 (20 ng/ml) and SCF (10 ng/ml) (PeproTech, Rocky Hill, NJ) and then in the presence of IL-3 (20 ng/ml). Cell purity (95– 97%) was confirmed by analyzing c-Kit expression by flow cytometry.

Transient transfection Transient transfection of RBL cells was performed as previously described (33). Briefly, 1.5 3 107 RBL cells were transfected with a total of 40–50 mg cDNAs by electroporation at 300 V, 1500 mF. The cells were immediately replated in tissue culture dishes containing growth medium. Transient transfection of BMMCs was performed with a Nucleofector 4D device (Amaxa, Koeln, Germany) using 1.5 3 106 BMMCs and a total of 5 mg cDNAs at program DV-100 using the P2 transfection kit (Amaxa), according to the manufacturer’s instructions.

Rab5 knockdown RBL cells (1.5 3 107 cells/ml) were transiently transfected with either 50 mg control pSilencer vector or with 25 mg Rab5A shRNA and 25 mg Rab5BC shRNA. Forty-eight hours later, total RNA was purified using the PerfectPure RNA purification system (5 Prime) according to the manufacturer’s instructions. For reverse transcription, cDNA was generated by using 2 mg total RNA with high-capacity reverse transcriptase (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions in a total volume of 20 ml. The expression levels of the Rab5 isoforms was determined by quantitative RT-PCR (ABI Prism 7900 SDS, Applied Biosystems), using a SYBR Green real-time PCR Kit (Applied Biosystems) according to manufacturer’s instructions in 10 ml reactions containing 0.4–60 ng DNA and 13 SYBR Green. The following primers were used: Rab5A, reverse primer 59-TGT GCA GGC TCA GTA AGG TC-39, forward primer 59-GCT AAG ACA TCA ATG AAT GTA AAT GAA-39; Rab5B, reverse primer 59-CTG CAT ATG CCT GAG CCT CT-39, forward primer 59ACA AAG CTG ACC TTG CCA AC-39; Rab5C, reverse primer 59-CAA ACT TGA CCG TTG TGT CG-39, forward primer 59-CCA GGA GAG CAC AAT TGG A-39.

FACS sorting of transfected cells RBL cells were transfected with 45 mg of either pEGFP or pEGFP-CA Rab5A cDNAs by electroporation at 300 V, 1500 mF. The cells were

Activation of RBL cells or BMMCs Cells were seeded in 24-well plates (5 3 105 RBL cells/well) or in 96-well plates (4 3 105 BMMCs/well) and incubated overnight with 1 mg/ml mouse anti-DNP–specific monoclonal IgE. Following three washes in Tyrode’s buffer (10 mM HEPES [pH 7.4], 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA), cells were stimulated in the same buffer for 30 min at 37˚C with the desired stimuli (i.e., a combination of calcium ionophore A23187 (Ion) and the phorbol ester TPA, or DNP-HSA [Ag]).

Secretion of b-hexosaminidase Activity of the SG-associated enzyme b-hexosaminidase was determined as described previously (33). Briefly, 10-ml aliquots of supernatants and cell lysates were incubated for 60 min at 37˚C with 50 ml substrate solution consisting of 1.3 mg/ml p-nitrophenyl-N-acetyl-b-D-glucosaminide in 0.1 M citrate (pH 4.5). Reactions were stopped by the addition of 150 ml 0.2 M glycine (pH 10.7). OD was measured at 405 nm. Results were expressed as percentage of total b-hexosaminidase activity present in the cells.

Secretion of NPY-mRFP Secretion of NPY-mRFP was measured as previously described (17). Briefly, the fluorescence of cell supernatants and cell lysates (200 ml), derived from cells transiently transfected with NPY-mRFP and activated as described above, was measured by an Infinite 200 (Tecan, Ma¨nnedorf, Switzerland) fluorescence plate reader, using a 590-nm, 20-nm bandwidth excitation filter and a 635-nm, 35-nm bandwidth emission filter. Auto fluorescence of nontransfected RBL cells was set as reference. The amount of secretion is presented as percentage of total cellular NPY-mRFP.

Immunostaining and confocal analyses RBL cells (4 3 105 cells/ml) or BMMCs (4 3 105 cells/ml) were grown on 12-mm round glass coverslips, washed three times with PBS, and fixed for 30 min at room temperature with 4% paraformaldehyde in PBS. Cells were then permeabilized for 30 min at room temperature with 0.1% Triton X-100, 5% FBS, and 2% BSA diluted in PBS. Cells were subsequently incubated for 1 h at room temperature with the primary Abs followed by three washes and 1 h incubation with the appropriate secondary Abs. After washing, cells were mounted (Golden Bridge Life science, Mukilteo City, WA) and analyzed by a Zeiss 510 laser confocal microscope (Zeiss, Oberkochen, Germany) or 3024 Nikon A1Rsi (Nikon, Tokyo, Japan) or Leica microscope (Leica Wetzlar, Germany), using a 363 oil/1.4 numerical aperture objective.

Time-lapse microscopy of living cells RBL cells were seeded at 2 3 105 cells/chamber in eight-well chamber borosilicate coverglass systems (Thermo Fisher Scientific Waltham, MA). Images were acquired by a Zeiss 510 laser confocal microscope, equipped with a heated chamber (37˚C) and CO 2 controller (4.8%) and a CApochromat 363/1.2 W Corr objective.

Quantitative analyses and image processing Deconvolution was carried out by Huygens deconvolution and analysis software (Scientific Volume Imaging, Hilverum, The Netherlands). Quantification of SG size and reconstruction of three-dimensional confocal images were carried by Imaris software (Bitplane, Zurich, Switzerland).

Tfn internalization RBL cells were seeded at 2 3 105 cells/chamber in eight-well chamber borosilicate coverglass systems, serum starved for 1 h at 37˚C in DMEM supplemented with 0.2% BSA and 50 mM HEPES (pH 7.4), followed by 1 h of incubation at 4˚C with Tfn Alexa Fluor 633 conjugate (50 mg/ml) to allow binding. Unbound Tfn was removed by washing with ice-cold PBS. To allow endocytosis, the cells were transferred to 37˚C for the desired time periods. Cells were subsequently processed for immunofluorescence analyses.

Acquisition of three-dimensional image Three-dimensional images of living RBL cells were obtained by successive scanning of two-dimensional confocal optical slices in the z-axes with optical slices ,0.7 mm. Images were deconvoluted and reconstructed into three-dimensional pictures.

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pEGFP-wt Rab constructs have been previously described (24, 25). Constitutively negative (CN) and constitutively active (CA) Rab mutants were prepared as described previously (26, 27) and subcloned into the pEGFP-C1 vectors (28). The nomenclature of the Rabs is according to Itoh et al. (26). pEF-T7-VAMP2 (29), pEF-FLAG-VAMP3, pEF-FLAG-VAMP8, pEFFLAG-VAMP7 (30), and pmSTR-CA Rab5A (31) were prepared essentially as described previously. NPY-mRFP was a gift from Dr. U. Ashery (Tel Aviv University, Tel Aviv, Israel), and pEGFP-CD63 was a gift from Dr. J.P. Luzio (University of Cambridge, Cambridge, U.K.). The pSilencer 2.1-U6 neo vector (Ambion, Austin, TX) encoding a mouse Rab5A short hairpin RNA (shRNA; 19-base target site: 59-GCACAGTCCTATGCAGATG-39) and Rab5B/C shRNA (19-base target site: 59-GTTTGAGATCTGGGACACA-39) was constructed as described previously (32).

immediately replated in tissue culture dishes containing growth medium. Cells were selected with 1 mg/ml G418 from Calbiochem (La Jolla, CA). Cells with positive GFP fluorescence signal were isolated by a FACSCalibur flow cytometer from BD Biosciences (San Jose, CA) and replated.

The Journal of Immunology Electron microscopy RBL cells, stably transfected with either pEGFP or pEGFP-CA Rab5A, were washed three times in PBS at pH 7.4, fixed with Karnovsky’s fixative (34) for 1 h at room temperature, washed twice with PBS, and postfixed with 1% OsO4 in the same buffer. Dehydration was carried out with graded ethanol and propylene oxide, and cells were embedded in Araldite kit (Merck, Rahway, NJ). Ultrathin sections (0.075 6 0.015 mm) were prepared on an LKB III Ultratome by using a diamond knife (Diatome, Nidau, Switzerland and SPI, West Chester, PA), and the sections were mounted on Formvar-coated 200-mesh nickel grids. Grids were double stained for 60 min with saturated uranyl acetate in 50% methanol and rinsed in 100% ethanol and for 17 min in 0.5% lead citratre solution and rinsed in doubledistilled water. All staining procedures were carried out at room temperature. The sections were examined in a JEOL-100CX transmission electron microscope at 80 kV.

Statistical analysis

Results Rab5 controls the size and number of the SGs in RBL mast cells To analyze the phenotypic and functional impact of Rab GTPases on mast cell exocytosis, we developed a novel methodology that is based on the coexpression of a GFP-fused Rab GTPase (wt or

FIGURE 1. Rab5 regulates the size of the SGs. RBL cells were transiently cotransfected with 15 mg NPYmRFP (magenta) and 30 mg pEGFP (green); or 15 mg NPY-mRFP and 30 mg pEGF-wt Rab5A (A); or 15 mg NPY-mRFP and 10 mg CN Rab5A, 10 mg CN Rab5B, and 10 mg CN Rab5C (B); or 15 mg NPY-mRFP and 50 mg pSillencer (CI); or 15 mg NPY-mRFP and 15 mg Rab5A shRNA and 15 mg Rab5BC shRNA (CII); or 50 mg pSilencer (open columns) or 25 mg Rab5A shRNA and 25 mg Rab5BC shRNA (close columns) (D); or with 15 mg NPY-mRFP and 30 mg pEGFP-CA Rab5A (E). Cells were analyzed by confocal microscopy after 24 h (A, B, E) or 48 h (C). Scale bars, 5 mm. Expression of the Rab5 isoforms was determined by quantitative RT-PCR after 48 h (D).

mutant) with NPY-mRFP as a reporter for exocytosis. We chose NPY-mRFP because it has been shown to recapitulate the behavior of endogenous SG markers in other systems (35), and the pH insensitivity of mRFP fluorescence allows visualization of the mast cell acidic SGs both in fixed and in living cells. Indeed, transient expression in RBL cells, our model mast cells, results in NPYmRFP expression and targeting to vesicular structures (Fig. 1A), which we have previously identified as the SGs based on the colocalization of NPY-mRFP with serotonin, the endogenous SG marker, and its regulated secretion, alongside the endogenous mediators serotonin and b-hexosaminidase, in response to FcεRI or calcium triggers (17). Similar results were obtained using BMMCs. Transiently expressed NPY-mRFP acquired a vesicular location, where it colocalized with serotonin (Supplemental Fig. 1A) and was released upon an Ag/IgE or Ca2+ Ion/phorbol ester (Ion/TPA) trigger, alongside b-hexosaminidase (Supplemental Fig. 1B). Therefore, these results validate the use of NPY-mRFP as an adequate marker of the mast cell SG and appropriate reporter for mast cell exocytosis. In this setup, GFP-wt Rab5A that was coexpressed with NPYmRFP in RBL cells (Fig. 1A) or BMMCs (Supplemental Fig. 1C) displayed a vesicular distribution that most likely reflected its well-known association with early endosomes (36). However,

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Data are expressed as means 6 SEM. The p values were determined by an unpaired two-tailed Student t test and Kolmogorov–Smirnov tests.

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FIGURE 2. Rab5 is dispensable for secretion but is required for controlling the size of SGs. (A) RBL cells were transiently cotransfected with 15 mg NPY-mRFP and either 30 mg pEGFP or 30 mg pEGFP-wt Rab5A or 10 mg pEGFP-CN Rab5A, 10 mg pEGFP-CN Rab5B, and 10 mg pEGFPCN Rab5C, or 15 mg Rab5A shRNA and 15 mg Rab5BC shRNA, as indicated. Forty-eight hours later cells were either left untreated or triggered with 1 mM Ca2+ ionophore and 50 nM TPA for the indicated time periods. Release of NPY-mRFP is presented as percentage of total. Data are means 6 SEM of three to six independent experiments. (B) RBL cells were cotransfected with 10 mg NPY-mRFP and either 30 mg pEGFP or 10 mg pEGFP-CN Rab5A, 10 mg pEGFP-CN Rab5B, and 10 mg pEGFP-CN Rab5C or 15 mg Rab5A shRNA and 15 mg Rab5BC shRNA. The volumes of SGs were calculated from confocal images by the Imaris software. A cumulative distribution histogram of SG volumes from .5000 granules is presented. (C) The mean volume of an SG and (D) the mean number of SGs per cell are presented. Data are means 6 SEM. SG volume: *Pv(CN Rab5A/ B/C) = 2.92 3 1028, *Pv(shRab5A/B/C) = 1.15 3 1028, *Pv(CA Rab5A) = 0.0046. Number of SGs: *Pv(CN Rab5A/B/C) = 3.9 3 1028, *Pv(shRNA Rab5A/B/C) = 0.0003, *Pv(CA Rab5A) = 9.97 3 1025 (determined by unpaired two-tailed Student t test). *Pv, *p value.

CA Rab5A-decorated endosomes, defined as vesicles that do not contain NPY-mRFP, was 25 mm3, suggesting that the SGs formed in the active Rab5 mutant-expressing cells were considerably larger than the enlarged endosomes. The remarkable increase in the SG size was linked with a significant reduction in their number, from an average of 166 SGs in the GFP-expressing cells to an average of 7 granules in CA Rab5A-expressing cells (Fig. 2D). Importantly, not only ectopic NPY-mRFP, but also endogenous cargos of the SGs localized to giant SGs in CA Rab5A-expressing cells. Specifically, we analyzed the location of serotonin, which we have previously shown to colocalize with NPY-mRFP in control, GFP-expressing RBL cells (17), and shown in the present study also to colocalize with NPY-mRFP in BMMCs (Supplemental

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we also noticed that in both the RBL cells and BMMCs, a fraction of wt Rab5A decorated NPY-mRFP containing SGs (Fig. 1A, Supplemental Fig. 1C). This close association between Rab5A and the SGs prompted us to investigate whether Rab5 plays a role in the regulated secretory pathway in mast cells. To this end, we first investigated whether inhibition of Rab5 function might influence exocytosis. Because mast cells express endogenously all three Rab5 isoforms (i.e., Rab5A, Rab5B, and Rab5C) (17, 37), we coexpressed NPY-mRFP, our reporter for exocytosis, with the three GDP-trapped CN Rab5 mutants (CN Rab5A/B/C) and analyzed the impact of expression of these mutants on the triggered secretion of NPY-mRFP. In a complementary approach, we coexpressed NPY-mRFP and a combination of shRNAs directed against Rab5A/B/C. Because BMMCs are more difficult to transfect, we focused on the RBL cells (Fig. 1B, 1C), in which evaluation of the efficacy of Rab5 knockdown by these vectors, determined by quantitative real-time RT-PCR, has demonstrated 80% reduction in Rab5A/B/C mRNA (Fig. 1D). Because changes in Rab5 expression or state of activation affect endocytosis and may thereby influence the cell surface expression of the FcεRI and consequently its triggered secretion (37), we focused on exocytosis stimulated by the combination of Ion/TPA, which act downstream of the receptor. The results of these experiments demonstrated that neither expression of CN Rab5A/B/C nor Rab5A/B/C knockdown had a significant impact on exocytosis (Fig. 2A). However, to our surprise, we noted that in cells expressing either CN Rab5A/B/C or shRNA Rab5A/B/C the SGs appeared smaller and their number increased when compared with control, GFP-expressing cells (Fig. 1B, 1C). To validate this notion, quantitative morphometric analyses of the SG distributions were performed. These analyses demonstrated that in control, GFP-expressing cells, 20% of the SGs were smaller than 1.1 mm3, the volumes of 60% of the SGs ranged between 1.1 and 2.8 mm3, and the volumes of the remaining 20% of SGs ranged between 2.8 and 5 mm3 (Fig. 2B). This pattern was markedly different in CN Rab5A/B/C or shRNA Rab5A/B/Cexpressing cells, in which the volume of 90% of the SGs was ,1.1 mm3 (Fig. 2B). Therefore, overall, the expression of CN Rab5A/B/C or shRNA Rab5A/B/C was associated with a 3- to 5-fold reduction in the average size of SGs, from 2.1 mm3 in GFP-expressing cells to 0.7 mm3 in cells expressing the CN Rab5 mutants and 0.4 mm3 in Rab5 knockdown cells (Fig. 2C). This reduction in size was accompanied by a 3- to 5-fold increase in the SG number, from an average of 166 SGs in a GFP-expressing cell to an average of 572 SGs in CN Rab5A/B/C-expressing cells and 800 SGs in Rab5 knockdown cells (Fig. 2D). Conversely, overexpression of wt Rab5A increased the mean volume of the SGs to 4.1 mm3 (Fig. 2C) while decreasing the number of SGs to an average of 100 per cell (Fig. 2D). Again, this alteration in the size and number of the SGs did not affect significantly the extent or the kinetics of exocytosis (Fig. 2A). In the endocytic system, Rab5A Q79L, the GTP-locked CA Rab5 mutant, facilitates considerably homotypic fusion of the early endosomes, giving rise to the generation of giant endosomes (38). Therefore, we also investigated how expression of this mutant would affect the SG size. As expected, expression of GFP-CA Rab5A resulted in the formation of giant Rab5-decorated vesicles (Fig. 1E). However, part of these vesicles also contained NPYmRFP (Fig. 1E), suggesting that the active mutant may distribute between enlarged endosomes and enlarged NPY-mRFP–containing SGs. Indeed, morphometric analyses of the NPY-mRFP–containing SGs demonstrated that their mean volume in CA Rab5Aexpressing cells reached the value of 44 mm3, which was .20fold larger than the mean volume of the SGs in the control GFPexpressing cells (Fig. 2C). Notably, the calculated mean volume of

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CA Rab5 stimulates SG fusion The inverse correlation between the size of the SGs and their number raised the possibility that Rab5 controls the size of the SGs by facilitating their fusion, in analogy to its well-established role in controlling the size of early endosomes (38). Consistent with this notion, close inspection of three-dimensional–reconstructed images of living cells coexpressing NPY-mRFP and CA Rab5A revealed a series of fusing NPY-mRFP containing SGs, some of which appeared to be connected by Rab5 bridges (Fig. 4A, arrow). This location of CA Rab5A to the boundaries between fusing granules is reminiscent of the “bridge” fusion mechanism by which Rab5 mediates endosomal homotypic fusion (39). Additionally, smaller CA Rab5-decorated structures, most likely corresponding to the early endosomes, were scattered among the giant SGs (Fig. 4A). Electron micrographs of control GFP-expressing cells depicted the previously reported heterogeneity of the SGs (40), showing larger and relatively lucid alongside smaller and denser granules (Fig. 4B). Although the granules were adjacent, we could rarely detect fusing granules (Fig. 4BI). In sharp contrast, the SGs in CA Rab5A-expressing cells appeared considerably larger and fusion events could be detected between two or more lucid granules as well as between a lucid and a smaller denser SG (Fig. 4BII, 4BIII). Occasionally, we could also detect fused SGs forming presumably a degranulation sac (Fig. 4BII), as is often the case during compound fusion (41). Therefore, these results suggested that active Rab5A may also stimulate fusion between SGs of different ages or SGs that contain distinct cargos. Indeed, close inspection of CA Rab5A localization in high-resolution confocal images revealed four distribution patterns: 1) Rab5A associated with giant granules, which contain both NPY-mRFP and serotonin; 2) giant granules, which contain NPY-mRFP and to which serotonin-containing granules were attached through a Rab5A bridge; 3) giant granules, which contain NPY-mRFP and to which NPY-mRFP–containing granules are attached through a Rab5A bridge; and 4) Rab5A-bound serotonin containing granules (Fig. 4C). Rab5A interacts with newly formed SGs In CA Rab5-expressing cells, most NPY-mRFP–containing SGs displayed a significantly increased size (Figs. 1E, 4A). However,

FIGURE 3. Triggered release from CA Rab5A-expressing cells. RBL cells were transfected with 45 mg pEGFP-CA Rab5A (green). Twenty-four hours later cells were transfected with 45 mg NPY-mRFP (magenta) and seeded in an eight-well chamber borosilicate coverglass system with (A) or without (B) 1 mg/ml IgE. Twenty-four hours later, cells were transferred to a heated chamber (37˚C) and CO2 controller (4.8%) for live cell imaging and stimulated with 50 ng/ml DNP-HSA (A) or 10 mM Ca2+ ionophore (B). Scale bars, 5 mm. Arrows point to NPY-mRFP–releasing SGs. (C) Cells were cotransfected with 15 mg NPY-mRFP and either 30 mg pEGFP or pEGFP-CA Rab5A, as indicated. Twenty-four hours later, cells were either left untreated or triggered with 1 mM Ca2+ ionophore and 50 nM TPA for the indicated time periods. Release of NPY-mRFP is presented as percentage of total. Data are means 6 SEM of six independent experiments. *p , 0.01.

not all giant SGs were decorated by CA Rab5A. This observation suggested that the interaction of Rab5 with the SGs might be transient. To explore this possibility and decipher the timing of this interaction, we took advantage of the fact that the enlarged SGs formed in CA Rab5A-expressing cells provide a unique tool to study in detail and in vivo SG protein interactions. Specifically, we monitored the spatiotemporal characteristics of NPY-mRFP on its route from the Golgi to the SG in living GFP-CA Rab5Aexpressing cells. For this purpose, cells were placed at 20˚C 2.5 h after their cotransfection with NPY-mRFP and CA Rab5A and kept at this temperature for an additional 2.5 h to prevent protein exit from the Golgi (42, 43). This period allowed the cells to recover from the transfection and to accumulate sufficient NPY-

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Fig. 1). Indeed, the large NPY-mRFP–containing vacuolar structures, which formed in the CA Rab5A-expressing cells and which could be easily detected by light microscopy, also contained serotonin (Supplemental Fig. 2A). We also examined the location of cathepsin D and CD63, two additional cargos of the mast cell SG, and found them to reside in giant vesicles in CA Rab5Aexpressing cells (Supplemental Fig. 2B). Therefore, representatives of all the SG cargo types (i.e., vasoactive amines, lysosomal enzymes, and membrane proteins) acquired a similar morphological pattern in the CA Rab5A-expressing cells. Monitoring by time-lapse microscopy the fate of NPY-mRFP in living GFP-CA Rab5A-expressing cells enabled us to demonstrate its release from the giant SGs in response to the addition of an Ag (DNP-HSA) to cells presensitized by the binding of DNP-specific IgE Abs to the FcεRI (Fig. 3A, Supplemental Video 1) or the addition of a Ca2+ ionophore (Fig. 3B, Supplemental Video 2). Therefore, these results indicated that the enlarged SGs retain their exocytosis competence. Moreover, cells expressing CA Rab5A displayed a 2fold enhancement in their initial rate of secretion (Fig. 3C). Large SGs were also formed upon the expression of CA Rab5B or CA Rab5C, suggesting that all three Rab5 isoforms (Rab5A, Rab5B, and Rab5C) that are endogenously expressed in RBL cells are functionally redundant in promoting the enlargement of the SGs (Supplemental Fig. 3A).

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mRFP in the Golgi for further analysis. Cells were then transferred to 37˚C (time 0) and visualized as a function of time. Forty-five minutes after delivery to 37˚C, NPY-mRFP was already detected in CA Rab5A-decorated vesicles (Fig. 5A). Similarly, three-dimensional–reconstructed and deconvoluted high-resolution confocal images revealed that at 2.5 h after Golgi exit most NPYmRFP resided in CA Rab5A-associated structures (Fig. 5B). This pattern contrasted its cellular location at 24 h after transfection, when NPY-mRFP was distributed between CA Rab5A-free and CA Rab5A-decorated structures (Fig. 4A). Indeed, quantitative analysis revealed that .75% of CA Rab5A colocalized with NPYmRFP at 0–2.5 h after Golgi exit; however, only 30% colocalization was noted after 24 h (Fig. 5C). These results have therefore corroborated the occurrence of a transient interaction between CA Rab5A and the mast cell SGs and demonstrated that this interaction took place shortly after the SGs budding from the Golgi. Because the giant SGs that formed in CA Rab5-expressing cells are exocytosis competent, we also used them as a model to ask at what time point do the SGs recruit Rab27B, a Rab GTPase that localizes to the mast cell SGs and is required for their fusion with the plasma membrane and degranulation (44, 45). In particular,

FIGURE 5. CA Rab5A interacts with newly formed SGs. (A) RBL cells were cotransfected with 15 mg NPY-mRFP and 30 mg pEGFP-CA Rab5A and 2.5 h later the cells were placed in 20˚C and kept at this temperature for an additional 2.5 h. Cells were then transferred to 37˚C (time 0) and visualized as a function of time. At 45 min after their transfer to 37˚C, cells were fixed and visualized by confocal microscopy. Scale bars, 5 mm. (B) High-resolution confocal images of living cells were captured 2.5 h after their delivery to 37˚C. Images were deconvoluted and three-dimensional images were reconstructed by the Imaris software. Scale bar, 5 mm. (C) Quantitative analysis of 26 living cells analyzed at 0–2.5 h or at 24 h after their Golgi exit. Data are means 6 SEM. *p = 0.0006 (determined by unpaired two-tailed Student t test). (D) RBL cells were cotransfected with 15 mg pEGFP-wt Rab27B (green), 15 mg NPY-mRFP (magenta), and 15 mg pmSTR-CA Rab5A (blue). Twenty-four hours later, high-resolution confocal images of living cells were captured, deconvoluted, and threedimensionally reconstructed. For spectral separation, l stack (10.7-nm intervals) of the cells was assigned for unmixing. Images from cells expressing either NPY-mRFP or pmSTR-CA Rab5A were obtained for emission fingerprinting. Scale bars, 5 mm. (E) RBL cells were cotransfected with 15 mg FLAG-wt Rab27B, 15 mg NPY-mRFP (magenta), and 15 mg GFP-CA Rab5A (green). Twenty-four hours later, cells were triggered with 1 mM Ca2+ Ion and 50 nM TPA (Ion/TPA) for 10 min. Cells were subsequently fixed and stained with anti FLAG Abs, followed by HiLyte Plus 647–conjugated secondary Abs (blue). The arrow points to a Rab27B decorated, NPY-mRFP–releasing SG. Scale bars, 10 mm.

because CA Rab5 interacted preferably with Golgi budding granules, whereas Rab27B is presumably localized to mature SGs, we asked what was the time line of these Rab interactions with the SGs. Indeed, the enlarged SGs retained their capacity to recruit Rab27B (Fig. 5D). However, Rab27B bound selectively to the Rab5A-free granules (Fig. 5D), suggesting its preferable binding to mature SGs, subsequent to their Rab5-mediated fusion and

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FIGURE 4. CA Rab5A induces giant SGs. (A) Confocal images of living cells 24 h after cotransfection with 15 mg NPY-mRFP and 30 mg pEGFP-CA Rab5A were deconvoluted by Huygens and three-dimensionally reconstructed by the Imaris software. Part of the NPY-mRFP–containing granules appears to be embedded within Rab5A (arrowheads). Others are naked or bridged with Rab5A (arrows). Small SGs appear to fuse with larger ones (I, III, arrow). A higher power view is shown in the right panels (II, III). Scale bars, 5 mm. (B) RBL cells, transfected with 45 mg pEGFP (I) or pEGFP-CA Rab5A and sorted (II, III), were fixed and processed for electron microscopy as described in Materials and Methods. Some of the SGs are indicated by arrows. Regions of homotypic fusion of SGs are marked by arrowheads. Deg, degranulation sac; Nu, cell nucleus. (C) RBL cells cotransfected with 30 mg pEGFP-CA Rab5A (green) and 15 mg NPY-mRFP (magenta) were immunostained with Abs against serotonin (blue) and analyzed by confocal microscopy and processed by the Imaris software. A three dimensional analysis of a deconvoluted image (original magnification 3160) is presented, illustrating four types of patterns: (I) a giant SG, which contains both serotonin and NPY-mRFP, and is decorated by Rab5A; (II) NPY-mRFP–containing giant SGs, decorated by Rab5A, to which serotonin-containing SGs are attached; (III) two NPYmRFP–containing SGs, bridged by Rab5A; and (IV) small serotonincontaining SGs to which Rab5A is attached. The insets are enlargements of the boxed areas.

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Overexpressed VAMP8 inhibits Rab5-mediated fusion and consequent enlargement of the SGs The giant SGs that form in CA Rab5-expressing cells offer an excellent opportunity to explore and identify the v-SNARE that is involved in their Rab5-mediated enlargement. Because vesicle fusion depends on the precise coupling of the SNARE proteins that zipper into stable membrane-bridging (trans) complexes, overexpression of a relevant SNARE may disturb the fusion process (47–49). Therefore, we overexpressed members of the VAMP family that were previously implicated in playing a role in mast cell exocytosis (49–53) and analyzed their impact on the Rab5stimulated enlargement of SGs. Consistent with their implicated role in controlling mast cell exocytosis (49, 53), VAMP2, VAMP3, VAMP7, and VAMP8 colocalized with NPY-mRFP in the SGs of the CA Rab5A-expressing cells (Supplemental Fig. 4A). However, whereas overexpression of neither VAMP2 nor VAMP3 or VAMP7 had any impact on the SG size (Supplemental Fig. 4A), in cells overexpressing VAMP8 most NPY-mRFP resided in small SGs, despite the expression of CA Rab5A (Supplemental Fig. 4A). Quantitative analyses of the SGs depicted in three-dimensional high-resolution confocal image stacks (Fig. 6A) revealed that the mean volume of an SG in cells coexpressing CA Rab5A and VAMP8 was 13.4 mm3 (Fig. 6B), hence 3-fold smaller than the mean volume of an SG in a cell expressing CA Rab5A alone. Accordingly, the number of SGs increased by 2.9-fold to an average of 20 SGs/cell (Fig. 6C). Therefore, overexpression of VAMP8 selectively interfered with the CA Rab5A-mediated enlargement of the SGs. Notably, overexpression of VAMP8 did not impair the ability of SGs to fuse with the plasma membrane as indicated by the fact that neither the overexpression of VAMP8 alone nor its coexpression with CA Rab5A had any impact on exocytosis triggered by Ion/TPA (Supplemental Fig. 4B). Endosomes are intermediates in the Rab5-mediated enlargement of the SGs Although our results support strongly a role of Rab5 in promoting homotypic SG fusion, they do not exclude the possibility of Rab5mediated fusion between newly formed granules and endosomes as an intermediate step in the biogenesis of the SGs that is followed by the budding of enlarged SGs. In such a scenario, the increased

FIGURE 6. VAMP8 coordinates Rab5A-mediated SGs fusion. (A) RBL cells were cotransfected with 15 mg NPY-mRFP (magenta) and 30 mg pEGFP-CA Rab5A (green) (I) or with 15 mg NPY-mRFP, 15 mg pEGFPCA Rab5A, and 15 mg FLAG-VAMP8 (II). Twenty-four hours after transfection Z-stacked deconvoluted images of living cells were acquired and reconstructed by the Imaris software. Scale bars, 5 mm. (B) The mean SG volume and (C) mean number of SGs per cell was calculated by the Imaris software. Data are means 6 SEM. *p = 0.05 (B), *p = 0.014 (C) (determined by unpaired two-tailed Student t test).

fusion capacity of CA Rab5 combined with the increased surface area of the endosomes in such cells leads to the formation of larger but fewer SGs. To examine the feasibility of this model, we analyzed the potential interactions between endosomes and SGs by monitoring the transport of Tfn with respect to NPY-mRFP in wt Rab5-expressing cells. Indeed, following 5 min of internalization, Tfn was distributed between Rab5-decorated, NPY-mRFP–free endosomes (Fig. 7A, type I) and NPY-mRFP–containing SGs, some of which were also associated with Rab5 (Fig. 7A, type II). However, with increasing time of endocytosis (i.e., 40 min), and consistent with its targeting to the recycling endosomes, Tfn segregated from Rab5, and largely separated from the NPYmRFP–containing SGs, which at this time point no longer colocalized with Tfn or Rab5 (Fig. 7A, type III). Therefore, these results supported strongly the occurrence of a transient Rab5mediated fusion between early endosomes and SGs that is followed by the segregation of the SG. To further substantiate these results, we also analyzed the spatiotemporal relationships of Tfn and NPY-mRFP in CA Rab5A-expressing cells. To this end, we subjected the transfected cells to a Golgi block and at the same time allowed them to bind Tfn. We then transferred the cells to 37˚C and monitored the localization of NPY-mRFP, CA Rab5A, and Tfn in living cells. Three minutes after their delivery to 37˚C, endocytosis was indicated by the formation of CA Rab5A- and Tfn-associated structures that seemed to bud from the plasma membrane (Fig. 7B, type I). At the same time, CA Rab5A- and NPY-mRFP–associated structures were detected in close proxim-

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dissociation. Notably, in Ca2+ ionophore/TPA–triggered cells, CA Rab5A decorated smaller and NPY-mRFP–free vesicles that most likely corresponded to early endosomes, whereas residual NPYmRFP mainly localized to large Rab27B-decorated vesicles (Fig. 5E). Hence, although not proven in the present study, these results are consistent with the premise of the replacement of Rab5 by Rab27B in mature exocytosis-competent SGs. We also analyzed the cellular distribution of additional endocytosis-regulating Rabs that have been implicated previously by others and us in regulating exocytosis in RBL cells or other cells of the immune system. Such is the case for the recycling regulating Rab11A, which plays a role in exocytosis of CTL granules (46). Previously we have shown that in analogy to CTLs, Rab11A localizes to the SGs in triggered RBL cells (17). Consistent with this finding, Rab11A resided at a perinuclear location, presumably at the endocytic recycling compartment, in resting CA Rab5Aexpressing cells, but it translocated to the large SGs upon Ag trigger (Supplemental Fig. 3B). Finally, Rab7, which we have previously shown to localize to the SGs (17), also localized to the large SGs (Supplemental Fig. 3C). Overall, these results indicated that the large SGs shared the characteristics of regular mature SGs with respect to their capacity to recruit Rab27B and Rab7 and recruit Rab11A in response to cell trigger.

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FIGURE 7. Rab5 mediates fusion between SGs and early endosomes and is required for cargo integration. (A) RBL cells were cotransfected with 15 mg NPY-mRFP (magenta) and 30 mg pEGFP-wt Rab5A (green). Twenty-four hours later cells were allowed to internalize 50 mg/ml Alexa Fluor 633-Tfn (blue) for the indicated time periods, as described in Materials and Methods. Following 5 min of internalization, Tfn distributed between Rab5-decorated and NPY-mRFP–free early endosomes (I) and Rab5-decorated structures that contained NPY-mRFP (II). The insets are the enlargements of the boxed areas merging Tfn and Rab5 (upper left corner) and Tfn, NPY-mRFP, and Rab5, showing their overlap (lower left corner). Following 40 min of internalization, Tfn is no longer detected in the type I structures that correspond to early endosomes, and only a minor amount is detected in type II structures that presumably reflect fusion between early endosomes and SGs. Most NPY-mRFP is contained in Tfnfree SGs (III). (B) RBL cells were cotransfected with 20 mg NPY-mRFP (magenta) and 30 mg pEGFP-CA Rab5A (green) and 2.5 h later the cells were placed in 20˚C and kept at this temperature for an additional 2.5 h. For the last hour of incubation cells were also exposed to 50 mg/ml Alexa Fluor 633-Tfn (blue). Cells were then transferred to 37˚C for 3 min and analyzed by confocal microscopy and processed by Imaris software. A three-dimensional analysis of a deconvoluted image is presented, illustrating Rab5A-associated Tfn-containing endosomes (I), Rab5A-associated and NPY-mRFP–containing SGs (II), and Rab5A-associated, Tfn- and NPY-mRFP–containing structures most likely reflecting fusing endosomes and SGs (III). The insets are the enlargements of the indicated areas. Scale bars, 1 mm. (C) RBL cells were cotransfected with 30 mg GFP-CD63 (green) and either 20 mg pSilencer or 10 mg Rab5A shRNA and 10 mg Rab5B/C shRNA, as indicated. Twenty-four hours after transfection cells were transfected with 45 mg NPY-mRFP (magenta) and visualized 24 h later by confocal microscopy. The inset is the enlargement of the boxed area showing GFP-CD63–decorated and NPY-mRFP–containing SGs.

ity to the Golgi (Fig. 7B, type II). However, structures associated with CA Rab5A, NPY-mRFP, and Tfn were clearly detected, thus supporting further the notion of fusion between endosomes and SGs (Fig. 7B, type III). Fusion between the SG and an early endosome during the SG biogenesis process may play a role in the integration of cargos that are delivered directly from the Golgi and cargos that travel to the

Secretion of multiple inflammatory mediators is the primary tool by which mast cells exert their broad biological functions in health, that is, innate and adaptive immunity (2–4), and disease, that is, allergy, chronic inflammation, autoimmunity, and cancer (1, 55). However, very little is known about the mechanisms underlying the biogenesis of the SGs in which these mediators are prestored and their exocytosis. Exploring the molecular mechanisms of mast cell exocytosis is challenging because of the difficulties involved with functional genomic screening approaches in primary mast cells. However, we have recently overcome this obstacle by using RBL cells as a model system and by employing a coexpression approach in which a reporter for exocytosis (i.e., NPY-mRFP) is cotransfected with a gene of interest (17). Indeed, ectopically expressed NPY-mRFP colocalizes with and is released alongside endogenous SG mediators not only in RBL cells (17), but also in primary BMMCs, thus confirming its suitability as a genuine reporter of mast cell exocytosis. Using this approach, in the present study we identified the small GTPase Rab5 as a key regulator of the mast cell fusion of SGs during their biogenesis process. Homotypic fusion was already implicated as playing a role in the biogenesis of SGs in neuroendocrine cells (56–58) as well as in mast cells (59, 60). However, although the evidence in support of homotypic fusion is strong, the underlying mechanisms behind this process largely remain to be defined. We show that in RBL cells, knockdown of the endogenous Rab5 isoforms by shRNAs or expression of constitutively negative Rab5 mutants result in the formation of 3- to 5-fold smaller SGs, whereas expression of a constitutively active Rab5 mutant generates giant SGs, whose size is 20-fold larger. Moreover, an inverse correlation exists between the size of SGs and their number, therefore implicating Rab5-mediated fusion of the SGs as the mechanism by which Rab5 controls their size. Consistent with this notion is the detection of active Rab5 at the boundaries between fusing SGs and the increased abundance of fusing SGs observed in electron micrographs of active Rab5-expressing cells. Strikingly, the interaction of Rab5 with the SGs is transient and restricted to newly formed SGs, shortly after Golgi exit. Rab5 is then replaced by Rab27B, a known regulator of mast cell exocytosis (44). Therefore, Rab5-mediated SG fusion is an intermediate step during the biogenesis and maturation of the mast cell SGs. We also provide conclusive evidence for the occurrence of Rab5-mediated fusion between newly formed SGs and early endosomes. Hence, this mechanism is distinct from the biogenesis of other types of lysosome-related organelles, such as melanosomes that are derived from the endocytic system and mature to become melanosomes (61). This mechanism is also different from the genesis of synaptic vesicles that does not involve fusion with an endosome

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Discussion

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FIGURE 8. Model for Rab5-mediated fusion of mast cells SGs. According to this model, granules that bud from the Golgi (unit granules [UG]) may fuse with early endosomes (EE) to form a transient hybrid organelle in which Golgi and endosome cargos will mix. The latter include endogenous cargo that travels through the plasma membrane (PM) and EE, such as CD63, or exogenous cargo such as Tfn. Cargo destined to regulated secretion will then segregate from endocytic cargo to be packaged into an SG that will separate by budding from the hybrid organelle. The thusly formed SG may then fuse again, in an Rab5-dependent fashion, with additional UGs or early endosomes, thereby increasing in size and changing in cargo composition. UGs may also fuse homotypically to form larger granules that are devoid of endocytic cargo, thereby giving rise to a heterogeneous population of exocytosis competent SGs.

for adaptation to environmental insults. Our current findings thus place the transient and preferable interaction of Rab5 with Golgibudding granules as central to the capacity of mast cells to face environmental needs. Rab5 is not the only Rab GTPase whose function was linked with the regulation of the SG size during biogenesis. Previous studies have implicated Rab3D in playing such a role. Hence, the SG size in exocrine pancreatic and parotid glands was doubled in Rab3D knockout mice (66). However, recent studies in PC12 cells demonstrate the involvement of Rab3D in controlling removal of cargo from the immature SG during the maturation process (58). Therefore, whereas both Rab5 and Rab3D regulate the SG size, their underlying mechanisms are distinct, whereby Rab5 facilitates SG fusion but Rab3D controls cargo recycling from the maturing granule. In agreement is our previous observation that knockdown of synaptotagmin 3, whose function is associated with the regulation of recycling (67, 68), displays the same phenotype of increased sized SGs (67). We also began to characterize the fusion machinery that mediates Rab5-induced fusion of the SGs and identified VAMP8 as involved in this process. Interestingly, although only the overexpression of VAMP8 functionally interfered with Rab5-mediated fusion, VAMP2, VAMP3, and VAMP7 also localized to the SGs. Therefore, whereas VAMP8 is presumably directly involved in mediating the homotypic SG fusion, VAMP2, VAMP3, VAMP7, and possibly also VAMP8 itself might be involved in the SG fusion with the plasma membrane or the endocytic system (49–52). In particular, interactions with the endocytic system might be amplified in cells expressing the CA Rab5A mutant. It is also interesting to note that VAMP8 has recently been implicated as playing a role in homotypic SG fusion during genesis (69) and compound exocytosis in pancreatic acinar cells (70). We observed an enhanced rate of exocytosis in CA Rab5A-expressing cells. Hence, although these accelerated kinetics might be accounted for by the larger volume of the SG that would lead to increased cargo discharge upon fu-

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but rather recycling through an endosome, and accordingly expression of CN Rab5 increases the vesicle size (62). However, in common with the genesis of synaptic vesicles, the intermediate step of fusion between Golgi-derived granules and endosomes is followed by the budding of an SG, which in the mast cell is composed of a number of Golgi-derived granules. We base this argument on the transient nature of the association between the CA Rab5 mutant and the SG. Thus, under conditions that Rab5 remains bound to the endosome, the enlarged SG maintains its capacity to separate and become an enlarged, Rab5-free SG. The latter is capable of recruiting Rab proteins, such as Rab27B, which is normally associated with the SGs and not endosomes, and can undergo regulated exocytosis. Hence, to our knowledge our results provide for the first time an explanation as to how external cargo that is internalized by endosomes gains access to the mast cell SG. For example, FITC-dextran was recently shown to serve as an adequate reporter for granule exocytosis in RBL cells (19). Notably, knockdown of Rab5A/B/C, which diminishes the number of endosomes (63), or expression of CN Rab5A/B/C results in the formation of numerous small SGs that fully retain their exocytosis competence. Therefore, fusion with endosomes is not pivotal for the genesis of functional SGs. In its absence, Golgi-derived granules continue to form and to maintain their secretory functionality, indicating that the fusion machinery responsible for the fusion of the SGs with the plasma membrane is independent of Rab5. Indeed, knockdown of Rab5A was previously shown to enhance Ag-induced secretion (37). However, Rab5A knockdown also increased the cell surface expression of the FcεRI, and this may account for the observed enhancement of secretion (37). Consistent with such a notion, BMMCs derived from RabGEF1/ Rabex-5, an Rab5 GEF, knockout mice, display reduced internalization of the FcεRI and enhanced Ag-induced responses (64). However, in agreement with our findings, these cells do not display any significant differences in Ion/TPA-induced responses (64). Interestingly, note that unlike the isoform-specific functions of Rab5 in regulating endocytosis of the FcεRI (37), the Rab5 isoforms are functionally redundant in prompting fusion of the SGs. Collectively, our results are compatible with a model (Fig. 8) whereby the association of Rab5 with newly generated granules provides them with the capacity to fuse with other granules or with endosomes by acquiring a fusogenic apparatus. The subsequent dissociation of Rab5 or the budding of a fused SG from the endosome will then deprive the mature SGs from fusion capacity, presumably to preserve the system’s robustness. However, mature SGs will still be able to fuse with newly generated granules and/or endosomes and thereby incorporate new cargo such as membrane proteins that travel to the SG by passing through the plasma membrane and endosomes (e.g., CD63), or endocytosed cargo. Consistent with this premise is the pattern of Rab5 distribution depicted in high-resolution confocal images, which demonstrates fusion between SGs that contain both serotonin and NPY-mRFP as well as fusion between NPY-mRFP–containing SGs and granules that contain only serotonin and most likely correspond to preexisting mature SGs. Interestingly, this model is highly compatible with the model that we have previously proposed based on morphometric analyses of electron micrographs of mast cells (65). In that model, fusion between newly formed unit granules to generate larger granules provides a mechanism for storing large amounts of material “ready to go,” and at the same time fusion between already fused, mature SGs with newly formed granules allows also integrating cargo that is synthesized to accommodate the latest environmental requirements. Therefore, such a mechanism would provide the most reliable communicating machinery

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Acknowledgments We thank Drs. U. Ashery and J. P. Luzio for the gifts of cDNAs. We thank Drs. L. Mittleman, M. Shaharbani, and Y. Zilberstein for invaluable assistance with microscopy and image analyses. We thank Dr. C.H. Kuo and A. Collins for assistance with BMMC culture generation. We also thank Dr. Joseph Orly for critical reading of this manuscript.

Disclosures The authors have no financial conflicts of interest.

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The Journal of Immunology

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