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The Journal of Immunology, 2003, 171: 5345-5352.
Copyright © 2003 by The American Association of Immunologists

Mast Cell Degranulation Requires N-Ethylmaleimide-Sensitive Factor-Mediated SNARE Disassembly 1

Niti Puri*, Michael J. Kruhlak*, Sidney W. Whiteheart{dagger} and Paul A. Roche2,*

* Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and {dagger} Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells are specialized secretory cells that play an important role in allergic inflammation. Following engagement of their high affinity FcR for IgE (Fc{epsilon}RI), mast cells release histamine, serotonin, and other inflammatory mediators (1, 2). Although the earliest steps of the cascade from Ag binding to calcium influx have been widely studied, the machinery required for the final steps leading to intracellular secretory granule exocytosis remains largely unknown. This complex process involves granule translocation, granule docking, and ultimately granule fusion with the plasma membrane. Because perturbation of any of these vital steps in exocytosis can alter mast cell function, there is intense interest in unraveling the molecular processes that lead to mast cell degranulation.

As in all eukaryotic cells, membrane trafficking events in mast cells require close apposition between vesicle and target membranes before the two lipid bilayers can fuse. This is mediated, in part, by the integral membrane proteins called soluble NSF attachment proteins (SNAREs) 3 (3, 4). SNAREs are present on both vesicle membranes (vesicle SNAREs, or v-SNAREs) and target membranes (target SNAREs, or t-SNAREs). v-SNAREs are small; type 2 integral membrane proteins that make up the vesicle-associated membrane protein (VAMP)/synaptobrevin family.t-SNAREs can exist as heterodimers consisting of one protein of the syntaxin family and one from the SNAP-23 family. Either immediately preceding, or concurrent with, membrane fusion three cognate SNARE proteins on opposing membranes assemble, via hydrophobic interactions in their coiled-coil domains, into a parallel, four-helical bundle (5, 6). This so-called trans complex is minimally required for membrane fusion (7), and upon fusion, the SNAREs exist in the same membrane in a thermodynamically stable cis configuration (8).

Regulating the assembly and disassembly of SNARE complexes is a critical aspect of vesicular trafficking. Various chaperones, i.e., Munc18, are involved in regulating the assembly of trans complexes from SNARE monomers (9). Other proteins such as N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins are thought to be required to disassemble cis-SNARE complexes to recycle monomers for subsequent fusion events (10, 11, 12). Soluble NSF attachment proteins are adaptor proteins that bind SNARE complexes, mediate binding of NSF, and stimulate the ATPase activity of NSF required to initiate complex disassembly (13, 14). The ATPase activity of NSF is essential to this process because ATP-hydrolysis-deficient mutants of NSF fail to complete the process in vitro (15, 16).

This model for NSF function agrees well with in vitro studies using recombinant proteins (16, 17). It has been shown that highly purified monodisperse synaptic vesicles possess stable SNARE complexes, and that these SNARE complexes (containing VAMP-2, syntaxin, and SNAP- 25) can be disassembled by NSF and soluble NSF attachment proteins in an ATP-dependent manner (8, 18). Thus, cis complexes can form and be disassembled while anchored in the same membrane. SNARE proteins involved in cis complexes are unavailable for the formation of trans complexes with plasma membrane SNAREs; therefore, the role of NSF as a complex helicase is critical to prime SNAREs for subsequent fusion events (19). A number of elegant studies in Drosophila and in vitro have revealed an important role for NSF in SNARE priming (20, 21); however, the importance of SNARE priming has not been directly investigated in nonneuronal mammalian cells.

A number of SNARE proteins have been identified in mast cells as well as the widely used rat mast cell line RBL (rat basophilic leukemia)-2H3 (hereafter referred to as RBL). SNAP-23 and syntaxin 4 are both localized on the plasma membrane, while syntaxin 3, VAMP-2, VAMP-7, and VAMP-8 are found on intracellular granules (22, 23, 24, 25). Despite data showing that perturbing either syntaxin (23) or SNAP-23 (22, 24) can affect mast cell function, the role for NSF-mediated SNARE complex disassembly in mast cell degranulation has not been investigated. The aim of the present study was to determine whether NSF-mediated SNARE priming is required for regulated exocytosis from mast cells. Expressing a biochemically defined mutant of NSF that lacks ATPase activity resulted in a dramatic inhibition of stimulated mast cell exocytosis. Biochemical analysis revealed that introduction of this NSF mutant led to an accumulation of secretory granule-associated SNARE complexes, demonstrating that NSF-dependent SNARE-priming complex disassembly is required for efficient exocytosis from mast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids

cDNAs encoding c-myc-tagged wild-type NSF (NSFwt) or the NSF E329Q mutant (NSFmut) (15, 16) were amplified by PCR and subcloned into the mammalian expression vector pcDNA3 (Invitrogen Life Technologies, Carlsbad, CA). Yellow fluorescent protein-tagged VAMP-7 (YFP-VAMP-7) has been described previously (26) and was a gift of M. Nakanishi (Nagoya City University, Nagoya, Japan). Green fluorescent protein (GFP)-VAMP-7 was generated by ligating the insert from the YFP-VAMP-7 expression vector into the enhanced GFP expression vector pEGFP-C1 (Clontech, Palo Alto, CA). The cDNA-encoding rat syntaxin 3 was obtained as a gift from R. Scheller (Stanford University, Stanford, CA) and was subcloned into pcDNA3. The human growth hormone (hGH) full-length cDNA in pcDNA3 was the gift of J. Lang (Institut Européen de Chimie et Biologie, Pessac, France).

Antibodies

Polyclonal rabbit antiserum recognizing syntaxin 3 was a gift from A. Hubbard (Johns Hopkins University, Baltimore, MD). Anti-CD63/lysome-associated membrane protein (LAMP) 3 mAb AD1 was obtained as a gift from R. Siraganian (National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD). Anti-GFP rabbit IgG fraction from Molecular Probes (Eugene, OR) was used for immunoprecipitations, and Living Colors mAb A.v. (Clontech) was used for immunoblotting. These Abs recognize GFP as well as its spectral variant YFP. Polyclonal rabbit antisera recognizing NSF were described previously (16). Mouse anti-c-myc mAb 9E10 was from Roche Diagnostics (Indianapolis, IN). Anti-DNP IgE mAb SPE-7 was obtained from Sigma-Aldrich (St. Louis, MO). Rabbit anti-hGH antisera were from DAKO (Carpenteria, CA).

Immunoprecipitation and immunoblotting

Immunoblot analysis of cell lysates or immunoprecipitation from cell lysates was performed, as previously described (27). Briefly, cells were washed with HBSS and lysed at 4°C for 1 h with 1 ml of lysis buffer (1% Triton X-100, 10 mM Tris, 150 mM NaCl, pH 7.5) containing 1 mg/ml BSA and protease inhibitors (0.5 mM PMSF, 0.5 mM tosyl-lysine chloromethyl ketone, 5 mM iodoacetamide, 10 µg/ml aprotinin, and 5 µg/ml leupeptin). The lysates were centrifuged at 13,000 rpm at 4°C to remove cell debris, and precleared lysates were used for immunoprecipitations. The specific immunoprecipitations were conducted for 6 h at 4°C. Immunoprecipitated proteins or lysates were analyzed by separation on 10.5% SDS-PAGE gels, transferring to Sequi-blot polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and immunoblotting with specific Abs. Secondary Abs included peroxidase-labeled protein A (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for rabbit primary Abs and HRP-conjugated goat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL) for mouse mAb primary Abs. The bands were revealed with Western Lightening Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA). The intensity of the bands was determined by densitometry using a Molecular Dynamics (Sunnyvale, CA) densitometer. The significance of any differences was calculated using a one-tailed distribution in a two-sample equal variance Student’s t test.

Transfection of RBL and HeLa cells

HeLa cells were maintained as adherent cultures in DMEM with 10% FBS. Subconfluent HeLa cells were transfected using LipofectAmine Reagent (Invitrogen Life Technologies), as per the manufacturer’s specifications, and analyzed 24 h later. The rat RBL mast cell line was a gift from J. Rivera (National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD) and was maintained as an adherent culture in a medium containing equal parts of MEM with Earle’s salts and IMDM and supplemented with 25 mM HEPES, 50 µg/ml gentamicin sulfate, and 20% FBS (RBL complete medium) in a humidified atmosphere containing 5% CO2 at 37°C. RBL cells (10 x 106/0.5 ml serum-free medium) were transfected by electroporation (310 mV, 960 µF) with 10 µg DNA. Immediately after electroporation, the cells were plated in 10 ml complete medium in 10-cm tissue culture dishes and analyzed 24 h later. For immunofluorescence microscopy studies, the transfected cells were plated in tissue culture dishes containing up to five coverslips. The coverslips were then analyzed, as described below.

Stimulation of RBL cell exocytosis

Exocytosis in RBL mast cells was triggered by using 1 µM ionomycin (Sigma-Aldrich) alone, 10 nM PMA (Invitrogen Life Technologies), and 1 µM ionomycin, or by cross-linking the high affinity Fc{epsilon}RI. For stimulation by IgE cross-linking, subconfluent RBL cells were sensitized with DNP-specific IgE (clone SPE-7; 1 µg/ml) in RBL complete medium overnight at 37°C. After a washing step, the cells were stimulated with 100 ng/ml DNP-BSA (a gift from D. Segal, National Cancer Institute, National Institutes of Health, Bethesda, MD) in phenol red-free RPMI medium. Degranulation by PMA and ionomycin was induced, as described earlier (24). Aliquots of the supernatant were withdrawn at the indicated times after induction with either reagent. Mock degranulation studies were conducted in parallel by using the vehicle in medium alone. At the end of the assay, cells were lysed with Triton X-100 in RPMI medium. To determine the released or residual cell-associated {beta}-hexosaminidase activity, aliquots of supernatants and cell lysates, respectively, were incubated with substrate solution, as described earlier (24). In case of the transfected cells, the amounts of hGH in the cell supernatants or in the cell lysates were determined using a hGH ELISA kit from Roche Diagnostics, as per the manufacturer’s instructions. All samples were assayed in triplicate to determine the amount of {beta}-hexosaminidase or hGH present. Each independent experiment was repeated at least three times. Statistical calculations were made using a one-tailed distribution in a two-sample equal variance Student’s t test.

Confocal microscopy

RBL cells with or without transfection were seeded on 10-mm-diameter coverslips 18 h before analysis. For indirect immunofluorescence analysis, the cells were either fixed with 4% paraformaldehyde in PBS for 30 min and excess paraformaldehyde quenched with 50 mM NH4Cl in PBS or the cells were fixed with cold methanol at -20°C for 4 min. After washing, the paraformaldehyde-fixed cells were permeabilized with 1% Nonidet P-40 in presence of 3% normal goat serum and 0.05% saponin in PBS. The paraformaldehyde-fixed as well as methanol-fixed cells were then incubated with 3% normal goat serum and 0.05% saponin in PBS for 1 h to prevent nonspecific protein binding. Primary Abs diluted in the same buffer were added to the cells, and incubation was conducted for 2 h. After washing, this was followed by 30-min incubation in the presence of secondary goat Abs conjugated to Alexa Fluor 488 (green) or Alexa Fluor 546 (red) (Molecular Probes). An irrelevant isotype-matched Ab was used as a control. In some experiments, the cells were incubated with 1 µg/ml of the lipophilic dye 1,1' dihexadecyl-3, 3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) in DMEM on ice for 30 min. Following staining, the cells were washed three times in PBS containing 5% FCS before fixation with paraformaldehyde. Coverslips were mounted in Fluoromount G (Southern Biotechnology Associates).

Confocal images were collected with a Zeiss (Oberkochen, Germany) 510 META laser-scanning microscope using a x100 Plan-Apochromat (N.A. 1.4) lens, 100 nm/pixel xy-sampling, and a pinhole diameter set to provide an optical slice thickness of 1.0 µm. Image z-stacks were collected through the depth of the cell using 0.21 µm step size. Individual channels were aligned within single pixels based on image stacks collected of 4.0 µm TetraSpeck beads (Molecular Probes) using the same image acquisition settings. Colocalization analysis was done for each plane of the individual image stacks using the colocalization analysis feature of the Zeiss LSM 510 software version 3.2. Briefly, individual channels were thresholded to include the structures of interest; regions of interest were then drawn to encompass the structures, resulting in scatter plots being generated and colocalization coefficients calculated. The colocalization coefficients are based on the relative number of colocalizing pixels in each respective channel compared with the total number of pixels above the chosen threshold level (28). The colocalization coefficients represent colocalization in the green channel with respect to the red channel. Single images were exported from the Zeiss LSM 510 software and organized into figures using Adobe Photoshop version 6.0 (Adobe Systems, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of a reporter assay for regulated exocytosis from RBL mast cells

In our goal to identify a role for NSF in SNARE disassembly and regulated exocytosis from mast cells, we used a transient transfection approach in which we overexpressed either NSFwt or the ATPase-deficient NSFmut together with an exocytosis reporter. The rationale behind this approach was that it allows for the transient expression of large amounts of NSF protein acutely in the cells, thereby minimizing potential nonspecific effects of additional NSF on cell growth and viability.

As our exocytosis reporter we chose to use hGH. Introduction of hGH in rat neuroendocrine cells allows easy identification of transfected cells and has elucidated the role of various secretion mediators on the exocytosis process (29, 30). Despite the fact that mast cells do not express growth hormone, we transiently transfected hGH cDNA into RBL cells. Confocal immunofluorescence microscopy revealed partial colocalization of the expressed hGH with the secretory granule marker LAMP 3 (Fig. 1A), and quantitative analysis of a number of images gave a colocalization coefficient of 0.73 ± 0.08 (n = 33). To determine whether hGH could be released from these cells in a regulated way, we stimulated the cells to degranulate and compared the release of the transfected hGH with that of the endogenous granule marker {beta}-hexosaminidase. Triggering exocytosis from these cells using PMA/ionomycin or by cross-linking Fc{epsilon}RI with IgE and polyvalent Ag gave secretion profiles of hGH and {beta}-hexosaminidase that were very similar (Fig. 1B). Despite the fact that our transient transfection efficiency was routinely less than 50%, this strategy effectively allows us to specifically monitor secretion from the transfected subpopulation of cells in the culture.



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  		FIGURE 1. Release of hGH from transfected RBL mast cells. A, RBL cells were transiently transfected with hGH, fixed with methanol, permeabilized, and labeled with anti-hGH Ab (green) and mAb AD1 against the granule membrane marker CD63/LAMP 3 (red). The distribution of each marker was examined by confocal fluorescence microscopy. An overlay (yellow) of the two images is also shown. The inset in each overlay shows an enlarged region of the cell (indicated by the arrow in the overlay). There was no detectable staining for hGH in mock-transfected RBL cells (not shown). The bar represents 10 µm. B, RBL mast cells transiently transfected with hGH were mock stimulated or stimulated for various times with PMA and ionomycin, or with IgE cross-linking, as described in the text. The release of transfected hGH or endogenous {beta}-hexosaminidase into the cell supernatant was assayed, as described in Materials and Methods. The data shown are mean ± SD of three independent experiments.

 
Expression of dominant-negative NSFmut inhibits regulated exocytosis from RBL mast cells

To examine the role of NSF in regulated mast cell exocytosis, RBL cells were transiently transfected with a trace amount of hGH cDNA together with an excess of expression vector containing no insert, wild-type NSF (NSFwt), or an ATPase-defective NSF mutant (NSFmut). Control studies showed that under the transfection conditions used in these studies, we routinely obtained 90% coexpression of hGH with various test proteins. Whereas overexpression of NSFwt had no effect on PMA/ionomycin-induced mast cell degranulation, overexpression of NSFmut dramatically inhibited regulated exocytosis from transfected mast cells (Fig. 2, A and B). Examination of a time course of PMA/ionomycin-induced exocytosis demonstrated that the presence of NSFmut inhibited exocytosis at all time points examined; however, the effect was most pronounced at early times of stimulation (Fig. 2C). Titration studies revealed that overexpression of NSFmut could inhibit the amount of hGH secreted after 5 min of PMA/ionomycin treatment by 90% (Fig. 2D) in a dose-dependent manner.



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  		FIGURE 2. Expression of NSFmut inhibits exocytosis from transiently transfected RBL mast cells. RBL cells transfected with 2 µg hGH together with 8 µg of either empty vector alone, NSFwt, or NSFmut were stimulated with PMA and ionomycin or by cross-linking surface Fc{epsilon}RI (IgE cross-linking). At the indicated times, the amount of hGH secreted into the cell supernatant was determined. A, The amount of hGH released after 15 min of stimulation under each condition was calculated as a percentage of the total amount of hGH present in the cells. The data shown are mean ± SD of four independent experiments, and asterisks indicate statistically significant differences between controls vs test samples (*, p < 0.005). B, The net hGH secretion of the samples shown in A is shown in this panel. The net secretion is defined as the amount of hGH secreted after 15 min in PMA and ionomycin minus the amount secreted after 15 min in medium alone. C, At various times after stimulation with medium alone or with PMA and ionomycin, aliquots of the cell supernatant were assayed for release of hGH. The secretion from these cells at various time points is shown as mean ± SD of three independent experiments. D, RBL cells were transfected with 2 µg hGH DNA and various amounts of NSFmut, as indicated. The total amount of DNA per transfection was held constant at 10 µg by the addition of empty vector. The amount of hGH released after only 5 min of stimulation with PMA and ionomycin is shown as mean ± SD of three independent experiments. E, RBL cells transfected with hGH together with empty vector alone or with NSFmut were stimulated by cross-linking surface Fc{epsilon}RI (IgE cross-linking) or by treating the cells with ionomycin. After 15 min, the net release of hGH was determined. Each data point represents the mean ± SD of three independent experiments, and the asterisks indicate statistically significant differences between NSFmut and empty vector-transfected RBL cells (*, p < 0.005).

 
To ensure that the inhibitory effects of NSFmut on PMA/ionomycin-induced exocytosis were physiologically relevant, we stimulated cells for exocytosis by cross-linking Fc{epsilon}RI with polyvalent Ag. Triggering degranulation by cross-linking surface Fc{epsilon}RI demonstrated that overexpression of NSFmut inhibited mast cell degranulation (Fig. 2E). In addition, stimulation with ionomycin alone, which triggers degranulation in a signal transduction-dependent manner (31), also revealed the inhibitory role of NSFmut on exocytosis (Fig. 2E). These data support the results obtained using PMA/ionomycin and show that NSFmut inhibits mast cell degranulation induced by physiological stimuli. Together these data reveal a role for ATP hydrolysis by NSF in the regulation of exocytosis from mast cells.

RBL mast cells express syntaxin 3 and YFP-VAMP-7 on secretory granules

Because the role of NSF in exocytosis is thought to involve the disassembly, or priming of cis-SNARE complexes present on cellular membranes, we attempted to determine whether overexpression of NSF altered the stoichiometry of SNARE complexes in RBL mast cells. Because our study hinges on the effect of transiently transfected NSF with reporter molecules, we set out to identify transiently expressed, tagged SNAREs in RBL cells and determine which of these are expressed on secretory granules. It has been reported that VAMP-8 and VAMP-7 are present on RBL granules (23). However, we found that while GFP-VAMP-8 was localized to vesicles, these structures did not colocalize with the secretory granule markers serotonin or LAMP 3 (data not shown).By contrast, YFP-VAMP-7 was clearly expressed on LAMP 3-containing secretory granules in RBL cells (Fig. 3A; colocalization coefficient 0.92 ± 0.01, n = 11), a finding that is consistent with the lysosomal localization of VAMP-7 in other cell types (32) and the lysosomal nature of these granules (33). Furthermore, both YFP-VAMP-7 and GFP-VAMP-7 almost completely colocalized with the t-SNARE syntaxin 3 on the lysosome-like secretory granules in RBL mast cells (Fig. 3B). The colocalization coefficient of YFP-VAMP-7 with syntaxin 3 was 0.91 ± 0.02 (n = 12), and that of GFP-VAMP-7 with syntaxin 3 was 0.94 ± 0.03 (n = 5). As anticipated, YFP-VAMP-7 and GFP-VAMP-7 exhibited the same subcellular distribution. We chose to generate and express GFP-VAMP-7 in addition to YFP-VAMP-7 because GFP is a more optimal partner with Alexa fluor 546 (red) for quantitative colocalization analyses. Subsequently, we found that under the imaging conditions used, YFP-VAMP-7 was also a suitable partner for these analyses. In each case, it was clear that tagged VAMP-7 colocalized with syntaxin 3. By comparison, YFP-VAMP-7 did not colocalize with the plasma membrane fluorescent lipid dye DiI (Fig. 3C; colocalization coefficient 0.16 ± 0.11 (n = 5)). Thus, the t-SNARE syntaxin 3 and the v-SNARE YFP-VAMP-7 are coexpressed on the lysosome-like secretory granules in RBL cells.



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  		FIGURE 3. Colocalization of YFP-VAMP-7 and syntaxin 3 on RBL mast cell secretory granules. RBL mast cells were transfected with YFP-VAMP-7 (green) or with GFP-VAMP-7 (green), as indicated in each panel. The cells were counterstained for the indicated marker and imaged by confocal microscopy. Also shown is an overlay of each image (colocalization appears yellow). The inset in each overlay shows an enlarged region of the cell (indicated by the arrow in the overlay). The bar represents 10 µm. A, YFP-VAMP-7-expressing cells were fixed with methanol, permeabilized, and stained with mAb against the granule marker LAMP 3 (red). No significant staining was observed in the red channel when using an isotype control Ab. B, Either YFP-VAMP-7- or GFP-VAMP-7-expressing cells were fixed with methanol, permeabilized, and stained with an Ab specific for syntaxin 3 (red). No significant staining was observed in the red channel when using an irrelevant Ab. C, RBL mast cells expressing YFP-VAMP-7 (green) were incubated on ice with the plasma membrane lipid dye DiI (red) before paraformaldehyde fixation and examination by confocal microscopy.

 
Syntaxin 3 and YFP-VAMP-7 form SNARE complexes in RBL mast cells

Previous studies have shown that cis-SNARE complexes containing syntaxin, VAMP, and SNAP-25 are present on the membranes of synaptic vesicles (8, 18). To determine whether granule-associated syntaxin 3 and VAMP-7 are similarly bound to each other in cis-SNARE complexes, we performed coimmunoprecipitation studies using extracts of YFP-VAMP-7-expressing RBL cells. Immunoblotting of anti-YFP immunoprecipitates clearly revealed the presence of syntaxin 3-VAMP-7 complexes in these cells (Fig. 4A). In agreement with data derived from analysis of yeast SNAREs (34), we found that a very small proportion of the total pool of VAMP-7 and syntaxin 3 was present in these SNARE complexes (data not shown). Because essentially all YFP-VAMP-7 and syntaxin 3 are present on RBL granule membranes, these results demonstrate that SNARE complexes, most likely representing cis-SNARE complexes, are present on mast cell granules.



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  		FIGURE 4. YFP-VAMP-7 is bound to syntaxin 3 in RBL mast cells. A, RBL cells were transfected with syntaxin 3, empty enhanced GFP vector, or YFP-VAMP-7 in various combinations, as indicated. Two independent transfections of RBL cells with YFP-VAMP-7 with syntaxin 3 are shown in this figure. Cell lysates were prepared, and anti-GFP immunoprecipitates of each transfection condition were probed by immunoblotting using anti-syntaxin 3 and anti-GFP Abs. The lysates were also analyzed for total syntaxin 3 by immunoblotting aliquots of the lysates with specific Abs recognizing syntaxin 3. B, RBL cells transfected with YFP-VAMP-7 were lysed alone or in presence of a 50-fold excess of untransfected RBL cells. Cell lysates were immunoprecipitated using anti-GFP Abs, and the immunoprecipitates and lysates were probed, as described in A. C, The various band intensities from B were quantified by densitometry and are expressed in relative units. Note that the relative band intensities are plotted on a log scale.

 
SNARE proteins bind to each other by coiled-coil interactions, and studies have revealed promiscuous associations of various syntaxin and VAMP isoforms in vitro (35, 36). Therefore, an important consideration for coimmunoprecipitation experiments is to demonstrate conclusively that any SNARE complexes observed are present in living cells and are not formed during or after cell lysis. To examine this, we lysed YFP-VAMP-7-transfected RBL cells alone or in the presence of a 50-fold excess of untransfected cells before examining each condition for syntaxin 3-YFP-VAMP-7 complexes. Despite the presence of a remarkable excess of total syntaxin 3 present in the cell lysate, there was no increase in the amount of syntaxin 3 present in YFP-VAMP-7 immunoprecipitates (Fig. 4, B and C). Control experiments revealed that YFP-VAMP-7 was not completely bound in SNARE complexes in these cells, as overexpression of syntaxin 3 in YFP-VAMP-7-expressing cells could lead to generation of even more YFP-VAMP-7 containing SNARE complexes (Fig. 4A). Because SNARE complex formation was not altered even under conditions in which one of the partners (syntaxin 3) was present in a large excess, we conclude that the cis-SNARE complexes containing syntaxin 3 and YFP-VAMP-7 were present in the live cells before lysis.

Expression of dominant-negative NSFmut leads to an accumulation of SNARE complexes in RBL mast cells and HeLa cells

To correlate the effects of NSFmut on regulated exocytosis with alterations in SNARE complex assembly, we cotransfected RBL cells with YFP-VAMP-7 and either empty expression vector, NSFwt, or NSFmut. As in our cotransfection studies using NSF and hGH, cotransfection with YFP-VAMP-7 increases our possibility of analyzing transfected cells that express both proteins. SNARE complexes containing syntaxin 3 and YFP-VAMP-7 were detected in RBL cell lysates of cells transfected with DNA for YFP-VAMP-7 and the empty expression vector alone (Fig. 5A). Coexpression of NSFwt did not alter the amount of SNARE complexes isolated from these cells, and immunoblotting for NSF revealed that the total amount of NSF expressed in these cells was dramatically increased. Despite the relatively modest expression of NSFmut as compared with NSFwt, coexpression with the dominant-negative mutant of NSF led to a marked accumulation of SNARE complexes (Fig. 5, A and B).



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  		FIGURE 5. Expression of NSFmut leads to an accumulation of syntaxin 3-YFP-VAMP-7 complexes in RBL mast cells. A, RBL cells were transiently transfected with 5 µg YFP-VAMP-7 together with 5 µg of either empty vector, NSFwt, or NSFmut. Cell lysates were prepared, and immunoprecipitations with anti-GFP Abs were conducted. Aliquots of cell lysates and anti-GFP immunoprecipitates were probed by immunoblotting with the indicated Abs. B, Band intensities of syntaxin 3 bound to YFP-VAMP-7 were quantified by densitometry. The relative amount of syntaxin 3 present in each anti-YFP-VAMP-7 immunoprecipitate was normalized for the total amount of YFP-VAMP-7 in the sample. Each data point represents the mean ± SD of six independent experiments, and the asterisks indicate statistically significant differences between NSFmut and empty vector-transfected RBL cells (**, p < 0.0005).

 
To examine the biochemical effects of NSFmut on syntaxin 3-YFP-VAMP-7 assembly in another cell type, we used the human cervical carcinoma HeLa cell line. Transient transfection efficiencies are high in HeLa cells, and these cells are routinely used to examine various aspects of protein trafficking. Because HeLa cells express relatively low levels of endogenous SNARE proteins (N. Puri, unpublished observation), the cells were cotransfected with cDNAs encoding syntaxin 3 and YFP-VAMP-7. Immunofluorescence microscopy revealed colocalization of YFP-VAMP-7 with a population of syntaxin 3-containing intracellular granules even in HeLa cells (data not shown). SNARE complexes containing syntaxin 3 and YFP-VAMP-7 were detected in lysates of HeLa cells transfected with DNA for both proteins (Fig. 6A), and coexpression of NSFwt resulted in a significant decrease in the amount of these complexes (Fig. 6, A and B). In agreement with our data obtained using RBL cells, coexpression of NSFmut resulted in a considerable increase in the amount of complexes (Fig. 6, A and B). These results, together with those shown above, demonstrate that overexpression of a dominant-negative ATPase-deficient mutant of NSF inhibits regulated exocytosis from mast cells that is accompanied by an NSFmut-dependent accumulation of cis-SNARE complexes.



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  		FIGURE 6. Expression of NSFmut leads to an accumulation of syntaxin 3-YFP-VAMP-7 complexes in HeLa cells. A, HeLa cells were transiently transfected with 1 µg YFP-VAMP-7 and 1 µg syntaxin 3 together with 4 µg of empty vector alone, with NSFwt, or with NSFmut. Cell lysates were prepared and anti-GFP immunoprecipitates were probed by immunoblotting with syntaxin 3 Abs. The lysates were also analyzed for total amounts of the indicated protein by immunoblotting of aliquots of cell lysates with specific Abs recognizing GFP, syntaxin 3, c-myc (recognizing transfected NSF), and total NSF. B, The relative band intensities of syntaxin 3 bound to YFP-VAMP-7, total YFP-VAMP-7, and total syntaxin 3 were quantified by densitometry. Each data point represents the mean ± SD of three independent experiments, and the asterisks indicate statistically significant differences between NSFmut and empty vector-transfected HeLa cells (**, p < 0.0005).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it is well known that NSF plays an important role in extremely rapid exocytosis of synaptic vesicles in neurons (20, 37), there are little data regarding the importance of this protein in regulated exocytosis from nonneuronal cells such as mast cells. In neurons, the primary role of NSF in exocytosis is thought to arise from its helicase-like activity in untwisting coiled-coil SNARE complexes, protein complexes that are widely believed to directly mediate the fusion of synaptic vesicles with the presynaptic plasma membrane (38). The ability of NSF to untwist and dissociate SNAREs is called SNARE priming (19). This process is thought to liberate individual SNARE proteins from cis-SNARE complexes (i.e., SNARE complexes present on the same membrane), making them available to form fusogenic trans SNARE complexes between opposing membranes. However, in neurons, the constitutive rapid recycling of synaptic vesicles makes elucidation of the precise membrane target of NSF difficult to dissect; e.g., is NSF untwisting cis-SNARE complexes that exist on the plasma membrane before endocytosis of synaptic vesicles or is NSF untwisting cis-SNARE complexes that exist on synaptic vesicles themselves?

The RBL mast cell line has been widely used as a model system to study the mechanism of mast cell degranulation (23, 24, 39, 40, 41). In addition, there is no obvious role for membrane recycling as a prerequisite to exocytosis, allowing a more clear understanding of the mechanism by which NSF can affect function in these cells. Given the focus on SNAREs as regulators of membrane fusion, there has been intense interest in identifying the SNAREs that modulate secretory granule fusion with the plasma membrane in mast cells and in identifying proteins that regulate SNARE assembly/disassembly in these cells. From our work and that of others, we know that the t-SNARE syntaxin 3 and the v-SNAREs VAMP-7 and VAMP-8 are present on mast cell vesicles (24, 25). Because our study relied on coexpression of NSF with tagged proteins, we examined the distribution of GFP-tagged SNAREs with secretory granules in transiently transfected RBL cells. Although it has been reported that endogenous VAMP-8 is present on RBL secretory granules (23), we found that the expression pattern of GFP-VAMP-8 was indeed vesicular, but these vesicles did not colocalize with the secretory granule marker LAMP 3. By contrast, GFP- and YFP-tagged versions of the lysosomal v-SNARE VAMP-7 were present on these granules and completely colocalized with endogenous syntaxin 3 on these organelles. In studies similar to those that reported a role for syntaxins in degranulation (23), we found that overexpression of VAMP-7 also inhibited RBL degranulation, strongly implicating this v-SNARE in regulated exocytosis from RBL mast cells (data not shown). When mast cells were lysed, we readily detected complexes of syntaxin 3 with YFP-VAMP-7, similar to the cis-SNARE syntaxin 1-VAMP-2 complexes found on synaptic vesicle membranes in earlier studies (8, 18). These syntaxin 3-YFP-VAMP-7 complexes formed in the RBL cells themselves and did not assemble during and/or after cell lysis, as demonstrated by our cell-mixing studies.

Having identified these cis-SNARE complexes, we investigated whether the accumulation of these complexes was detrimental to mast cell degranulation by introducing an ATPase-deficient mutant of NSF into the mast cells. Our initial attempts to generate stable lines of RBL cells overexpressing large amounts of NSFmut were not successful, a result that was most likely due to cellular toxicity, as is seen in temperature-sensitive NSF mutants in Drosophila and yeast (42, 43). Therefore, we developed a transient transfection method and used a reporter to examine the effect of NSF on secretion from RBL cells. We serendipitously found that hGH was packaged in secretory granules and released with kinetics that were very similar to that of the endogenous granule marker {beta}-hexosaminidase in RBL cells. Others have used similar cotransfection systems for studying the mechanism of exocytosis from neuronal cells (29, 30), but to our knowledge this is the first use of a secretion reporter to study the regulation of exocytosis in RBL mast cells. Another distinct advantage of this system is that coexpression of a test protein together with hGH allows one to examine the role of a test protein on mast cell degranulation even under conditions in which transfection efficiency is relatively low.

Transient overexpression of NSFmut in RBL mast cells inhibited exocytosis in response to PMA and ionomycin, ionomycin alone, or following cross-linking of surface Fc{epsilon}RI by polyvalent Ag. By contrast, overexpression of NSFwt had no effect on exocytosis, suggesting that the levels of endogenous NSF in RBL cells are not limiting for efficient SNARE priming and exocytosis. The inhibitory effect of NSFmut was not on the early signaling events necessary for exocytosis, because the characteristic membrane ruffling that occurs after Ag stimulation of RBL cells was observed regardless of whether the cells expressed NSFwt or NSFmut (data not shown). NSF is a hexamer of identical subunits, and functional studies in vitro have demonstrated that the introduction of even one mutant subunit of NSF into the hexamer inhibits NSF function (15). Therefore, in these studies, NSF is acting as an in vivo dominant-negative mutant and is able to inhibit even the activity of the endogenous wild-type protein. Although NSFmut-mediated inhibition of exocytosis was significant, it was not complete, which could be explained by: 1) incomplete cotransfection of NSFmut with hGH in a fraction of the cells; 2) insufficient amounts of NSFmut being expressed to completely inactivate NSF hexamers; or 3) the presence of wild-type NSF hexamers that were present before transfection with NSFmut.

This study is the first example of a biochemically defined mutant of NSF altering mammalian cell physiology. The mutant of NSF used in these studies was identified as an inhibitor of SNARE disassembly in vitro (16), and we have now shown that introduction of this mutant into RBL cells inhibits SNARE complex disassembly and, most importantly, inhibits regulated exocytosis. Because NSF is thought to regulate vesicle-trafficking events throughout the entire secretory pathway (3, 44), it is likely that the effects of the NSFmut observed in our studies are not specifically targeted to the process of secretory granule/plasma membrane fusion. However, expression of the NSFmut does not alter the packaging of the hGH secretion reporter in LAMP 3-positive secretory granules (N. Puri, unpublished observations). Because hGH trafficking appears normal in NSFmut-expressing RBL cells, our data highlight the importance of NSF-mediated disassembly of SNARE complexes in secretory granule exocytosis. It is also possible that the dominant-negative effect of NSFmut on exocytosis may be related to a failure to disassemble not only syntaxin 3-VAMP-7 complexes, but other cis-SNARE complexes required for secretory granule/plasma membrane fusion. Although NSF is likely to function at multiple steps in the secretory pathway, our data reveal a clear role for NSF in mast cell degranulation.

The cascade of signaling events that ultimately leads to mast cell degranulation is exceedingly complex, involving various protein and lipid kinases, phosphatases, and cytoskeletal rearrangements (45, 46). We now add an additional layer of regulation to the process of exocytosis, that of modulating the disassembly or priming of cis-SNARE complexes on secretory granules. It is interesting to speculate that even NSF activity is regulated during mast cell signaling; however, there are only limited data to suggest that this occurs (47). It is also important to point out that even if NSF activity were regulated during exocytosis, our data do not allow us to discern whether the importance of SNARE priming is an early event in exocytosis or is one of the events proximal to granule-plasma membrane fusion. Although this may be difficult to evaluate, it is nevertheless clear that NSF activity is required for mast cell degranulation, and our biochemical studies suggest that the inhibition in secretion is a consequence of the inhibition of cis-SNARE priming by NSF.


   Acknowledgments
 
We thank Drs. Juan Rivera and Pierre Henkart for critical reading of this manuscript. We thank the Experimental Immunology Branch Microscope Facility for support in confocal imaging. We thank our many colleagues for the generous gifts of Abs, cDNAs, and other reagents used in this study.


   Footnotes
 
1 This work was supported in part by a grant from the National Institutes of Health (HL56652) to S.W.W. Back

2 Address correspondence and reprint requests to Dr. Paul A. Roche, National Institutes of Health, Building 10, Room 4B36, Bethesda, MD 20892. E-mail address: paul.roche{at}nih.gov Back

3 Abbreviations used in this paper: SNARE, soluble NSF attachment protein receptor; DiI, 1,1' dihexadecyl-3, 3,3', 3'-tetramethylindocarbocyanine perchlorate; GFP, green fluorescent protein; hGH, human growth hormone; LAMP, lysome-associated membrane protein; NSF, N-ethylmaleimide-sensitive factor; NSFmut, E329Q ATPase-deficient mutant of NSF; NSFwt, wild-type NSF; RBL, rat basophilic leukemia; t-SNARE, target SNARE; v-SNARE, vesicle SNARE; VAMP, vesicle-associated membrane protein; YFP, yellow fluorescent protein. Back

Received for publication April 11, 2003. Accepted for publication September 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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