The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baram, D.
Right arrow Articles by Sagi-Eisenberg, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baram, D.
Right arrow Articles by Sagi-Eisenberg, R.
The Journal of Immunology, 1998, 161: 5120-5123.
Copyright © 1998 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Ca2+-Dependent Exocytosis in Mast Cells Is Stimulated by the Ca2+ Sensor, Synaptotagmin I1

Dana Baram*,{dagger}, Michal Linial{ddagger}, Yoseph A. Mekori{dagger} and Ronit Sagi-Eisenberg2,*

* Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; {dagger} Allergy and Clinical Immunology Unit, Sapir Medical Center, Kfar Saba, Israel; and {ddagger} Department of Biological Chemistry, Life Sciences Institute, Hebrew University in Jerusalem, Jerusalem, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Mast cells secrete a variety of biologically active substances that mediate inflammatory responses. Synaptotagmin(s) (Syts) are a gene family of proteins that are implicated in the control of Ca2+-dependent exocytosis. In the present study, we investigated the possible occurrence and functional involvement of Syt in the control of mast cell exocytosis. Here, we demonstrate that both connective tissue type and mucosal-like mast cells express Syt-immunoreactive proteins, and that these proteins are localized almost exclusively to their secretory granules. Furthermore, expression of Syt I, the neuronal Ca2+ sensor, in rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, resulted in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Therefore, these findings implicate Syt as a Ca2+ sensor that mediates regulated secretion in mast cells to calcium ionophore.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Mast cells are specialized secretory cells that release a variety of biologically active substances in a process of regulated exocytosis; these substances mediate inflammatory and allergic reactions (1). As with nearly all secretory cells, exocytosis in mast cells can be evoked by Ca2+ ionophores, indicating a role for the elevation of cytosol Ca2+ in the regulation of exocytosis from these cells (2). However, the identity of the Ca2+ receptor has remained obscure.

In the synapse, the role of the Ca2+ sensor has been ascribed to synaptotagmin (Syt)3, which is a Ca2+-binding, synaptic vesicle membrane protein (reviewed in 3 . However, the finding that Syt belongs to a gene family of differentially expressed proteins, some of which are ubiquitously distributed (4), has raised the possibility that Syt may serve the role of a general Ca2+ sensor, regulating exocytosis in both neuronal and nonneuronal secretory cells (5). Therefore, in the present study, we decided to explore the hypothesis that Ca2+-dependent exocytosis in mast cells may be controlled by a protein homologue of Syt.


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

The Abs used included polyclonal serum directed against the cytoplasmic domain of Syt I (a generous gift of Dr. T. C. Sudhof, Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas, TX, (6)) and affinity purified Abs derived from this serum.

Isolation and growth of mast cells

Bone marrow-derived mast cells (BMMCs) were obtained as described previously (7). Rat peritoneal mast cells (RPMCs) were also obtained as described previously (8). Rat basophilic leukemia (RBL) cells (RBL-2H3) were maintained in adherent cultures in DMEM supplemented with 10% FCS in a humidified atmosphere of 6% CO2 at 37°C.

Preparation of cell lysates

Mast cells (1 x 106) were washed in PBS and resuspended in 30 µl of lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.1% SDS, 50 mM NaF, 10 mM NaPPi, 2 mM NaVO4, 1 mM PMSF, and 10 µg/ml leupeptin) and immediately centrifuged at 12,000 x g for 15 min at 4°C. The cleared supernatants were mixed with 5x Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting. For the preparation of the rat brain homogenate, rat brains were homogenized in PBS at 4°C using a Polytron (Kinematica, GmbH, Switzerland).

Subcellular fractionation of RBL cells

RBL-2H3 cells (2 x 108 cells) were fractionated over a discontinuous sucrose gradient as described previously (8).

Stimulation of RBL cells

Exocytosis in the RBL-2H3 cells was triggered as described previously (9). Secretion was allowed to proceed for 30 min at 37°C, and aliquots from the supernatants were taken for measurements of released ß-hexosaminidase activity.

Assay of ß-hexosaminidase activity

Aliquots (20 µl) of supernatants and cell lysates were incubated for 90 min at 37°C with 50 µl of the substrate solution consisting of 1.3 mg/ml p-nitrophenyl-N-acetyl-ß-D-glucosaminide (Sigma, St. Louis, MO) in 0.1 M citrate (pH 4.5). The reaction was stopped by the addition of 150 µl of 0.2 M glycine (pH 10.7). OD was read at 405 nm in an ELISA reader. Results were expressed as the percentage of total ß-hexosaminidase activity present in the cells.

Cell transfection

A full-length rat Syt I cDNA (generously provided by Dr. R. H. Scheller, Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University Medical Center, CA) was subcloned into the EcoRI site of the pCDNA3 expression vector (Invitrogen, San Diego, CA). RBL-2H3 cells (8 x 106) were transfected with 20 µg of pCDNA3-Syt I DNA or pCDNA3 alone by electroporation (0.25 V, 960 µF). Cells were immediately replated in tissue culture dishes containing growth medium (supplemented DMEM). G418 (1 mg/ml) was added at 24 h after transfection, and stable transfectants were selected within 7 days.

Immunofluorescence and berberine staining

Purified RPMCs (2.5 x 104 cells/ml) were allowed to adhere to glass coverslips for 2.5 h at 37°C in a humidified incubator. The cells were subsequently washed twice with PBS and fixed for 30 min at room temperature in 3% paraformaldehyde/PBS. Next, cells were washed three times with PBS and permeabilized for 3 min on ice with a cold permeabilization buffer (10 mM PIPES (pH 7.0), 100 mM NaCl, 300 mM sucrose, 1 mM EGTA, 1 mM PMSF, and 0.5% Triton X-100). For Syt immunostaining, the coverslips were washed with PBS containing 0.05% Tween 20 and incubated with the primary Ab (anti-Syt I at a 1/50 dilution) for 30 min. The coverslips were then washed three times in PBS/Tween 20 and incubated with rhodamine-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1/400 dilution for 30 min in the dark. For the berberine staining, the coverslips were subsequently washed in water for 10 min and stained for 20 min with 0.02% berberine-chloride (Sigma) in double-distilled water acidified to pH 4.0 by 1% citric acid. Coverslips were then washed in acidified water for 5 min and mounted with Slow-Fade mounting medium (Molecular Probes, Eugene, OR). Samples were analyzed using a Zeiss laser confocal microscope (Oberkochen, Germany).

SDS-PAGE and immunoblotting

Samples (normalized according to protein content or number of cells) were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed. Immunoreactive bands were visualized by the enhanced chemiluminescence method according to standard procedures.

Statistics

Statistical analysis was performed using the one-tailed Student t test for unpaired data. Data are presented as mean ± SEM of at least three independent experiments and three independent clones.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Western blot analyses using Abs directed against the cytosolic domain of the neuronal Ca2+ sensor Syt I revealed the presence of a major 65-kDa Syt immunoreactive protein; this protein comigrated with the Syt immunoreactivity present in a crude brain homogenate in whole cell lysates derived from RBL cells (RBL-2H3), which are a tumor analogue of mucosal mast cells, from primary mouse BMMCs, and from fully differentiated connective tissue-type RPMCs (Fig. 1Go). Brain homogenates as well as the RPMC and RBL cell homogenates also contained a 43-kDa immunoreactive protein, most probably corresponding to the well-documented proteolytic fragment of Syt that comprises its cytosolic domain (10).



View larger version (76K):
[in this window]
[in a new window]
  		FIGURE 1. Expression of Syt in mast cells. A crude rat brain homogenate (1.5 µg protein) (lane a) and whole lysates (1 x 106 cell equivalents) derived from RBL-2H3 cells (lane b), BMMCs (lane c), and RPMCs (lane d) were resolved by SDS-PAGE and immunoblotted using Abs directed against the cytoplasmic domain of Syt I (6).

 
The subcellular distribution of the Syt-like immunoreactivity in the RPMCs was subsequently examined by indirect immunofluorescence and confocal microscopy (Fig. 2Go). Syt (red) was found in a granular pattern (Fig. 2GoA), which suggested that it was localized to the secretory granules (SGs). To investigate this possibility further, the mast cells were also stained with berberine, for which fluorescence represents the content of heparin stored in the SGs of connective tissue-type mast cells (11). Indeed, staining with berberine (green) was remarkably similar to that of Syt immunoreactivity (Fig. 2GoB); most of it colocalized with Syt immunoreactivity, as indicated by the yellow stain (Fig. 2GoC).



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 2. Subcellular distribution of Syt in RPMCs and RBL cells. RPMCs were fixed, permeabilized, and incubated with anti-Syt I Abs followed by rhodamine-conjugated donkey anti-rabbit Abs. The cells were subsequently stained with berberine, and pictures were taken by confocal microscopy. A, Anti-Syt I (red); B, Berberine (green); C, Anti-Syt I and berberine (yellow). D, RBL cells were fractionated into SGs, cytosol, and PM fractions (8). A total of 80 µg of protein from each fraction was resolved by SDS-PAGE and immunoblotted with the anti-Syt I Abs.

 
The subcellular localization of Syt was also analyzed in RBL cells by separating the SGs from the cytosol and plasma membranes (PMs) as described previously (8). Most (~94%) of the 65-kDa Syt immunoreactive protein (RBL-Syt) was present in the histamine-containing SGs; only minor amounts were localized to the PM-enriched fraction (Fig. 2GoD and Table IGo). However, the latter could be accounted for by granular contamination, as this fraction also contained 5% of the total cellular histamine content (Table IGo). As expected, the 43-kDa protein was localized exclusively to the cytosolic fraction (Fig. 2GoD). Because all types of mast cells tested expressed the major 65-kDa Syt-like protein, we have chosen to focus on RBL cells, a cell line that could be genetically manipulated.


View this table:
[in this window]
[in a new window]
  		Table I. Distribution of protein, histamine, and Syt immunoreactivity in fractions of RBL cell sonicates1

 
To gain insight into the functional role of Syt, we stably transfected RBL cells with a full-length cDNA encoding the neuronal Ca2+ sensor Syt I. Lysates derived from the G418-resistant clones also expressed a smaller (~60-kDa) Syt immunoreactive protein in addition to the major, endogenous, 65-kDa protein (Fig. 3GoA, lanes a, b, and f). This protein, as well as the major 65-kDa Syt immunoreactive protein, was also detected when immunoblots of SGs were probed with anti-Syt I Abs that had been affinity-purified over recombinant Syt I, thus establishing the Syt immunospecificity as well as the SG location of both the endogenous and newly expressed Syt proteins (Fig. 3GoB).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 3. Expression of Syt I in RBL transfectants. A, Whole lysates derived from pCDNA3-Syt I-transfected RBL clones (1 x 106 cell equivalents) were probed with the anti-Syt I Abs. B, Samples of SGs (80 µg of protein) derived from G418-resistant RBL cells stably transfected with either the pCDNA3-Syt I recombinant vector (lane a), an empty pCDNA3 vector (lane b), or control RBL cells (lane c) were probed using affinity-purified anti-Syt I Abs (20 µg/ml). Arrowheads indicate the position of the new 60-kDa band.

 
To examine whether Syt I could modulate Ca2+-dependent exocytosis of the RBL cells, the Ca2+ ionophore (A23187)-induced release of ß-hexosaminidase, which is a marker enzyme for exocytosis, was measured both in control cells expressing the endogenous 65-kDa RBL-Syt protein and in Syt I-transfected cells. The latter cells expressed the new 60-kDa Syt-immunoreactive protein in addition to similar levels of RBL-Syt. In the absence of any stimulus, both control and Syt I-expressing cells released <=4% of their total ß-hexosaminidase, suggesting that basal release was not affected by the expression of Syt I. However, with increasing concentrations of the Ca2+ ionophore alone or in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), release from the Syt I-expressing cells was significantly elevated when compared with control cells (Fig. 4GoA). Secretion was dependent upon the concentration of the external Ca2+ (Fig. 4GoB). However, in contrast to its pronounced effect on the extent of exocytosis, the expression of Syt I had only a marginal effect on Ca2+ dependency; half-maximal responses were achieved at concentrations of 200 and 80 µM for control and the Syt I-expressing cells, respectively (Fig. 4GoB). Consequently, these results support the model (12), suggesting that Syt I may act in concert with an additional, high-affinity Ca2+ sensor.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4. Ca2+ ionophore-induced release from control and Syt I-expressing cells. Empty vector (open symbols) and pCDNA3-Syt I-transfected (closed symbols) RBL cells were incubated for 30 min at 37°C with either indicated concentrations of the Ca2+ ionophore A23187 alone (squares) or together with 50 nM TPA (circles) (A); 1 µM of the Ca2+ ionophore A23187 and 50 nM TPA in the presence of indicated concentrations of external Ca2+ (B); 10 µM of the Ca2+ ionophore A23187 alone (C); or 1 µM of the Ca2+ ionophore A23187 and 50 nM TPA (D) for the indicated time periods. The extent of the release is presented as the percentage of total ß-hexosaminidase activity. The data points presented are for the means of 16–20 determinations and include four independent clones stably transfected with the empty pCDNA3 vector and four independent clones stably transfected with the Syt I-pCDNA3 vector (A) and for 6–10 determinations that included three control and three Syt I-expressing independent clones (B–D). **, p < 0.005; *, p < 0.05.

 
The expression of Syt I accelerated the rate of Ca2+-dependent exocytosis triggered by the Ca2+ ionophore either alone (Fig. 4GoC) or in the presence of TPA by four- to fivefold (Fig. 4GoD). These results demonstrated that increasing the number of Syt molecules on the SGs could shift the kinetics and amount of released granules, probably by direct interactions of Syt I with endogenous effector proteins of mast cells. Because RBL cells express SNARE proteins (D.B., M.L., Y.A.M., and R.S.-E., unpublished observations), the latter are likely candidates to serve as these putative effectors.

In conclusion, our results substantiate the postulate that Syt serves as a general Ca2+ sensor, rather than as a regulator of neuroexocytosis alone. Our findings strongly suggest that Ca2+-dependent exocytosis in mast cells is controlled by a machinery that is similar to that of synaptic transmission, with Syt serving the role of a positive regulator in the process of ionophore-induced, Ca2+-dependent exocytosis. As such, our findings unveil a novel, previously unappreciated regulator of mast cell biology.


   Acknowledgments
 
We thank Dr. L. Mitelman for his help in all the microscopy studies and Drs. Y. Zick, I. Hammel, and D. Neumann for helpful discussions and critical reading of this manuscript. We also thank Drs. T. C. Sudhof and R.H. Scheller for their generous gifts of Abs and cDNA.


   Footnotes
 
1 This work was supported by grants from the Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities) and the Fritz Thyssen Stiftung (to R.S.-E.). Back

2 Address correspondence and reprint requests to Dr. Ronit Sagi-Eisenberg, Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978 Israel. E-mail address: Back

3 Abbreviations used in this paper: Syt, synaptotagmin; BMMC, bone marrow-derived mast cell; SG, secretory granule; RBL, rat basophilic leukemia; RPMC, rat peritoneal mast cell; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back

Received for publication June 25, 1998. Accepted for publication September 9, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Galli, S. J., J. R. Gordon, B. K. Wershil. 1991. Cytokine production by mast cells and basophils. Curr. Opin. Immunol. 3:865.[Medline]
  2. Foreman, J. C., J. L. Mongar, B. D. Gomperts. 1973. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature 245:249.[Medline]
  3. Sudhof, T. C., J. Rizo. 1996. Synaptotagmins: C2-domain proteins that regulate membrane traffic. Neuron 17:379.[Medline]
  4. Li, C., B. Ullrich, J. Z. Zhang, R. G. Anderson, N. Brose, T. C. Sudhof. 1995. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375:594.[Medline]
  5. Lang, J., M. Fukuda, H. Zhang, K. Mikoshiba, C. B. Wollheim. 1997. The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic ß-cells: action of synaptotagmin at low micromolar calcium. EMBO J. 16:5837.[Medline]
  6. Perin, M. S., V. A. Fried, G. A. Mignery, R. Jahn, T. C. Sudhof. 1990. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345:260.[Medline]
  7. Katz, H. R., E. T. Dayton, S. F. Levi, A. C. Benson, K. F. Austen, R. L. Stevens. 1988. Coculture of mouse IL-3-dependent mast cells with 3T3 fibroblasts stimulates synthesis of globopentaosylceramide (Forssman glycolipid) by fibroblasts and surface expression on both populations. J. Immunol. 140:3090.[Abstract]
  8. Sagi-Eisenberg, R., J. C. Foreman. 1984. Fractionation of mast cell components for studies of ligand-receptor binding at the plasma membrane. Immunol. Lett. 8:43.[Medline]
  9. Gat-Yablonski, G., R. Sagi-Eisenberg. 1990. Differential down-regulation of protein kinase C selectively effects IgE-dependent exocytosis and inositol trisphosphate formation. Biochem. J. 270:679.[Medline]
  10. Hausinger, A., W. Volknandt, H. Zimmermann. 1995. Calcium-dependent endogenous proteolysis of the vesicle proteins synaptobrevin and synaptotagmin. Neuroreport 6:637.[Medline]
  11. Enerback, L.. 1974. Berberine sulphate binding to mast cell polyanions: a cytofluorometric method for the quantitation of heparin. Histochemistry 42:301.[Medline]
  12. DeBello, W. M., H. Betz, G. J. Augustine. 1993. Synaptotagmin and neurotransmitter release. Cell 74:947.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
B. Textor, A. H. Licht, J. P. Tuckermann, R. Jessberger, E. Razin, P. Angel, M. Schorpp-Kistner, and B. Hartenstein
JunB Is Required for IgE-Mediated Degranulation and Cytokine Release of Mast Cells
J. Immunol., November 15, 2007; 179(10): 6873 - 6880.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
Y. Atiya-Nasagi, H. Cohen, O. Medalia, M. Fukudan, and R. Sagi-Eisenberg
O-glycosylation is essential for intracellular targeting of synaptotagmins I and II in non-neuronal specialized secretory cells
J. Cell Sci., April 1, 2005; 118(7): 1363 - 1372.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y.-N. Lee, J. Tuckerman, H. Nechushtan, G. Schutz, E. Razin, and P. Angel
c-Fos as a Regulator of Degranulation and Cytokine Production in Fc{epsilon}RI-Activated Mast Cells
J. Immunol., August 15, 2004; 173(4): 2571 - 2577.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. E. McRory, J. Hamid, C. J. Doering, E. Garcia, R. Parker, K. Hamming, L. Chen, M. Hildebrand, A. M. Beedle, L. Feldcamp, et al.
The CACNA1F Gene Encodes an L-Type Calcium Channel with Unique Biophysical Properties and Tissue Distribution
J. Neurosci., February 18, 2004; 24(7): 1707 - 1718.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Kapp-Barnea, S. Melnikov, I. Shefler, A. Jeromin, and R. Sagi-Eisenberg
Neuronal Calcium Sensor-1 and Phosphatidylinositol 4-Kinase {beta} Regulate IgE Receptor-Triggered Exocytosis in Cultured Mast Cells
J. Immunol., November 15, 2003; 171(10): 5320 - 5327.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Puri, M. J. Kruhlak, S. W. Whiteheart, and P. A. Roche
Mast Cell Degranulation Requires N-Ethylmaleimide-Sensitive Factor-Mediated SNARE Disassembly
J. Immunol., November 15, 2003; 171(10): 5345 - 5352.
[Abstract] [Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
N. Kimura, S.-i. Shiraishi, K. Mizunashi, H. Ohtsu, and I. Kimura
Synaptotagmin I Expression in Mast Cells of Normal Human Tissues, Systemic Mast Cell Disease, and a Human Mast Cell Leukemia Cell Line
J. Histochem. Cytochem., March 1, 2001; 49(3): 341 - 346.
[Abstract] [Full Text]

Home page
 JEMHome page
D. Baram, R. Adachi, O. Medalia, M. Tuvim, B. F. Dickey, Y. A. Mekori, and R. Sagi-Eisenberg
Synaptotagmin II Negatively Regulates Ca2+-triggered Exocytosis of Lysosomes in Mast Cells
J. Exp. Med., May 17, 1999; 189(10): 1649 - 1658.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baram, D.
Right arrow Articles by Sagi-Eisenberg, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baram, D.
Right arrow Articles by Sagi-Eisenberg, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS