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Published as doi: 10.1096/fj.07-8333com.
 (The FASEB Journal. 2008;22:194-206.)
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(The FASEB Journal. 2008;22:194-206.)
© 2008 FASEB

Synaptotagmin VII splice variants {alpha}, β, and {delta} are expressed in pancreatic β-cells and regulate insulin exocytosis

Benoit R. Gauthier*,1, Dominique L. Duhamel*, Mariella Iezzi*, Sten Theander*, Frédéric Saltel*, Mitsunori Fukuda{dagger}, Bernhard Wehrle-Haller* and Claes B. Wollheim*

* Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland, and

{dagger} Laboratory of Membrane Trafficking Mechanisms; Department of Developemental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi, Japan

1 Correspondence: Department of Cell Physiology and Metabolism, University Medical Center, 1211 Geneva 4, Switzerland. E-mail: benoit.gauthier{at}medecine.unige.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Synaptotagmins (SYT) are calcium-binding proteins that participate in regulated exocytosis. Although SYTI to IX isoforms are expressed in insulin-producing cell lines, hitherto only SYTIX has been associated with native β-cell insulin granules and implicated in exocytosis. SYTVII was also proposed to regulate insulin exocytosis, but its subcellular location and number of alternative splice variants produced remain controversial. Only transcripts of SYTVII {alpha}, β, and a novel splice variant {delta} are expressed in β-cells and INS-1E cells. Western blotting revealed that INS-1E cells predominantly produced SYTVII {alpha} and low levels of SYTVII β, whereas SYTVII {delta} was undectable. The protein colocalized with insulin granules but not with synaptic-like microvesicles. Overexpression of SYTVII {alpha} resulted in decreased insulin granule content with a concomitant translocation of the variant to the plasma membrane, while SYTVII β retained largely a granular pattern. Overexpressed SYTVII {delta} exhibited a distribution different to that of insulin granules and inhibited exocytosis when assessed by whole cell patch clamp capacitance recording. Silencing of SYTVII {alpha} by targeted RNA interference suppressed secretion, while repression of β slightly increased release. Our results demonstrate that SYTVII is expressed on insulin granules and that only SYTVII {alpha} is implicated in exocytosis under physiological conditions.—Gauthier, B. R., Duhamel, D. L., Iezzi, M., Theander, S., Saltel, F., Fukuda, M., Wehrle-Haller, B., Wollheim. C. B. The synaptotagmin VII splice variants {alpha}, β, and {delta} are expressed in pancreatic β-cells and regulate insulin exocytosis


Key Words: islet β-cells • calcium-induced exocytosis • INS-1E cells • membrane capacitance • RNAi


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BLOOD GLUCOSE HOMEOSTASIS in mammals is controlled by the regulated exocytosis of insulin from pancreatic islet β-cells. Coupling between glucose metabolism and insulin secretion is mediated by an increase in cytosolic ATP levels, resulting in the closure of ATP-sensitive K+ channels, with the subsequent depolarization of the plasma membrane and opening of voltage-sensitive Ca2+ channels (1) . The consequential rise in cytoplasmic free Ca2+ concentration triggers exocytosis through the concerted action of several components including SNARE proteins, which force the two membranes (vesicular and plasma) into close proximity. However, SNARE proteins are insufficient to account for Ca2+-regulated exocytosis, indicating the existence of a sensor, which accomplishes fusion on calcium entry (2) .

The leading candidates to serve as Ca2+ sensors that control vesicular fusion are members of the synaptotagmin (SYTs) gene family (2 , 3) . Most of the 15 members share a common gene structure comprising 14 exons. The encoded proteins are composed of a short intravesicular NH2-terminal region, a single membrane-spanning domain, a lysine- and arginine-rich region, as well as two homologous C2 domains (C2A and C2B) located in the cytoplasmic tail (3 , 4) . The C2 domains were first characterized in protein kinase C (5) are implicated in Ca2+-dependent phospholipid binding and interact with other essential exocytotic proteins (2 , 6) . SYTs I-VI and IX-XIII are predominantly expressed in brain, while SYTs VII-VIII and XIV-XV are mainly expressed in non-neuronal tissues such as heart, kidney, and pancreas (3 , 4 , 7) . Multiple synaptotagmins are often coexpressed within the same cell suggesting that different isoforms may accomplish distinct functions (8) . Biochemical studies performed mainly on SYTI and II indicate that these proteins, at high calcium concentrations, interact with the phospholipid bilayer of the plasma membrane as well as with SNARE proteins to coordinate and control fusion pore formation (9) . Furthermore, SYTI was also demonstrated to be implicated in recycling of synaptic vesicles subsequent to exocytosis (10) . Disruption of the SYTI gene in the mouse selectively abolishes the initial rapid phase of exocytosis for example in hippocampal synapses (11) and in chromaffin cells (12) . However, the restricted neuronal expression of SYTI and II suggests that additional isoforms may act as Ca2+ sensors in regulated exocytosis in neurons and insulin-secreting cells (8 , 13 , 14) .

Pancreatic islets as well as insulin-producing cell lines express multiple SYT isoforms (14 15 16 17) . However, to date, only SYTV and IX have been clearly shown to reside on large dense core vesicles (LDCV) and demonstrated by RNA interference (RNAi) experiments to mediate Ca2+-evoked insulin secretion in the insulinoma INS-1E cell line. Interestingly, both these SYTs are expressed in rat islets; however, SYTIX is found only in β-cells, while SYTV is expressed in {alpha}-cells (18 19 20) . We and others have also reported the expression of SYTVII in insulin-producing cells (15 , 16 , 21) . Experiments using permeabilized primary β-cells showed that introduction of a recombinant peptide containing the C2 domains of SYTVII inhibited Ca2+-stimulated insulin release (16) . Furthermore, overexpression of SYT VII in RINm5F cells caused amplification of carbachol-induced insulin secretion from intact cells (15) . Interestingly, SYTVII was found to produce several alternative splice variants derived mainly from within the linker domain (between the transmembrane and C2A domains) and that multiple variants were expressed in various tissues (21 , 22) . It was proposed that these splice variants differentially regulate synaptic vesicle recycling in neurons (23) . Alternatively, SYTVII was proposed to have a more ubiquitous function, coupling Ca2+ signaling to exocytosis of lysosomes for membrane resealing after injury as well as delivering lysosomal membrane to nascent phagosomes (24 , 25) . The subcellular localization of SYTVII as well as number of splice variants and function remains to be elucidated in β-cells. Indeed, in neuronal tissue, SYTVII was shown to be associated with the synaptic membrane, while in fibroblasts and potentially β-cells SYTVII was found on vesicles (15 , 22 , 26) . Surprisingly, two independent studies have also reported that overexpression of SYTVII in PC12 cells results in the distribution of the protein to either the plasma membrane or to the LDCV (21 , 22) .

The aim of the current study was to establish the expression pattern, localization, and function of SYTVII splice variants in insulin-producing cells. We show that SYTVII {alpha}, β, and a novel spliced variant {delta} lacking exons 6 to 14 are the only transcripts expressed in islet β-cells as well as in INS-1E cells whereas glucagon-producing {alpha}-cells express predominantly SYTVII {alpha}. Repression of splice variants by RNAi revealed that only SYTVII {alpha} stimulates glucose induced-insulin secretion. We propose that SYTVII, in concert with SYTIX, is the major Ca2+ sensor for insulin exocytosis in islet β-cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The expression vector encoding the green fluorescent protein was purchased from Clontech (Basel, Switzerland). The rabbit polyclonal antibody directed against the N-terminal domain of SYTVII was prepared as described previously (21) . Alternatively, a second rabbit polyclonal antibody directed against the amino acid sequence 46 to 133 of SYTVII was purchased from Synaptic Systems (Gottingen, Germany). The guinea pig antibody against insulin was supplied by Dr. P. Meda (University of Geneva, Geneva, Switzerland). Monoclonal antibodies against the c-myc epitope and synaptophysin (clone SVP-38) were obtained from Invitrogen (Basel, Switzerland) and Sigma (Buchs, Switzerland), respectively. The polyclonal antibody generated against carboxypeptidase H was a gift from J. Parkinson (Sheffield Hallam University, Sheffield, UK). Finally, streptolysin-O was expressed and purified as described previously (27) .

Cell culture and islet isolation
INS-1E cells (28) were cultured in RPMI 1640 (Invitrogen) supplemented with 5% fetal calf serum (FCS; Brunschwig AG, Basel, Switzerland) and other additions as described previously (29) . Islets were isolated from 7-week-old male Wistar rats (Elevage Janvier, Le Genest-St-Isle, France) by collagenase digestion (30) , handpicked, and cultured for 24 h in 11.5 mM glucose/RPMI 1640 supplemented with, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin (Sigma). Rat islet β- and {alpha}-cells were purified by FACS subsequent to autofluoresence activation using 20 mM glucose (31) .

Reverse transcript-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from rat brain as well as islets and INS-1E cells using the Trizol reagent (Invitrogen), and 2 µg were converted into cDNA as described previously (32) . Primers located in exon 3 (EX3–5') and exon 9 (EX9–3') of SYTVII were used to amplify by PCR all potential spliced variant found within these two exons (22) . The same EX3–5' primer was used in combination with a primer nested within exon 5 (EX5–3') to amplify truncated isoforms of SYTVII. Primer sequences are provided in Table 1 . Additional PCR reactions were performed with primers located in exon 6 to 14 along with a second primer positioned within exon 5 (EX5–5'). Quantitative real-time RT-PCR was executed on an ABI 7000 SDS (Applera Europe, Rotkreuz, Switzerland) using SYTVII isoform specific primers (33) . Primer sequences, listed in Table 1 , were designed using the Primer Express Software (Applera Europe, Rotkreuz, Switzerland).


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  		Table 1. Primers used for PCR analysis and RNA interference. Only the targeted sequences within the various SYTVII splice variants are provided for the RNA interference

Plasmid construction
cDNAs for rat {alpha}, β, and {delta} synaptotagmin VII were amplified from reverse transcribed RNA prepared from INS-1E cells. A common 5'-oligonucleotide spanning the ATG start codon (5'-GACGAAGGGGACCATGTACC-3') was employed in combination with two 3'-oligonucleotides; the first primer, located in exon 14 (5'-CGGCTTTCAGCTGGTGCCACTG-3') was used to amplify the {alpha} and β variants, while the second primer, situated in exon 5, was employed to amplify the {delta} varaint (5'-AACGACGGAGGAAGAGCGGG-3'). SYT VII amplicons were gel purified, cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), and confirmed by sequencing. EcoRI fragments were then gel purified and subcloned into the expression vector pcDNA3.1/myc-His (Invitrogen) in frame with the myc epitope. For the production of SYTVII C2AB recombinant peptides, a SpeI/EcoRI fragment harboring the C2AB domain was isolated from pEF-T7-GST-FLAG-SytVII-cyto (34) and subcloned into the SpeI and EcoRI sites of pGEX-2T (Amersham Biosciences Europe, Otelfingen, Switzerland). The GST-tagged SYTVII C2AB recombinant peptide was purified according to the manufacturer’s instructions (Amersham Biosciences Europe), and purity was confirmed by SDS-PAGE.

Cell transfection
Transient transfection of INS-1E or HEK cells was performed using the Lipfectamine 2000 reagent according to the manufacturer’s guidelines (Invitrogen).

Electrophysiological measurements
Cells were seeded on glass coverslips, transfected with appropriate SYTVII expression constructs along with a GFP construct, and maintained in culture for 2 days. Patch clamp capacitance recordings were performed as described by Iezzi et al. (35) .

Protein extracts preparation and Western blotting
Whole cell extracts as well as cytosolic and membrane protein fractions of INS-1E or HEK cells were prepared as described previously (36) . Homogenates (15 and 30 µg) were resolved on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20, and 5% milk powder and then incubated with the appropriate primary antibodies. Immunoreactive products were revealed by enhanced chemiluminescence (SuperSignal West Pico, Pierce, Rockford, IL, USA) using horseradish peroxidase coupled secondary antibodies.

Sucrose gradient
Subcellular fractionation of INS-1E was performed as described previously (19 , 37) . The specific distribution within the gradient of synaptic-like microvesicles (SVP38) and insulin-containing secretory granules (carboxypeptidase H) was assessed by Western blotting. The amount of insulin was monitored by radioimmunoassay (RIA).

Immunofluorescence studies
INS-1E and primary β-cells were cultured on glass cover slips precoated with polyornithine (Sigma). For immunodetection of endogenous SYTVII, SYTIX, insulin, and SVP-38 proteins, cells were fixed for 25 min in a 4% paraformaldehyde-PBS solution and permeabilized for an additional 60 min in PBS containing 0.1% saponin and 5% horse serum. Preparations were then processed as described by Iezzi et al. (35) . Alternatively, SYTVII/insulin costaining were analyzed using objective type, double color total internal reflection fluorescence microscopy (TIRF) performed on a Zeiss Axiovert 100M (Carl Zeisse AG, Feldbach, Switzerland) equipped with a combined epi-fluorescence/TIRF adapter (TILL-photonics, Gräfelfing, Germany) and a high numerical aperture objective (x100 NA 1.45; Carl Zeiss AG, Feldbach, Switzerland). Fluorophores where excited either with the 488nm line of a 150 mW argon-ion laser (Reliant 150 m, Laserphysics) using an exitation filter (488/10), dichroic mirror (DCLP500) and band pass emission filter (BP525/50) or exited with a 535 nm 20 mW Diode laser (Compass 215M-20; Coherent AG, Lübeck, Germany) and analyzed with a XF2062 (555DRLP, Omegafilters) dichroic mirror and bandpass emission filter XF3022 (580DF30, Omega filters). Images were aquired with a 12-bit CCD camera (Orca 9742–95; Hamamatsu, Japan). Laser shutters, camera, and microscope set up were controlled by the Openlab software (Improvision, Basel, Switzerland).

RNAi
RNAi experiments were performed as described previously using a novel expression vector that we created using the Adeno-X Tet-On expression system from Clontech (BD Clontech, Basel, Switzerland; ref. 38 ). Four 21-nucleotide SYTVII hairpin RNA structures (shRNA) with a 6-nucleotide loop were synthesized, annealed and ligated to the PmeI and XbaI sites of pDLDU6. The targeted sequences, listed in Table 1 , were 1) siEX5, targeted to a sequence located in exon 5, 2) siEX9, aimed at a common region of SYTVII {alpha} and β located in exon 9, 3) siEX39, located at the splice junction of exon 3 and 9 of SYTVII {alpha}, and 4) siEX4 targeted to exon 4 of SYTVII β and {delta}. Impact of the various constructs on protein levels was initially verified in HEK cells transfected with the individual SYTVII splice variants. The effect of siEX5, siEX9, siEX39, and siEX4 on endogenous mRNA levels of SYTVII {alpha}, β as well as on SYTIX in INS-1E cells was determined by real time RT-PCR.

Secretion assays
Preparation of intracellular buffers, permeabilization of INS-1E cells (1.5x105 cells/20 mm/well) with streptolysin-O and Ca2+-induced exocytosis in the presence of either 100 nM GST or GST-SYTVIIC2AB recombinant peptides were performed as described (14) . Secreted insulin levels were determined by radioimmunoassay (33) . Cellular insulin content was determined after extraction with acid ethanol as described previously (39) . Alternatively, hormone secretion was monitored using the well established human growth hormone assay as a reporter of secretion in RNAi experiments (19) . INS-1E cells were transiently cotransfected with a plasmid encoding the human growth hormone along with pDLDU6, siEX5, siEX39, siEX9 or siEX4. Glucose-induced hormone release was measured as detailed by Iezzi et al. 72 h post-transfection (19) .

Statistical analysis
Results are expressed as mean ± SE. Where indicated, the statistical significance of the differences between groups was estimated by Student’s unpaired t test. Statistical significance is P < 0.05 (*) and P < 0.01 (**).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Synaptotagmin VII {alpha}, β, and {delta} are expressed in β-cells
Several alternative splice variants of SYTVII are expressed in the neuronal PC12 cell line as well as in neurons (21 , 22) . These were shown to arise from alternative splicing spanning exons 4 to 8 located between the transmembrane region and the C2A/B domains (Fig. 1 A). To establish the alternative splicing variants formed in insulin-producing cells, we amplified this spacer region by RT-PCR using primers located in exon 3 and exon 9 (forward, EX3–5' and reverse EX9–3'). We also devised a third primer located in exon 5 (reverse EX5–3') to specifically detect, in combination with EX3–5', the truncated variants described by Sugita et al. (22) . As shown in Fig. 1B , three PCR products were detected in INS-1E cells, FACS purified rat β-cells and in the control rat brain sample. Sequence analysis revealed that two of these fragments corresponded to the splice variants SYTVII {alpha} and β (21) , while the third fragment was identified as the truncated variant comprising exons 1 to 5 inclusively (22) . Glucagon-producing {alpha}-cells were found to express predominantly SYTVII {alpha} as well as low levels of the {delta} transcript (Fig. 1B ). To exclude the possibility that other alternative splice variants were produced at lower levels, we repeated these experiments using the EX3–5' primer along with reversed primers nested within exons 6 to 8. Additional transcripts were never detected indicating that alternative splicings between exon 3 or 4 and exons 6, 7, and 8 are rare events in insulin-producing cells (data not shown). To delineate the exon configuration of the truncated variant, we also performed PCR using a forward primer located in exon 5 (EX5–5') in combination with reverse primers located in exons 6 to 14. In contrast to a previous study (22) , we were unable to amplify the truncated form using primers extending beyond exon 5 indicating that our transcript is only composed of exons 1 to 5 (data not shown). We thus named this novel alternative splice variant SYTVII {delta}. We next evaluated the relative abundance of the three splice variants by quantitative RT-PCR and found that levels of SYTVII {alpha}, β, and {delta} mRNAs in INS-1E cells were ~11 ± 2, 3.8 ± 1.0, and 0.5 ± 0.1, respectively, as compared to cyclophilin (Fig. 1C ). Although a similar ratio in splice variant transcripts was evaluated in purified primary rat β-cells, overall levels were much lower than in INS-1E cells (Fig. 1C ). Western blotting analysis using an antibody raised against the N terminus of SYTVII and previously shown to be highly specific (21) confirmed that SYTVII {alpha} (~60 kDa) was the predominant form expressed in INS-1E cells. A fainter band with a molecular mass of ~66 kDa, corresponding to SYTVII β, was also detected in extracts, while the {delta} variant was never discerned (Fig. 1D ). As expected, SYTVII was only found in the crude membrane protein fraction. We previously demonstrated that SYTIX and SYTV were critical for calcium-dependent insulin exocytosis in INS-1E cells (19) . However, primary β-cells express only SYTIX (19 , 20) , while SYTV was only found in {alpha}-cells (18 , 19) . We thus evaluated the relative expression levels of SYTVII (including all splice variants) and IX in insulin-producing-cells by quantitative RT-PCR. We found that SYTVII was consistently higher than SYTIX in INS-1E cells (9.8±2.0 vs. 3.0±0.5), islets (2.0±0.2 vs. 0.75±0.10), and FACS purified β-cells (3.8±0.1 vs. 0.75±0.10) as compared to the housekeeping gene cyclophilin (Fig. 1E ).


Figure 1
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  		Figure 1. Insulin-producing cells express only synaptotagmin VII {alpha} and β as well as a novel splice variant {delta}. A) Schematic representation of SYTVII mRNA structure depicting all potential exons. Five alternative spliced exons (4 to 8) between transmembrane region (exon 2, represented in black) and C2AB domains (exons 9 to 14) are shown in gray. B) Structure of SYTVII {alpha}, β, and {delta} and corresponding RT-PCR amplification performed with RNA isolated from rat brain, INS-1E cells and islets as well as from purified β- and {alpha}-cells. Plasmids containing either SYTVII {alpha} or β cDNA were used as control templates. Fragments were analyzed by 2% agarose gel electrophoresis and confirmed by DNA sequencing. C) Quantitative real-time RT-PCR analysis of SYTVII mRNA splice variants steady-state levels in INS-1E cells (left panel) and purified rat β-cells (right panel). For each sample (20 ng), 3 distinct amplifications were performed in parallel and mean values for each splice forms were normalized to the mean value of the reference housekeeping gene cyclophilin. Results are expressed as relative transcript levels and are mean ± SE of at least 3 independent experiments. D) Membrane and cytosolic protein fractions of INS-1E cells were separated by SDS-PAGE and subjected to immunoblotting with anti-SYTVII antibody. E) Quantitative real-time RT-PCR analysis of SYTVII and SYTIX mRNA steady-state levels in INS-1E cells, islets, and purified β-cells. Results are expressed as in C.

SYTVII predominantly localizes on LDCV of insulin-producing cells
Several studies executed in PC12 cells have indicated that SYTVII is localized to the plasma membrane, while others detected the protein on LDCV (22 , 40 , 41) . Consistent with the latter, SYTVII was also found on mature dense lysosomes of fibroblasts (42) . Discrepancies in the localization of SYTVII may originate from the fact that most studies were performed using cell lines overexpressing SYTVII, which may cause inappropriate targeting of the recombinant protein. To circumvent this caveat, we determined the subcellular localization of endogenous SYTVII by immunofluorescence in INS-1E and primary β-cells (Fig. 2 ). Insulin-producing cells contain predominantly LDCV and GABA-containing synaptic-like microvesicles (SLMV; refs. 37 , 43 ), while lysosomes account for ~0.4% of the total vesicular population (44) . In view of the negligible lysosomal population, we focused on colocalization of SYTVII with the former two types of vesicles. TIRF analysis revealed that insulin granules in close proximity to the basal plasma membrane in INS-1E cells as well as in primary β-cells partially stained for SYTVII (Fig. 2A ). Interestingly, LDCVs appear to be associated with stress fibers in well attached INS-1E cells, while they are randomly distributed in primary β-cells that do not adhere strongly to the substratum. To reinforce the notion that SYTVII is found on insulin granules, we performed coimmunofluorescence analysis by conventional confocal microscopy using SYTIX as another marker of insulin granules (19 , 20) . Consistent with TIRF analysis, SYTVII colocalized with SYTIX in both INS-1E and primary β-cells (Fig. 2B ). Of note, SYTVII was also detected on vesicles devoid of insulin (Fig. 2A, B ). However, SYTVII was not located on organelles containing synaptophysin (Fig. 2C ). The latter is used as marker for SLMV, which are typically found in the perinuclear region (37) and therefore were examined by conventional confocal microscopy.


Figure 2
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  		Figure 2. SYTVII preferentially colocalizes with insulin and SYTIX but not with synaptophysin in INS-1E and rat islet β-cells. A) Total internal reflection fluorescence images of insulin and SYTVII protein obtained from immunostained INS-1E cells (top panel) or primary β-cells (bottom panel). Dispersed rat islet cells or INS-1E cells were cultured for 3 days on coated glass bottom wells, fixed, and stained with anti-insulin antibodies followed by FITC coupled secondary antibodies (green) and anti-SYTVII antibody (Synaptic Systems) followed by Texas-Red conjugated secondary antibodies (red). Merged image reveals SYTVII/insulin double positive vesicles (inset). B) Confocal microscopy images acquired from either INS-1E cells or dispersed pancreatic β-cells colabeled with an antibody against SYTVII (green; Fukuda et al., 2002) together with an anti-SYTIX (red). C) Double immunofluorescence with antisynaptophysin (SVP38, red) and anti-SYTVII (green) in INS-1E cells (top panel) and in a rat pancreatic β-cell (bottom panel). Dotted circles delineate nuclei.

To further characterize the intracellular distribution of SYTVII, we performed a continuous sucrose density gradient, which permits the separation of the two predominant vesicle populations in INS-1E cells (19) . Insulin vesicles were mainly detected in the fractions 12–17 (Fig. 3 , top panel) along with the LDCV resident protein carboxypeptidase H, while synaptophysin was concentrated in fractions 7–11 (Fig. 3 , bottom panels). SYTVII was only found in fractions 12–17 consistent with its association with LDCV and not the plasma membrane. Of note, the SYTVII antibody recognized a protein complex of ~110 kDa corresponding to the SDS-resistant SYTVII homodimer (45) . However, we were unable to detect either SYTVII β or {delta} (refer to Fig. 1C ).


Figure 3
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  		Figure 3. Subcellular fractionation of SYTVII in INS-1E cells by a sucrose density gradient. Aliquots of individual fractions were analyzed by Western blotting using antibodies directed against synaptophysin (SVP38), carboxypeptidase H (CPH), and SYTVII. Top panel displays amount of insulin present in fractions corresponding to the distribution of insulin-containing granules. Fractions in which plasma membrane partitioned are denoted on top of autoradiograph. Sucrose concentration was calculated from refractive index of fractions.

Overexpression of myc-tagged SYTVII {alpha}, β, and {delta} in INS-1E cells results in distinct cellular localization of the three recombinant proteins
We next sought to define the subcellular distribution of the three alternative splice variants in INS-1E cells. Since the antibody is unable to distinguish the various SYTVII proteins due to their common N-terminal sequence, we cloned the cDNAs corresponding to SYTVII {alpha}, β, and {delta} from INS-1E cells and constructed myc-tagged recombinant proteins (myc-SYTVII {alpha}, β, and {delta}). These were then individually transfected in INS-1E cells and visualized by immunostaining of the myc-epitope. Double labeling experiments using anti-insulin as well as anti-myc sera revealed that myc-SYTVII {alpha} was predominantly targeted to the plasma membrane (Fig. 4 A). Furthermore, a consistent decrease in insulin staining was noted in the presence of myc-SYTVII {alpha}. Although similar plasma membrane redistribution was noticed in INS-1E cells overexpressing myc-SYTVII β, this splice variant was predominantly localized with LDCV (Fig. 4A , middle panel). Interestingly, overexpression of myc-SYTVII β did not decrease insulin staining. However, insulin granules appeared to concentrate in the peri-nuclear region. In contrast, myc-SYTVII {delta} was only found on vesicular structures distinct from insulin granules (Fig. 4A , bottom panel) or SLMV (data not shown). The astonishing disparity in the subcellular distribution of the three recombinant proteins could be attributable to variation in their expression levels. However, as estimated by immunoblotting using anti-SYTVII, myc-SYTVII {alpha}, β, and {delta} (predicted molecular mass of ~25 kDa) were expressed at similar levels, thus excluding this possibility (Fig. 4B ). Interestingly, Myc-SYTVII {alpha}/GFP+ INS-1E cells contained ~35% less insulin as compared to either SYTVII {alpha}/GFP– cells or to SYTVII β and {delta} transfected cells (Fig. 4C ), indicating that overexpression of the alpha splice variant may promote exocytosis or decrease the insulin content of individual granules. To discriminate between these two possibilities, we measured human growth hormone (hGH) release as well as cellular content of cells cotransfected with individual SYTVII splice variants and a plasmid encoding the hGH. The latter is targeted to insulin-containing granules and can be used to monitor secretion as an insulin substitute in the subpopulation of transfected cells (46) . Cells were maintained in 11.5 mM glucose to stimulate exocytosis. A 30% increase in hGH was measured in the culture media of cells overexpressing SYTVII {alpha}, which was accompanied by a 25% decrease in cellular content of growth hormone. No significant changes in hGH release or content were detected in INS-1E cells overexpressing either SYTVII β or {delta} (Fig. 4D ). Taken together, these results suggest that SYTVII {alpha} is involved in glucose-induced insulin secretion rather than modulating hormone content of vesicles.


Figure 4
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  		Figure 4. Overexpression of SYTVII splice variants in INS-1E cells results in distinct subcellular localization: A) Confocal microscopy images of transfected cells expressing myc-SYTVII {alpha}, β, and {delta}. The cells were costained with anti-insulin ({alpha}, β, green; {delta}, red) together with the anti-c-myc serum ({alpha}, β red; {delta}, green). Arrows indicate cells coexpressing insulin and transfected SYTVII. B) Western blotting performed with anti-SYTVII ref. (21 ) showing equal amounts of SYTVII {alpha}, β and {delta} proteins in transfected INS-1E cells. *Potential multimer of myc-SYTVII {delta}. C) Insulin content of INS-1E cells cotransfected with a GFP expressing vector as well as SYTVII {alpha}, β or {delta} was determined in FACS purified GFP– (white bars) and GFP+ (black bars) cells. Results are depicted as % of GFP– cells of 3 independent experiments. D) hGH released into culture medium as well as cellular content of INS-1E cells during 24 h after transfection with equal amounts of SYTVII {alpha}, β, {delta}, or control vector (24 h post-transfection). Glucose concentration in media was 11.5 mM. Amount of hGH (ng/ml) was determined by ELISA from at least 3 independent experiments. *P < 0.05.

Overexpression of myc-SYTVII {delta} but not {alpha} or β reduces calcium-induced exocytosis in INS-1E cells
Disparities in the subcellular distribution of the endogenous SYTVII as compared to the recombinant proteins as well as increased basal insulin secretion in cells overexpressing SYTVII {alpha} reveal potential functional differences of the three splice variants in regulating exocytosis and/or endocytosis in INS-1E cells. To appraise this possibility, we performed membrane capacitance measurements of INS-1E cells overexpressing individual myc-SYTVII variants. This technique detects overall changes in membrane surface with time thus reflecting both vesicle fusion and recycling. No alterations in resting capacitance were observed among groups indicating that overexpression of myc-SYTVII did not modify net membrane surface at steady state (data not shown) 48 h post-transfection. We next recorded changes in membrane capacitance induced by infusion of 2 µM Ca2+ through the patched pipette. A monotonous increase in membrane capacitance, similar in magnitude to previously reported values in INS-1E cells and native β-cells (47 , 48) , was observed in control cells (Fig. 5 A). An average capacitance rate of 5 fF/s was calculated from these measurements. This rate was not statistically altered in INS-1E cells transfected with either myc-SYTVII {alpha} or β, while a significant decrease of 60% was detected in INS-1E cells overexpressing myc-SYTVII {delta} (Fig. 5B ).


Figure 5
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  		Figure 5. Overexpression of SYTVII {delta} alters the rate of membrane capacitance induced by Ca2+. INS-1E cells were transfected with equal amounts of SYTVII {alpha}, β, or {delta} and capacitance measurements were estimated 48 h later using the whole cell patch configuration. A) Representative traces subsequent to a rise in cytosolic Ca2+. Time of patch rupture is set at 0. B) Capacitance rates were derived from slopes calculated from initial 40 s of recording and are depicted as a bar graph. Number of experiments ranged from 9–11. *P < 0.05.

SYTVII regulates glucose-induced exocytosis in INS-1E cells
In a previous study, we reported the potential involvement of SYTVII in Ca2+-dependent insulin secretion in rat islet cells (16) . To substantiate these findings, we performed similar experiments in INS-1E cells in which the effect of a recombinant fragment corresponding to the SYTVII C2AB domain on Ca2+-induced insulin secretion was evaluated in streptolysin-O permeabilized cells. In control experiments, we stimulated cells with the slowly hydrolysable GTP analog GTP{gamma}S, which exerts its effect independent of Ca2+ (49) . The fusion recombinant peptide, GST-SYTVII C2AB, was efficiently expressed in bacteria and was not degraded or sequestered in inclusion bodies (Fig. 6 A). Addition of this fragment to permeabilized cells resulted in 30% inhibition of Ca2+-induced exocytosis, while secretion in the presence of 100 mM GTP{gamma}S was unaffected (Fig. 6B ). Although, these results clearly indicate that the SYTVII C2AB domain is proficient in interfering with Ca2+-induced exocytosis, the peptide may impede the general exocytotic machinery, preventing discrimination between the effect of SYTVII and SYTIX on insulin secretion (19) . To circumvent this caveat, RNAi was performed to specifically suppress SYTVII. As endogenous protein levels of the SYTVII splice variants are too low in INS-1E cells for adequate quantification of their suppression, HEK cells were cotransfected with individual myc-SYTVII constructs along with the siEX5 and siEX9 and inhibition was estimated by Western blot analysis using the myc antibody. The results demonstrate that siEX5 inhibited by ~50% SYTVII {delta}, while having no effect on either {alpha} or β whereas siEX9 drastically suppressed {alpha} and β but not {delta} (Fig. 6C ). Transcript levels were then measured in INS-1E cells cotransfected with either siEX5 or siEX9 along with GFP. Subsequent to FACS purification, SYTVII {alpha} and β were reduced by 70 and 50%, respectively, in INS-1E cells coexpressing GFP and siEX9 (Fig. 6D ). In contrast, no reduction was observed in cells transfected with either the empty vector or siEX5. Transcript levels for SYTVII {delta} were to low to accurately allow estimation of repression of this variant (data not shown). However, more importantly, repression was specific to SYTVII since mRNA levels for SYTIX were unaltered (Fig. 6E ). We also found that insulin as wells as STAT1 transcripts were unaltered indicating that cells were not undergoing apoptosis as described previously by Sledz and colleagues (50 ; data not shown). We next evaluated the effect of the two shRNA on glucose-induced hGH secretion in transfected INS-1E cells. Basal secretion at 2.5 mM glucose was not different among the various experimental groups (Fig. 6F ). When hormone secretion was induced with 16.5 mM glucose, there was a 50% inhibition for cells expressing siEX9, while levels for siEX5 were similar to those found in control cells (Fig. 6F ).


Figure 6
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  		Figure 6. SYTVII promotes insulin secretion in INS-1E cells. A) Analysis of purified GST-SYTVII C2AB recombinant fusion peptide by SDS-PAGE and coomassie staining. B) INS-1E cells were permeabilized with streptolysin-O and subsequently exposed to basal or stimulatory concentrations of Ca2+ (0.1 or 10 µM) or 100 mM GTP{gamma}S (at 0.1 µM Ca2+) in the presence of GST fusion proteins. Insulin secretion was measured by radioimmunoassay, and results are expressed as percentage of insulin release as compared to control samples without the addition of recombinant proteins. Results are mean of 3 independent experiments and are expressed as % released as compared to GST fragment alone. C) Selective decrease of SYTVII expression by RNAi. HEK cells were transiently transfected with myc-tagged SYTVII {alpha}, β, or {delta}. Degree of silencing was then determined by cotransfecting cells with either an empty pDLDU6 vector (control) or with a pDLDU6 vector containing a shRNA directed against exon 5 (siEX5) or 9 (siEX9) of SYTVII. After 72 h cells were homogenized and equal amounts of protein were analyzed by Western blotting using an antibody directed against the myc epitotpe. D) INS-1E cells were cotransfected with an empty pDLDU6 vector (U6, control) or with siEX5 or siEX3/9 along with an expression vector for EGFP. GFP– (white bars) and GFP+ (black bars) cells were purified by FACS, RNA extracted and endogenous SYTVII {alpha}, β as well as SYTIX (E) mRNA levels were evaluated by QT RT-PCR. Results are the mean of least 4 independent experiments performed in triplicates and are expressed as fold changes as compared to the empty vector. F) Effect of SYTVII silencing on hormone secretion in INS-1E cells. Cells were transiently cotransfected with an expression vector encoding hGH along with the shRNA vector described in C. Seventy two hours later, the cells were incubated for 30 min in 2.5 mM glucose (white bars) and subsequently stimulated with 16.5 mM glucose (black bars). Amount of hGH released (ng/ml) from 3 independent experiments was determined by ELISA. **P < 0.01; *P < 0.05.

SYTVII {alpha} but not β is involved in glucose-induced exocytosis in INS-1E cells
Since SYTVII {alpha} and β mRNAs are relatively abundant as compared to SYTIX mRNA (Fig. 1E ), we evaluated the contribution of each variant on insulin secretion. To repress SYTVII {alpha}, a shRNA overlapping the splice junction of exon 3 and 9 was designed (siEX3/9), while a shRNA targeted to exon 4 was generated to inhibit SYTVII β (siEX4). Western blotting analysis performed on transfected HEK cells revealed that siEX3/9 completely repressed SYTVII {alpha} whereas β was only slightly inhibited by the siRNA. However, the {delta} splice variant was unaffected by siEX3/9 (Fig. 7 A). In contrast, siEX4 inhibited both exon 4-bearing β and {delta} splice variants whereas the {alpha} form remained unaltered (Fig. 7A ). Taken together, these results show that we can efficiently repress individual splice variants of SYTVII. Consistent with transfection studies in HEK cells, 80% repression of endogenous SYTVII {alpha} transcript was achieved in GFP+ INS-1E cells expressing siEX3/9, while a small but significant 20% inhibition of the β variant was also detected with this shRNA most likely due to the common exon 9 sequence (51) (Fig. 7B ). However, siEX4 specifically interfered with SYTVII β resulting in 50% repression of the transcript in GFP+ cells (Fig. 7C ). Hormone secretion was then measured in INS-1E cells cotransfected with either siEX3/9 or siEX4 along with the hGH plasmid. In control cells, a 4-fold increase in hGH was observed when 16.5 mM glucose was added to the medium (Fig. 7D ). Expression of siEX3/9 completely abrogated this increase (Fig. 7D ), while cells harboring siEX4 exhibited increased glucose-induced hGH release (Fig. 7E ).


Figure 7
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  		Figure 7. Repression of SYTVII {alpha} by RNAi abolishes glucose stimulated hormone release whereas decrease in SYTVII β has no effect. Attempts to inhibit SYTVII {alpha} and β mRNA were made by creating shRNAs targeted to either the splice junction of exons 3 and 9 (siEX3/9) or to exon 4 (siEX4). (A) Efficiency of protein repression was then evaluated in HEK cells cotransfected with individual splice variants along with either siEX3/9 or siEX4. After 72 h cells were homogenized and equal amounts of protein were analyzed by Western blotting using an antibody directed against the myc epitotpe. Subsequent to cotransfection with either siEX3/9 (B) or siEX4 (C) along with a GFP expressing vector, INS-1E cells were FACS purified into GFP– (white bars) and GFP+ (black bars) subgroups, RNA extracted and transcript levels for SYTVII {alpha} and β were determined by QT RT-PCR. Results are mean of least 3 independent experiments performed in triplicates and are expressed as fold changes as compared to the pDLDU6 empty vector (U6). hGH release in response to glucose was measured in INS-1E cells transfected with either siEX3/9 (D) or siEX4 (E). Amount of hGH released (ng/ml) from at least three independent experiments was determined by ELISA. **P < 0.01; *P < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of synaptotagmins as calcium sensors in regulated exocytosis is irrefutable (2 , 52 , 53) . However, the exact contribution and localization of individual family members such as SYTVII in endocrine cells have been a matter of perpetual debates (15 , 16 , 22 , 40 , 41 , 54) . This has been further compounded by the controversial presence of alternative splice variants as seen for SYTVII (21 22 23) . Our thorough RT-PCR analysis using a combination of nested primers within individual exons revealed that only 3 splice variants are generated in rat brain as well as in islet β-cells. The predominant form of SYTVII expressed in INS-1E cells is produced from a splicing event between exon 3 and 9 and corresponds to the {alpha} variant previously described by Fukuda et al. (21) . SYTVII β arises from the alternative splicing of exon 4 to 9 and is expressed at lower levels as compared to SYTVII {alpha} (2.5 fold lower). Similar differences were obtained at the protein level indicating no post-transcriptional regulation of the two transcripts. Interestingly, in contrast to previous studies we found that the third splice variant corresponding to a truncated form of SYTVII did not extend beyond exon 5 (22) . This novel SYTVII variant, denoted as {delta}, lacks exons 6 to 14. A stop codon located in exon 5 generates a SYTVII peptide devoid of the cytoplasmic C2 domains. Such truncation was shown to increase vesicle recycling in PC12 cells (23) . Transcript levels of this variant were found to be 22-fold lower than those of SYTVII {alpha} in INS-1E cells. Western blotting failed to reveal the SYTVII {delta} protein, suggesting that either levels are below detection limits or that the transcript may not produce a stable protein (22) . Although we cannot exclude that additional alternative splice variant are produced during development, our results suggest that mature islet β-cells appear to exclusively express SYTVII {alpha}, β, and possibly {delta}. Interestingly, we found that overall mRNA levels of SYTVII were consistently higher than SYTIX in INS-1E, islets and purified β-cells indicating that the participation of SYTVII in exocytosis may be at least as important as SYTIX (19 , 20) .

We assessed the subcellular distribution of the endogenous SYTVII by confocal and TIRF microscopy as well as by continuous sucrose density gradient. All approaches clearly demonstrated colocalization of the protein with insulin granules but not with either SLMV or the plasma membrane. Our results are consistent with the targeting of SYTVII to LDCVs controlling exocytosis in insulin-producing-cells (15) and PC12 cells (41 , 55) as well as to lysosomes in fibroblast (26) . Interestingly, a very recent study localized SYTVII to the endosomal/lysosomal pathway in INS-1E cells (54) . However, SYTVII was not found on insulin granules. Intriguingly, the insulin granule resident membrane protein SV2C (35) was also not detected (54) . The concept that endogenous SYTVII is found at the plasma membrane is, however, strongly weakened by the current findings (22) . In contrast, only overexpression of myc-SYTVII {alpha} in INS-1E cells resulted in the translocation of the protein to the plasma membrane, while myc-SYTVII β and {delta} predominantly retained a vesicular pattern. The major difference between the various splice variants is the inclusion of exon 4 within the spacer region of SYTVII β and {delta}. Interestingly the spacer region of SYTIV was shown to be essential for Golgi localization (56) . In depth analysis of exon 4 both at the nucleotide and amino acid levels, however, did not reveal any identifiable motifs involved in cellular localization. Given that this exon is not conserved among SYT family members, a potential explanation for the distinct subcellular distribution of SYTVII splice variants subsequent to overexpression may be attributable to this sequence. Furthermore this could provide a reconcilable rationalization of discrepancies of SYTVII localization reported pending the choice of splice variant used for transfection.

The astonishing findings that distinct localizations of SYTVII variants arise subsequent to overexpression provide valuable insight into the potential distinct regulatory function of these in exocytosis and/or endocytosis. Indeed, stimulated hormone release with a concomitant translocation of SYTVII {alpha} to the plasma membrane suggests that increased expression of this variant may promote exocytosis, while overexpression of SYTVII β is less efficient. Consistent with this hypothesis, an earlier study has shown a marked decrease in the number of vesicles in synapses overexpressing SYTVII {alpha} and to a lesser extent with SYTVII β (23) . Furthermore, this same study demonstrated that a truncated SYTVII accelerated endocytosis. Substantiating these observations we find that overexpression of SYTVII {delta} results in reduced membrane capacitance indicative of either decreased exocytosis or increased endocytosis. Consistent with the latter, SYTVII {delta} was found to reside on vesicles distinct from insulin or SLMV. However, preliminary experiments using the membrane dye FM4–64 failed to detect any differences in endocytosis in INS-1E cells overexpressing SYTVII {delta} subsequent to glucose stimulation (data not shown). Thus further work will be required to substantiate the functional impact of this low abundance truncated splice variant on endocytosis in insulin-producing cell.

Evidence for the potential role of SYTVII in secretagogue-induced insulin exocytosis was provided earlier in stable clones overexpressing the protein. However, although these cells exhibited enhanced Ca2+-stimulated insulin exocytosis in a permeabilize preparation, K+ did not evoke increased insulin secretion in intact cells. Thus, long-term expression of the recombinant SYTVII may interfere with other components of the secretory pathway such as SYTIX leading to aberrant exocytosis. Furthermore, the physiological secretagogue of insulin, glucose, was not evaluated in that study (15) . Here, we clearly demonstrate the specific impact of SYTVII on glucose-stimulated hormone secretion of β-cell granules by RNAi. We use a novel RNAi approach in which a shRNA targets intron/exon boundaries in order to discriminate between splice variants. This allowed us to show that only SYTVII {alpha} could effectively control secretion, while repression of SYTVII β did not interfere with this process. However, we discovered that silencing of SYTVII β consistently resulted in enhanced glucose-induced hormone secretion by ~35% (refer to Fig. 7D ). Furthermore, although not statistically significant, we found that the capacitance rate of cells overexpressing myc-SYTVII β was consistently lower than myc-SYTVII {alpha}. It is therefore tempting to speculate that SYTVII β could act as a regulator of exocytosis by inhibiting the positive role of SYTVII {alpha} on the rate of fusion. Furthermore, SYTVII β could interfere with recruitment of granules to the plasma membrane, as we observed increased peri-nuclear insulin staining in INS-1E cells overexpressing this splice variant. The exact mechanism involved in this novel regulatory loop remains to be clarified but could implicate exon 4 of SYTVII β. Indeed, SYTVII promotes exocytosis through Ca2+-dependent multimerization with other synaptotagmins, which is mediated by the C2 domains (57) . We show that overexpression of SYTVII β induces only partial plasma membrane translocation of the protein. Thus, the relative rate at which SYTVII {alpha} and β oligomerize may regulate the number of vesicles predestine for exocytosis. Consistent with this hypothesis, truncated V2 vasopressin receptors generated from alternative splicing were shown to heterodimerize with wild type V2 receptor and inhibit function as well as cell surface trafficking (58) . The strong attenuation of hormone release observed after repression of SYTVII {alpha} argues against a unique role of SYTIX in insulin secretion. However, SYTVII and IX were shown to heterodimerize in PC12 cells on Ca2+-stimulation suggesting that interference with a particular synaptotagmin or splice variant destroys the intricate balance between the isoforms ultimately leading to impaired exocytosis (4 , 41) .

Genome-wide analysis of alternative splicing indicates that ~40–60% of human genes undergo such an event, suggesting that alternative splicing has an extremely important role in expanding protein diversity (59) . Salient examples for which profound biological consequences have been attributed to alternative splicing of transcripts are genes encoding the calcium-activated potassium channel, slo or the neurexin family of neural proteins that act as receptors for neuropeptides and as adhesion molecules that participate in synaptogenesis (60 , 61) . However, for the majority of alternative splicing events associated with individual mRNA, the functional significance remains largely unknown (62) . Our study extends the current understanding of how individual splice variants may function differently. Indeed we show that insulin-producing cells express three SYTVII splice variants localized predominantly on LDCV. RNAi and electrophysiological studies reveal that SYTVII {alpha} is indispensable for insulin secretion, while SYTVII β and {delta} may have different function. Thus, in endocrine islet β-cells, SYTVII and IX may be the major Ca2+ sensors for insulin secretion and crosstalk between these two synaptotagmins could determine overall exocytosis (20) .


   ACKNOWLEDGMENTS
 
This work was supported by the Swiss National Science Foundation (grant 32–66907.01 to C.B.W. and B.R.G. and 3100A0–107682/1 to B.R.G.) and by the European Union (Integrated Project EuroDia; LSHM-CT-2006–518153 in the Framework Programme 6 of the European Community). This study was performed within the framework of the Geneva program in metabolic disorders (GeMet).

Received for publication March 2, 2007. Accepted for publication July 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wollheim, C. B. (2000) Beta-cell mitochondria in the regulation of insulin secretion: a new culprit in type II diabetes. Diabetologia 43,265-277[CrossRef][Medline]
  2. Sudhof, T. C. (2004) The synaptic vesicle cycle. Annu. Rev. Neurosci. 27,509-547[CrossRef][Medline]
  3. Fukuda, M. (2003) Synaptotagmins, Ca2+ and phospholipid-binding proteins that control Ca2+-regulated membrane trafficking. Recent Res. Devel. Chem. Phys. Lipids 1,15-51
  4. Sudhof, T. C. (2002) Synaptotagmins: why so many?. J. Biol. Chem. 277,7629-7632[Free Full Text]
  5. Coussens, L., Parker, P. J., Rhee, L., Yang-Feng, T. L., Chen, E., Waterfield, M. D., Francke, U., Ullrich, A. (1986) Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233,859-866[Abstract/Free Full Text]
  6. Hui, E., Bai, J., Wang, P., Sugimori, M., Llinas, R. R., Chapman, E. R. (2005) Three distinct kinetic groupings of the synaptotagmin family: candidate sensors for rapid and delayed exocytosis. Proc. Natl. Acad. Sci. U. S. A. 102,5210-5214[Abstract/Free Full Text]
  7. Marqueze, B., Berton, F., Seagar, M. (2000) Synaptotagmins in membrane traffic: which vesicles do the tagmins tag?. Biochimie (Paris) 82,409-420
  8. Li, C., Davletov, B. A., Sudhof, T. C. (1995) Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release. J. Biol. Chem. 270,24898-24902[Abstract/Free Full Text]
  9. Bai, J., Wang, C. T., Richards, D. A., Jackson, M. B., Chapman, E. R. (2004) Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions. Neuron 41,929-942[CrossRef][Medline]
  10. Nicholson-Tomishima, K., Ryan, T. A. (2004) Kinetic efficiency of endocytosis at mammalian CNS synapses requires synaptotagmin I. Proc. Natl. Acad. Sci. U. S. A. 101,16648-16652[Abstract/Free Full Text]
  11. Geppert, M., Ullrich, B., Green, D. G., Takei, K., Daniels, L., De Camilli, P., Sudhof, T. C., Hammer, R. E. (1994) Synaptic targeting domains of synapsin I revealed by transgenic expression in photoreceptor cells. EMBO J. 13,3720-3727[Medline]
  12. Han, W., Rhee, J. S., Maximov, A., Lin, W., Hammer, R. E., Rosenmund, C., Sudhof, T. C. (2005) C-terminal ECFP fusion impairs synaptotagmin 1 function: crowding out synaptotagmin 1. J. Biol. Chem. 280,5089-5100[Abstract/Free Full Text]
  13. Li, C., Ullrich, B., Zhang, J. Z., Andreson, R. G. W., Brose, N, Sudhof, T. C. (1995) Ca2+ -dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375,594-599[CrossRef][Medline]
  14. Lang, J., Fukuda, M., Zhang, H., Mikoshiba, K., Wollheim, C. B. (1997) The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic beta-cells: action of synaptotagmin at low micromolar calcium. EMBO J. 16,5837-5846[CrossRef][Medline]
  15. Gao, Z., Reavey-Cantwell, J., Young, R. A., Jegier, P., Wolf, B. A. (2000) Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet beta -cells. J. Biol. Chem. 275,36079-36085[Abstract/Free Full Text]
  16. Gut, A., Kiraly, C. E., Fukuda, M., Mikoshiba, K., Wollheim, C. B., Lang, J. (2001) Expression and localisation of synaptotagmin isoforms in endocrine beta- cells: their function in insulin exocytosis. J. Cell Sci. 114,1709-1716[Abstract]
  17. Monterrat, C., Boal, F., Grise, F., Hemar, A., Lang, J. (2006) Synaptotagmin 8 is expressed both as a calcium-insensitive soluble and membrane protein in neurons, neuroendocrine and endocrine cells. Biochim. Biophys. Acta 1763,73-81[Medline]
  18. Saegusa, C., Fukuda, M., Mikoshiba, K. (2002) Synaptotagmin V is targeted to dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells. J. Biol. Chem. 277,24499-24505[Abstract/Free Full Text]
  19. Iezzi, M., Kouri, G., Fukuda, M., Wollheim, C. B. (2004) Synaptotagmin V and IX isoforms control Ca2+-dependent insulin exocytosis. J. Cell Sci. 117,3119-3127[Abstract/Free Full Text]
  20. Iezzi, M., Eliasson, L., Fukuda, M., Wollheim, C. B. (2005) Adenovirus-mediated silencing of synaptotagmin 9 inhibits Ca2+-dependent insulin secretion in islets. FEBS Lett. 579,5241-5246[CrossRef][Medline]
  21. Fukuda, M., Ogata, Y., Saegusa, C., Kanno, E., Mikoshiba, K. (2002) Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human. Biochem. J. 365,173-180[CrossRef][Medline]
  22. Sugita, S., Han, W., Butz, S., Liu, X., Fernandez-Chacon, R., Lao, Y., Sudhof, T. C. (2001) Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis. Neuron 30,459-473[CrossRef][Medline]
  23. Virmani, T., Han, W., Liu, X., Sudhof, T. C., Kavalali, E. T. (2003) Synaptotagmin 7 splice variants differentially regulate synaptic vesicle recycling. EMBO J. 22,5347-5357[CrossRef][Medline]
  24. Andrews, N. W., Chakrabarti, S. (2005) There’s more to life than neurotransmission: the regulation of exocytosis by synaptotagmin VII. Trends Cell Biol. 15,626-631[CrossRef][Medline]
  25. Czibener, C., Sherer, N. M., Becker, S. M., Pypaert, M., Hui, E., Chapman, E. R., Mothes, W., Andrews, N. W. (2006) Ca2+ and synaptotagmin VII-dependent delivery of lysosomal membrane to nascent phagosomes. J. Cell Biol. 174,997-1007[Abstract/Free Full Text]
  26. Jaiswal, J. K., Andrews, N. W., Simon, S. M. (2002) Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. J. Cell Biol. 159,625-635[Abstract/Free Full Text]
  27. Weller, U., Muller, L., Messner, M., Palmer, M., Valeva, A., Tranum-Jensen, J., Agrawal, P., Biermann, C., Dobereiner, A., Kehoe, M. A., Bhakdi, S. (1996) Expression of active streptolysin O in Escherichia coli as a maltose-binding-protein–streptolysin-O fusion protein. The N-terminal 70 amino acids are not required for hemolytic activity. Eur. J. Biochem. 236,34-39[Medline]
  28. Merglen, A., Theander, S., Rubi, B., Chaffard, G., Wollheim, C. B., Maechler, P. (2004) Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 145,667-678[Abstract/Free Full Text]
  29. Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P. A., Wollheim, C. B. (1992) Establishment of 2-mercaptoethanol-dependent differentiated insulin- secreting cell lines. Endocrinology 130,167-178[Abstract/Free Full Text]
  30. Pralong, W. F., Bartley, C., Wollheim, C. B. (1990) Single islet beta-cell stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and secretion. EMBO J. 9,53-60[Medline]
  31. Van De Winkel, M., Pipeleers, D. (1983) Autofluorescence-activated cell sorting of pancreatic islet cells: purification of insulin-containing B-cells according to glucose-induced changes in cellular redox state. Biochem. Biophys. Res. Commun. 114,835-842[CrossRef][Medline]
  32. Gauthier, B., Robb, M., McPherson, R. (1999) Cholesteryl ester transfer protein gene expression during differentiation of human preadipocytes to adipocytes in primary culture. Atherosclerosis 142,301-307[CrossRef][Medline]
  33. Gauthier, B. R., Brun, T., Sarret, E. J., Ishihara, H., Schaad, O., Descombes, P., Wollheim, C. B. (2004) Oligonucleotide microarray analysis reveals PDX1 as an essential regulator of mitochondrial metabolism in rat islets. J. Biol. Chem. 279,31121-31130[Abstract/Free Full Text]
  34. Fukuda, M., Katayama, E., Mikoshiba, K. (2002) The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization. J. Biol. Chem. 277,29315-29320[Abstract/Free Full Text]
  35. Iezzi, M., Theander, S., Janz, R., Loze, C., Wollheim, C. B. (2005) SV2A and SV2C are not vesicular Ca2+ transporters but control glucose-evoked granule recruitment. J. Cell Sci. 118,5647-5660[Abstract/Free Full Text]
  36. Regazzi, R., Kikuchi, A., Takai, Y., Wollheim, C. B. (1992) The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. J. Biol. Chem. 267,17512-17519[Abstract/Free Full Text]
  37. Reetz, A., Solimena, M., Matteoli, M., Folli, F., Takei, K., De Camilli, P. (1991) GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J. 10,1275-1284[Medline]
  38. Brun, T., Duhamel, D. L., Hu He, K. H., Wollheim, C. B., Gauthier, B. R. (2007) The transcription factor Pax4 acts as a survival gene in the insulinoma INS1E cells. Oncogene 26,4261-4271[CrossRef][Medline]
  39. Regazzi, R., Kikuchi, A., Takai, Y., Wollheim, C. B. (1992) The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. J. Biol. Chem. 267,17512-17519[Abstract/Free Full Text]
  40. Han, W., Rhee, J. S., Maximov, A., Lao, Y., Mashimo, T., Rosenmund, C., Sudhof, T. C. (2004) N-glycosylation is essential for vesicular targeting of synaptotagmin 1. Neuron 41,85-99[CrossRef][Medline]
  41. Fukuda, M., Kanno, E., Satoh, M., Saegusa, C., Yamamoto, A. (2004) Synaptotagmin VII is targeted to dense-core vesicles and regulates their Ca2+ -dependent exocytosis in PC12 cells. J. Biol. Chem. 279,52677-52684[Abstract/Free Full Text]
  42. Martinez, I., Chakrabarti, S., Hellevik, T., Morehead, J., Fowler, K., Andrews, N. W. (2000) Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts. J. Cell Biol. 148,1141-1149[Abstract/Free Full Text]
  43. MacDonald, P. E., Obermuller, S., Vikman, J., Galvanovskis, J., Rorsman, P., Eliasson, L. (2005) Regulated exocytosis and kiss-and-run of synaptic-like microvesicles in INS-1 and primary rat beta-cells. Diabetes 54,736-743[Abstract/Free Full Text]
  44. Barg, S., Huang, P., Eliasson, L., Nelson, D. J., Obermuller, S., Rorsman, P., Thevenod, F., Renstrom, E. (2001) Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification. J. Cell Sci. 114,2145-2154[Abstract/Free Full Text]
  45. Fukuda, M., Kanno, E., Ogata, Y., Mikoshiba, K. (2001) Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains. J. Biol. Chem. 276,40319-40325[Abstract/Free Full Text]
  46. Iezzi, M., Regazzi, R., Wollheim, C. B. (2000) The Rab3-interacting molecule RIM is expressed in pancreatic beta-cells and is implicated in insulin exocytosis. FEBS Lett. 474,66-70[CrossRef][Medline]
  47. Hoy, M., Maechler, P., Efanov, A. M., Wollheim, C. B., Berggren, P. O., Gromada, J. (2002) Increase in cellular glutamate levels stimulates exocytosis in pancreatic beta-cells. FEBS Lett. 531,199-203[CrossRef][Medline]
  48. Wang, H., Iezzi, M., Theander, S., Antinozzi, P. A., Gauthier, B. R., Halban, P. A., Wollheim, C. B. (2005) Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells. Diabetologia. 48,720-731[CrossRef][Medline]
  49. Vallar, L., Biden, T. J., Wollheim, C. B. (1987) Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells. J. Biol. Chem. 262,5049-5056[Abstract/Free Full Text]
  50. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., Williams, B. R. (2003) Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5,834-839[CrossRef][Medline]
  51. Tomari, Y., Zamore, P. D. (2005) Perspective: machines for RNAi. Genes Dev. 19,517-529[Abstract/Free Full Text]
  52. Tucker, W. C., Weber, T., Chapman, E. R. (2004) Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science 304,435-438[Abstract/Free Full Text]
  53. Meldolesi, J., Chieregatti, E. (2004) Fusion has found its calcium sensor. Nat. Cell Biol. 6,476-478[CrossRef][Medline]
  54. Monterrat, C., Grise, F., Benassy, M. N., Hemar, A., Lang, J. (2007) The calcium-sensing protein synaptotagmin 7 is expressed on different endosomal compartments in endocrine, neuroendocrine cells or neurons but not on large dense core vesicles. Histochem. Cell Biol. 127,625-632[CrossRef][Medline]
  55. Tsuboi, T., Fukuda, M. (2007) Synaptotagmin VII modulates the kinetics of dense-core vesicle exocytosis in PC12 cells. Genes Cells 12,511-519[Abstract/Free Full Text]
  56. Fukuda, M., Ibata, K., Mikoshiba, K. (2001) A unique spacer domain of synaptotagmin IV is essential for Golgi localization. J. Neurochem. 77,730-740[CrossRef][Medline]
  57. Fukuda, M., Mikoshiba, K. (2001) Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains. J. Biol. Chem. 276,27670-27676[Abstract/Free Full Text]
  58. Zhu, X., Wess, J. (1998) Truncated V2 vasopressin receptors as negative regulators of wild-type V2 receptor function. Biochemistry 37,15773-15784[CrossRef][Medline]
  59. Modrek, B., Resch, A., Grasso, C., Lee, C. (2001) Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res. 29,2850-2859[Abstract/Free Full Text]
  60. Black, D. L. (1998) Splicing in the inner ear: a familiar tune, but what are the instruments?. Neuron 20,165-168[CrossRef][Medline]
  61. Missler, M., Fernandez-Chacon, R., Sudhof, T. C. (1998) The making of neurexins. J. Neurochem. 71,1339-1347[Medline]
  62. Graveley, B. R. (2001) Alternative splicing: increasing diversity in the proteomic world. Trends. Genet. 17,100-107[CrossRef][Medline]



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