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First published online 9 June 2004
doi: 10.1242/jcs.01179

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Synaptotagmin V and IX isoforms control Ca²⁺-dependent insulin exocytosis

¹ Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, 1211 Geneva 4, Switzerland
² Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

View larger version (37K): [in a new window]   Fig. 1. Expression of Syt V and Syt IX in insulin-secreting cells. (A) PCR amplification of Syt IX cDNA from rat brain, INS-1E cells and rat islets. PCR reactions in which reverse transcriptase was omitted are shown by (–). The DNA size markers (bp) are also indicated. (B) Homogenates (100 µg) of rat brain, INS-1E cells and rat islets were separated by SDS-PAGE and subjected to immunoblotting with anti-Syt V or anti-Syt IX antibodies.

View larger version (30K): [in a new window]   Fig. 2. Immunolabelling of Syt V in the INS-1E clone and pancreatic islet cells. INS-1E cells (A) and pancreatic endocrine cells (B) were analysed by confocal microscopy after double immunofluorescence with an antibody against insulin or glucagon (revealed using fluorescein-conjugated antibody) and with the anti-Syt V (detected using rhodamine-conjugated antibody). The overlapping images were obtained after superposition of the green and red channels.

View larger version (67K): [in a new window]   Fig. 3. Intracellular localization of Syt IX in insulin-secreting cells. INS-1E cells and pancreatic ß-cells were double labelled with an antibody against insulin (revealed by FITC-conjugated antibody) together with the anti-Syt IX (detected by rhodamine-conjugated antibody). Images were obtained by confocal microscopy. The upper panels show the localization of insulin, the middle panels the position of Syt IX and the lower panels the superposition of the green and red channels.

View larger version (21K): [in a new window]   Fig. 4. Subcellular fractionation of Syt V and Syt IX in INS-1E cells. Subcellular fractions of INS-1E cells were obtained using the sucrose density gradient described in Materials and Methods. Aliquots of each fraction of the gradient were analysed by western blotting using antibodies directed against Na+/K+-ATPase, synaptophysin (SVP38), carboxypeptidase H (CPH), Syt V and Syt IX. Sucrose concentration was calculated from the refractive index of the fractions. The top panel shows the amount of insulin present in the fractions corresponding to the distribution of insulin-containing secretory granules.

View larger version (46K): [in a new window]   Fig. 5. Concentration-dependent localization of Syt V and Syt IX after their overexpression in INS-1E cells. INS-1E cells were transiently transfected with 1 µg and 5 µg of cDNAs encoding myc-tagged Syt V (A) or myc-tagged Syt IX (B). Two days later, the cells were double stained with antibodies directed against insulin (revealed by FITC-conjugated antibody) and against the myc-epitope (detected by rhodamine-conjugated antibody). Images were obtained by confocal microscopy. Yellow images correspond to the overlay of the anti-insulin and anti-myc signals.

View larger version (28K): [in a new window]   Fig. 6. Effect of Syt V and Syt IX overexpression on insulin exocytosis. INS-1E cells were transiently cotransfected with a plasmid encoding human growth hormone (hGH) and 1 µg of the control pcDNA3 (control), the Syt V or the Syt IX vectors. Two days later, the cells were incubated in the presence of 2.5 mM glucose (open bars) or with 15 mM glucose (filled bars) for 30 minutes. The cells were then collected and the expression of the exogenous proteins was analysed by western blotting using anti-Syt V or anti-Syt IX antibodies (top panels). The same membranes were also blotted with an anti-myc antibody. The positions of endogenous Syt V and Syt IX and their transfected myc-tagged counterparts (loaded on duplicates) are indicated by the arrows. After incubation the amount of hGH secreted by the cells was measured by ELISA. The lower panel shows the mean+s.e.m. of three independent experiments performed in triplicate.

View larger version (21K): [in a new window]   Fig. 7. Selective decrease of Syt V and Syt IX expression by RNA interference. (A) INS-1E cells were transiently transfected with myc-tagged Syt V (a) or myc-tagged Syt IX (b). Silencing of the genes was determined by cotransfecting the cells either with an empty pSilencer vector (control) or with a pSilencer vector containing a sequence that directs the synthesis of Syt V-specific siRNA or Syt IX-specific siRNA (RNAi). Nontransfected cells are shown by (–). After three days the cells were homogenized and equal amounts of protein were analysed by western blotting using antibodies directed against Syt V or Syt IX. The same membrane was also blotted with an anti-myc antibody. The arrow indicates Syt IX unrelated proteins. (B) INS-1E cells were transiently cotransfected with GFP and with an empty vector (control) or with a vector containing either the Syt V or the Syt IX silencer sequences (RNAi). Three days later, GFP-expressing cells were enriched by FACS separation. The cells were then homogenized and the same amount of protein was separated by SDS-PAGE and immunoblotted with anti-Syt V (a) or anti-Syt IX (c) antibodies. The membranes (a) and (c) were then blotted respectively with anti-Syt IX (b) and anti-Syt V (d) antibodies.

View larger version (79K): [in a new window]   Fig. 8. Immunolabelling of Syt V and Syt IX suppression by RNAi in INS-1E cells. INS-1E cells were transiently transfected with GFP together with the Syt V silencer (A) or with the Syt IX silencer (B). After three days the cells were fixed and analysed by immunofluorescence with the antibodies against Syt V or Syt IX. Transfected cells were identified using GFP fluorescence. The arrows indicate the position of transiently transfected cells expressing the silencer.

View larger version (17K): [in a new window]   Fig. 9. Effect of Syt V and Syt IX silencing on hormone secretion in INS-1E cells. INS-1E cells were transiently cotransfected with a plasmid encoding hGH and with the empty vector (control) or with either the Syt V-specific siRNA (RNAi Syt V) or the Syt IX-specific siRNA (RNAi Syt IX). Three days later, the cells were incubated for 30 minutes with 2.5 mM glucose (open bars) or stimulated with 15 mM glucose (filled bars). The experiments depicted in the lower panel were performed in the presence of 100 nM PMA. The amount of hGH released during the incubation period was determined by ELISA. The results correspond to the mean+s.e.m. of three independent experiments performed in triplicate.

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