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First published online 20 June 2006
doi: 10.1242/jcs.03037

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Tomosyn-1 is involved in a post-docking event required for pancreatic ß-cell exocytosis

¹ Department of Cell Biology and Morphology, Rue du Bugnon 9, University of Lausanne, 1005 Lausanne, Switzerland
² Department of Clinical Sciences, Malmö, Lund University, Sweden
³ Laboratoire de Neurobiologie Cellulaire, Ecole Polytechnique Fédérale, Lausanne, Switzerland

View larger version (65K): [in a new window]   Fig. 1. Tomosyn-1 is expressed in rat pancreatic islets. Rat pancreatic sections were incubated with a polyclonal antibody directed against tomosyn-1 (Hatsuzawa et al., 2003) and a guinea pig antibody against insulin. (A) Expression of tomosyn-1 was assessed by confocal microscopy after labeling with an anti-rabbit antibody coupled to FITC. (B) Insulin-containing cells were visualized with an anti-guinea pig antibody coupled to Cy3. (C) Overlay.

View larger version (33K): [in a new window]   Fig. 2. Determination of the tomosyn-1 isoforms expressed in insulin-secreting cells. The three tomosyn-1 isoforms were amplified by RT-PCR from RNA extracts of rat brain, INS-1E and MIN6 cells and freshly isolated rat pancreatic islets using specific primers. b, b-tomosyn-1; m, m-tomosyn-1; s, s-tomosyn-1

View larger version (47K): [in a new window]   Fig. 3. Subcellular localization of tomosyn-1 in insulin-secreting cells. INS-1E cells were grown on glass coverslips coated with laminin and poly-L-lysine. Then, the cells were fixed with paraformaldehyde and incubated with a rabbit antibody against tomosyn and a guinea pig antibody against insulin. (A) The distribution of tomosyn was analysed by confocal microscopy after labeling with an anti-rabbit antibody coupled to FITC. (B) Secretory granules were visualized with an anti-guinea pig antibody coupled to Cy3. (C) Overlay of images A and B.

View larger version (46K): [in a new window]   Fig. 4. Analysis of the subcellular distribution of tomosyn-1 on a sucrose density gradient. A post-nuclear supernatant of INS-1E cells was loaded on a sucrose density gradient (0.45-2.0 M) and centrifuged for 18 hours at 110,000 g. Aliquots of the fractions containing 0.55-1.74 M sucrose were analyzed by western blotting using antibodies against tomosyn-1, granuphilin (secretory granule marker) and synaptophysin (synaptic-like vesicle marker).

View larger version (53K): [in a new window]   Fig. 5. Effect of short interfering RNAs on tomosyn-1 expression. (A) COS cells were transiently co-transfected with GFP-tagged tomosyn-1 and with empty pSUPER vector (control) or with vectors directing the sequence of two different short interfering RNAs (siRNA-i and siRNA-a). After 3 days the expression level of tomosyn-1 and GFP-tomosyn-1 was analysed by western blotting with a polyclonal antibody against tomosyn-1. The level of tubulin present in the same samples was tested in parallel as a control for protein loading. (B) INS-1E cells were transiently transfected with empty pSUPER vector (control), siRNA-i or siRNA-a. The amount of endogenous tomosyn-1 remaining in the cells after 3 days was assessed by western blotting with antibody against tomosyn-1. Western blotting with antibody against tubulin was performed in parallel as a control for protein loading.

View larger version (21K): [in a new window]   Fig. 6. Effect of up and down regulation of tomosyn-1 expression on exocytosis. (A) INS-1E cells were transiently co-transfected with a plasmid encoding hGH and with the empty pSUPER vector (control), the pEGFP-rat-tomosyn-1, siRNA-i or siRNA-a. After 3 days, the cells were incubated under basal condition or in the presence of stimulatory concentrations of glucose, K+, forskolin and IBMX. After 45 minutes, the incubation medium was collected. The fraction of hGH released by the cells under basal and stimulatory conditions were measured by ELISA. None of the constructs used in this study affected basal secretion (data not shown). Stimulated exocytosis was defined as the ratio of basal hGH release to the amount of hGh secreted in the presence of glucose, K+, forskolin and IBMX. The results are the mean of at least three independent experiments measured in triplicates. (B) INS-1E cells were transiently co-transfected with the pSUPER-GH plasmid, encoding both hGH and either the empty pSUPER vector (control) or the siRNA-a, and with either pEGFP or pcDNA3-mouse-tomosyn-1. As described above, cells were incubated under basal condition or in the presence of stimulatory concentrations of glucose, K+, forskolin and IBMX for 45 minutes. The incubation medium was then collected and the fraction of hGH released by the cells was measured by ELISA. The results are the mean of at least three independent experiments measured in triplicates.

View larger version (14K): [in a new window]   Fig. 7. Capacitance increase after reduction of tomosyn-1 expression. INS-1E cells were transiently transfected with a pSUPER-derived plasmid encoding both GFP and the control siRNA-i (gray) or siRNA-a (black). The transfected cells were identified by EGFP fluorescence and exocytosis was measured by capacitance recordings using the whole-cell configuration of the patch-clamp technique. Exocytosis was triggered by 10x300 ms voltage-clamp depolarizations from –70 mV to 0 mV. **P<0.01, significantly different (unpaired t-test).

View larger version (20K): [in a new window]   Fig. 8. Tomosyn-1-silencing affects exocytosis at a step beyond Ca2+ influx. (A) Charge/voltage (Q/V) relation recorded in siRNA-i-treated cells (control, gray squares) and siRNA-a treated cells (tomosyn-1-silenced, black circles). (B) Ca2+ evoked exocytosis. Left: Mean capacitance increase (C/t) in control (siRNA-i treated cells, gray bar) and tomosyn-1-silenced cells (siRNA-a, black bar). Exocytosis was elicited by intracellular dialysis of a patch pipette solution containing Ca2+-EGTA buffer (free [Ca2+]i 1.5 µM). Right: Examples of capacitance traces from one control and one tomosyn-1-silenced cell (siRNA-a) as indicated. The standard whole-cell configuration was established at t=0, indicated by the arrow. *P<0.05, significantly different (unpaired t-test).

View larger version (27K): [in a new window]   Fig. 9. Effect of tomosyn-1 silencing on the pool of secretory granules docked at the plasma membrane. INS-1E cells were grown on glass coverslips coated with laminin and poly-L-Lysine, and were transiently co-transfected with a plasmid encoding IAPP-Emerald and the pSUPER vector siRNA-i (control) or siRNA-a. Three days later the transfected cells were visualized by TIRF microscopy (A). Single IAPP-Emerald-EGFP-positive granules from nine control cells and nine cells transfected with siRNA-a were manually selected and counted. The number of IAPP-Emerald-EGFP-positive granules was then divided by the single cell area. (B) Results of the experiments in from A, expressed as means ± s.e.m. (C) Six control cells and six cells transfected with siRNA-a were incubated in the presence of stimulatory concentrations of glucose, K+, forskolin and IBMX. Single IAPP-Emerald-EGFP-positive granules were manually selected and counted after 0, 8 and 16 minutes of incubation in the presence of the stimuli. Data are expressed as means ± s.e.m. *P<0.01, significantly different (unpaired t-test).

View larger version (23K): [in a new window]   Fig. 10. Measurement of the interaction forces between the R-SNARE-like motif of tomosyn-1 and the Q-SNAREs syntaxin-1 and SNAP-25. A GST-fusion protein containing the SNARE-like motif of tomosyn-1 was fixed to the cantilever of the AFM. Syntaxin-1 (left panel) or the syntaxin-1-SNAP25 complex (right panel) were attached on the mica. The histograms show the frequencies of the forces of the specific events (gray bars). White bars indicate the frequencies and forces of nonspecific events measured using GST-covered mica. The mean unbinding forces were 152±9 and 237±13 pN for the interactions with syntaxin-1 and with the syntaxin-1-SNAP25 complex, respectively.