PMC

Munc18b gain-of-function mutant increases SM-activated SNARE complex formation, and INS-1 cells were transduced with AdMunc18 mutants, kept in nonstimulated (lanes 1–4) or simulated (lanes 5–8) condition as in and subjected to coimmunoprecipitation (left panel) with antibodies against Syn-1A (A), Syn-2 (B), or Syn-3 (C). Exogenous Munc18b (tagged with Myc) expression was detected by Myc antibody. Corresponding right panel: Input controls (25 μg protein, total INS-1 lysates) showing similar levels of indicated SNARE proteins. Results shown are representative of three independent experiments, with densitometry analyses shown in Supplementary Fig. 4. IP, immunoprecipitation.

Munc18b depletion in INS-1 cells disrupts SM-activated SNARE complex formation. INS-1 cells were transduced with Munc18b siRNA or control scrambled RNA and then kept at basal condition (0.8 mmol/L glucose) or simulated with 16.7 mmol/L glucose plus 10 nmol/L GLP-1 with IBMX (150 μmol/L) and then subjected to coimmunoprecipitation (left panel) with antibodies against Syn-1A (A), Syn-2 (B), or Syn-3 (C). Corresponding right panel showing “input” controls (25 μg protein, total INS-1 lysates) confirmed the reduction in Munc18b levels and similar levels of indicated SNARE proteins. Results shown are representative of three independent experiments, with densitometry analyses in Supplementary Fig. 3. IP, immunoprecipitation.

Munc18b depletion reduces primary and sequential exocytosis in rat β-cells. TEP images of single SG exocytosis and SG-SG fusions were analyzed from islets treated with lenti-shRNA/Munc18b-eCFP (67 cells, 8 islets) vs. lenti-eCFP (control, 48 cells, 8 islets) during 10-min period of stimulation with 20 mmol/L glucose plus 10 nmol/L GLP-1 and 150 μmol/L IBMX, and the following were calculated. A: Total number of exocytotic events. B: Number of exocytotic events at each indicated time interval in the 10-min recording. Right panel: Summation of the first 5 min and second 5 min of recording. C: Secondary exocytotic events considered to be sequential exocytosis expressed as a percentage of total number of exocytotic events assessed in A. Data shown as means ± SEM. *P < 0.05, ***P < 0.001.

Munc18b is a major mediator of GSIS in pancreatic islet β-cells. A: Pancreatic β-cells (rat islets, INS-1) express SM proteins and Syn. Rat pancreatic acini and brain were used as positive and negative controls. B: Immunofluorescence images showing cognate Munc18b and Syn-2 and Syn-3 are abundant in insulin SGs in rat β-cells. Cognate Munc18a and Syn-1A are abundant in the PM. These images are representative of four independent experiments. Scale bars, 10 μm. C: Munc18b depletion in rat islets by lenti-Munc18b shRNA/eCFP vs. eCFP control. Top panel: Representative blots. Bottom panel: Analysis of three experiments, shown as means ± SEM. *P < 0.05, **P < 0.01. D: Islet perifusion assays of 48 h lenti-shRNA/Munc18b-eCFP (vs. lenti-eCFP [control]) depletion of endogenous Munc18b in rat islets showing reduction of GSIS and GLP-1 (10 nmol/L) plus IBMX (150 µmol/L)-potentiated GSIS, shown as means ± SEM of three independent experiments, and AUC analysis; *P < 0.05, ***P < 0.001. G, glucose.

Munc18b gain-of-function mutant potentiates GSIS. A: AdMunc18b mutant (-WT, -KR, and -E59K) transduction of rat islets did not influence the expression of SNARE or other SM proteins. Shown are representative of three independent experiments; analysis of Munc18b proteins expression shown in bottom panel. B: Confocal imaging showing AdMunc18b mutants did not influence Syn-2 or Syn-3 targeting to insulin SGs in rat β-cells. Shown are representative of four independent experiments. Scale bars, 10 μm. C and D: Islet perifusion assays showing AdMunc18b mutant–transduced rat islets influencing GSIS (C) and 10 nmol/L GLP-1 plus 150 µmol/L IBMX–potentiated GSIS (D). Data shown are means ± SEM from three to four sets of experiments, with each experiment performed simultaneously in all of the four conditions. Bottom panels show quantification of AUC analysis of first-phase (encompassing 11–22 min) and second-phase (encompassing 22–40 min) GSIS. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. E: Effects of AdMunc18b mutants on GSIS and 10 nmol/L GLP-1 plus 150 µmol/L IBMX-potentiated GSIS in INS-1 cells. Results shown are means ± SEM from three independent experiments performed in duplicates or triplicates (n = 6–8). *P < 0.05 by ANOVA and Scheffé tests.

Munc18b mediates primary exocytosis and sequential SG-SG fusion in rat β-cells. A: Example of TEP imaging of single insulin SG exocytosis (single granule) in an AdGFP-transduced rat islet stimulated with 20 mmol/L glucose plus 10 nmol/L GLP-1 and 150 μm IBMX in a solution containing 0.3 mmol/L Alexa Fluor 594 hydrazide. An ω construction appeared when the SG(s) coalesced with the PM. B: Example of TEP imaging of sequential exocytosis (two granules) involving two SGs in an AdMunc18b-KR–transduced rat islet stimulated as in A. The Alexa fluorescence distinguishes several adjacent β-cells in the islet, along with small blood vessels (white arrowheads) and major blood vessels (white arrows) (i). White circle shows the position of an ω construction in a former panel (ii). An example of a single SG exocytosis shown in A with imaging interval of 1.2 s; in B, an example of 2-SG sequential events with imaging interval of 0.6 s. The numbers below each panel represent time after onset of exocytosis. Dashed circles show position of an ω construction in a former panel. Arrows point to the direction of SG fusion toward the cell interior. iii: Time courses of fluorescence at the area containing the exocytotic events. Vertical bars indicate the times when images in ii were acquired. The fluorescence value before exocytosis was set to zero. A and B: Scale bar represents 10 μm. C: Total number of exocytotic events calculated as number of exocytotic events/cell/min in β-cells from islets treated with AdGFP (8 islets, 68 cells), AdMunc18b-WT (7 islets, 44 cells), AdMunc18b-KR (6 islets, 58 cells), and AdMunc18b-E59K (5 islets, 75 cells) during a 10-min period of stimulation as in A. D: Fractions of total exocytotic events that are sequential exocytosis. The number of secondary exocytotic events considered to be sequential exocytosis is expressed as a percentage of total number of exocytotic events assessed in C. Data in C and D are shown as means ± SEM and analyzed by Steel-Dwass test for multiple comparisons using AdGFP as control. *P < 0.05, ***P < 0.001. A.U., arbitrary units.

Munc18b mediates long-chain sequential SG fusion. A: The ordinate represents the ratios for increasing chain length of sequential SG fusion events relative to total numbers of exocytotic events from >20 β-cells (>2 rat islets) transfected with AdGFP, AdMunc18b-WT, AdMunc18b-KR, and AdMunc18b-E59K. Here, we also assessed the distinct increase in fluorescence that predicts the sizes of SGs, which was ∼0.3 μm, shown in Supplementary Fig. 5. B: TEP imaging of long-chain sequential exocytosis of insulin SGs in a rat β-cell treated with AdMunc18b-KR. i: Sequential images for Supplementary Movie 1 of an islet stimulated with 20 mmol/L glucose plus 10 nmol/L GLP-1 and 150 μmol/L IBMX. Dashed circles show positions of an ω construction in a former panel. Arrows indicate the direction of oncoming SG undergoing sequential fusion. ii: Time course of fluorescent intensity within the region that contained all exocytotic events shown in i. *Independent single exocytotic event, occasionally seen close to compound SGs. Scale bars, 1 μm. C: EM demonstrating long-chain sequential exocytosis of insulin SGs in an islet treated with AdMunc18b-KR. An example of AdMunc18b-KR–transduced islet stimulated with 20 mmol/L glucose plus 10 nmol/L GLP-1 and 150 μmol/L IBMX that illustrates long-chain sequential fusion of SG-SG events up to six SGs in length. Left panel: 70-nm-thick sections cut serially, showing only four of eight consecutive slices containing the SG-SG fusions. G plus number (in red) indicates the numbered SGs involved in long-chain exocytosis, M (in red) indicates PM, and numbered asterisks (in green) indicate SG-SG fusion sites. Scale bar, 500 nm. Cartoon panels: SG-SG fusion events constructed from the EM images in left panel and converted into cartoons (first column) followed by reconstruction into a three-dimensional image (third column). The second column shows blowups of this three-dimensional image that can be rotated to selectively visualize fusion pores between indicated SGs, which correspond to raw EM images in the left panel. Blue arrows indicate locations of fusion pore openings. A second example of a 7-SG fusion is shown in Supplementary Fig. 7. D: Insulin SGs within 120 nm from PM calculated as SG/µm² area, expressed as means ± SEM of three to four independent experiments. AdGFP: 17 sections. AdMunc18b-KR: 16 sections. AdMunc18b-WT: 17 sections. AdMunc18b-E59K: 21 sections. *P < 0.05 and **P < 0.01. A.U., arbitrary units.