Fig 1: Stx2 flips from the inner to the outer surface of the cell membrane in human β-cells.a Schematic representation of the genetically encoded flip reporter for monitoring Stx2 flipping. b INS832/13 cells were infected with FAP-Stx2, stimulated with high glucose and visualized by TIRF microscopy. c Time-lapse images show the global Stx2 flip-out of the cell in b. Scale bar, 5 µm. Montages are shown at ~10-s intervals. d Representative time course of single Stx2 flipping events on INS832/13 cell surface (n = 3 independent experiments). e Assessment of Stx2 on the extracellular surface of single human β-cells analyzed by flow cytometry. f Quantification of Stx2 flip-out with the results from e (n = 4 independent experiments, box plots indicate median, 25th, 75th percentile and whiskers min/max limits). g Light micrograph of fresh human pancreatic tissue slice. h Simultaneous imaging of Stx2 flip-out (FAP) and exocytosis (aqueous tracer SRB) in human pancreatic tissue slices. Human pancreatic tissue slices infected with FAP-Stx2 for 48 h, then labeled with primary and secondary antibodies (see methods). ROI (dashed box indicated in g) was selected to speed up image acquisition, preferably at β cell-rich region (top left). A superimposed image of SRB fluorescence shows the distribution of exocytotic events in an intact islet stimulated with 16.7 mM glucose. The dots indicate sites at which exocytotic events occurred (top right), which coincided with the FAP-Stx2 hotspots. Pancreatic β cells (identified by HPi2+/HPa3−) were recognized by co-labeling with antibodies against HIC1-2B4 and HIC3-2D12 (bottom left) (n = 3 independent experiments). i Time-lapse images of the region indicated by the dashed box in (h, top right) shown that glucose-induced exocytosis visualized by SRB fluorescent hotspots occurred where the FAP signals accumulated. Montages are shown at 3.2-s intervals. The numbers indicate the multiple exocytotic events that matched the FAP hotspots. j Time course of the concurrent increase in the fluorescence of SRB and FAP-Stx2 of one of these matched hotspots, indicated by the blue arrowhead in (h, top right).
Fig 2: Modulating Stx2 flipping efficiency regulates insulin secretion.a Quantification of the flipping capability of the Stx2 mutants in INS832/13 cells by flow cytometry. Note that mSNARE increased Stx2 flipping whereas other mutants reduced Stx2 flipping, with the least flipping for the polyK mutant. b Insulin content (normalized to normal Stx2) remained unchanged in human islets after two-round coinfection with the indicated Stx2 mutants and Ad-shRNA-Stx2. c Infection with mutants targeted to affect Stx2 flipping affected glucose-stimulated insulin secretion. Human islets were treated for 1 h with basal (2.8 mM) glucose versus high (16.7 mM) glucose stimulation. Values shown were normalized to the Stx2 basal secretion. d Fold-change representation of stimulated over basal secretion in c, shown as highest (mSNARE with the highest flipping) to lowest secretion capability (polyK with the lowest flipping). e Enhanced Stx2 flipping mSNARE increased the formation of SNARE complexes for predocked (Stx1a immunoprecipitated (IP)) and newcomer (Stx3 IP) granules, whereas reduced Stx2 flipping polyK reduced these SNARE complexes. This is representative of four experiments, with the analysis shown in Supplementary Fig. 7a, b. f Flipping per se of the Stx2 mutants affects GSIS is shown by BoNT/C1-LC cleavage of extracellular Stx2 at near the C-terminal transmembrane domain. To negate or control for potential different expression levels, Insulin secretion was normalized to the total FAP levels after Triton X-100 permeabilization and then subject to 100 nM β GREEN-np. g Fold-change representation of 16.7 mM GSIS of BoNT/C1-LC-treated islets over the corresponding control untreated islets of each Stx2 mutants that were performed in f, which showed BoNT/C1-LC-induced enhancement of GSIS of the Stx2 mutants that were flipped efficiently vs. no enhancement in non-flipping polyK. Data shown as mean ± s.d. in a to g from four independent experiments. h T2D GK rat islets, known to have reduced levels of SNARE proteins11, were infected with Ad-Stx2-shRNA (knockdown efficiency shown in Supplementary Fig. 7e), which enhanced biphasic GSIS. The data shown are mean ± s.e.m. from four independent experiments, with area-under-the-curve (AUC) analysis of first and second-phase secretion shown in Supplementary Fig. 7f. i Summary model of the i-SNARE role of Stx2 in insulin SG exocytosis. Source data are provided as a Source Data file.
Fig 3: The diagrammatic presentation of the underlying mechanism of STX2 promoting PE development. STX2 activated the PI3K-AKT pathway by interacting with PI3K p85 to prompt the trophoblast proliferation, migration and invasion. Therefore, down-regulation of STX2 in placenta contributed to the development of PE.
Fig 4: STX2 promotes trophoblasts proliferation, migration and invasion through activating PI3K-AKT pathway. (A,B) Western blot analysis of p-AKT (ser473), AKT in indicated stably transfected HTR-8/SVneo and primary human trophoblast cells treated with LY294002 (PI3K inhibitor, 10 μM) or DMSO (negative control) for 48 h. (C) EdU assay and (D) colony forming assay were used to evaluated the proliferation of control and experimental cells in which STX2 was stably transfected with or without LY294002 treatment. (E) Transwell assays were used to evaluated the migration and invasion of control and experimental cells in which STX2 was stably transfected with or without LY294002 treatment. All the experiments were repeated three times independently. Data are represented as the mean ± SEM. Student’s t-test: ***P < 0.01.
Fig 5: Stx2 deletion enhances exocytosis by facilitating trans-SNARE complex assembly and cis-SNARE complex disassembly.a Stx2 deletion promotes the formation of profusion trans-SNARE complexes. SNARE complexes were co-immunoprecipitated (co-IP) from human islets lysates by antibody against Stx1a and Stx3. Native primary antibody-specific secondary antibodies were used to eliminate interference by the heavy- and light-chain IgG fragments of initial IP assay. Representative blots are shown in a and quantification shown in Supplementary Fig. 3a (n = 4 independent experiments). b Stx2 inhibits the transition of early assembly intermediates to fusogenic SNARE complexes by substituting for the fusogenic syntaxins (Stx1a, Stx3) as detected by native-PAGE. Samples were loaded with 5× native loading buffer into 12% non-denaturing polyacrylamide gel without SDS. This is because Stx2 directly bound not only cognate SNAREs but also the priming factors, Munc18a and Munc13 (in Supplementary Fig. 3b) (n = 3 independent experiments). c, d Stx2 deletion enhances disassembly of cis-SNARE complexes. Native SNARE complexes isolated from INS832/13 by antibody against VAMP2 or VAMP8 were disassembled by recombinant NSF and α-SNAP in the absence or presence of Stx2. 10 mM EDTA was used to inactivate ATP hydrolysis by chelating Mg2+. SDS-resistant complexes disassemble completely when the sample was heated to 100 °C before electrophoresis. SNARE complexes were visualized by SDS/PAGE and immunoblotting using antibody against SNAP25. Representative blots are shown in c and quantification in d. Values are mean ± s.d. from 4 independent experiments (two-tailed unpaired Student’s t-test). Source data are provided as a Source Data file.
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