Figure 8: FRET/2pFLIM with SNARE probes in the β cells in the pancreatic islets of Langerhans. | Nature Communications

Figure 8: FRET/2pFLIM with SNARE probes in the β cells in the pancreatic islets of Langerhans.

From: Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells

Figure 8

(a) A fluorescence image of islets transfected with mtq-SNAP25 by lentivirus (scale bar, 50 μm). The arrow indicates the typical cells investigated. (b) Schematic drawings of three FRET probes. (c,d,g,h,j,k) Fluorescence and A1 fraction images of mtq-SNAP25/Venus-syntaxin (Ven-Syx; c,d), mtq-Syx/Ven-VAMP2 (g,h) or mtq-SNAP25 and Ven-VAMP2 (j,k) (scale bars, 5 μm). ‘G’ denotes the Golgi apparatus, and ‘M’ in the right panels of images (d,h,k) denotes the masked cytosolic regions. (e,i,l) Lifetime decay curves of the plasma membranes of β cells transfected with mtq-SNAP25/Ven-Syx (b, e), mtq-Syx/Ven-VAMP2 (b, i), and mtq-SNAP25 and Ven-VAMP2 (b, l), which were obtained from 12, 12 and 11 cells, respectively. (f) The average A1 fractions of the lifetime decay of β-cell plasma membrane transfected with four different probes indicated. The A1 fractions of mtq-SNAP25 are −0.8±1.1% (10 cells; P=0.14), those mtq-SNAP25/Ven-Syx are 5.3±1% (22 cells; **P<0.01), those of mtq-Syx/Ven-VAMP2 are 0.8±2.3% (15 cells; P=0.82) and those of mtq-SNAP25/Ven-VAMP2 are 0.04±1.4% (21 cells; P=0.89) at the endogenous expression levels of Ven-Syx and Ven-VAMP2 (Supplementary Figs 3f,h and 8d,f), respectively. The P values were obtained using the Wilcoxon signed-rank test. The four groups were significant in analysis using the Kruskal–Wallis test with P<0.007. Steel’s test versus mtq-SNAP25 yielded P=0.014 for mtq-SNAP25/Ven-Syx, P=0.92 for mtq-Syx/Ven-VAMP2 and P=0.74 for mtq-SNAP25/Ven-VAMP2.

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