Fig. 3: Human GLP-1R localisation to ERMCSs. | Nature Communications

Fig. 3: Human GLP-1R localisation to ERMCSs.

From: GLP-1R associates with VAPB and SPHKAP at ERMCSs to regulate β-cell mitochondrial remodelling and function

Fig. 3

a Confocal microscopy analysis of SNAP/FLAG-hGLP-1R (red) co-localisation with ERMCSs (SPLICS Mt-ER Long P2A, green) in vehicle (Veh, left) versus exendin-4 (Ex-4, right)-stimulated INS-1 832/3 SNAP/FLAG-hGLP-1R cells; size bars, 10 μm; images representative of n = 3 biologically independent experiments. b SNAP/FLAG-hGLP-1R - SPLICS Mt-ER Long P2A co-localisation (Mander’s coefficient) in Veh versus Ex-4-stimulated conditions from data in (a); n = 5 cells from 3 independent experiments, p value as indicated by two-tailed unpaired t-test. c Time-lapse confocal microscopy analysis of SNAP/FLAG-hGLP-1R–ERMCS association, imaged as in (a); arrows indicate co-localised spots; time post-Ex-4 addition indicated; size bar, 10 μm; images representative of n = 3 biologically independent experiments. d Confocal microscopy analysis of EGFP-VAPB (blue), mitochondria (MitoTracker Red, green) and SNAP/FLAG-hGLP-1R (SNAP-Surface Alexa Fluor 647, red) in an Ex-4-stimulated INS-1 832/3 SNAP/FLAG-hGLP-1R cell; size bar, 10 μm; images representative of n = 3 biologically independent experiments. e Single confocal slice from an Ex-4-stimulated INS-1 832/3 SNAP/FLAG-hGLP-1R cell, with signals for EGFP-VAPB (green), SNAP/FLAG-hGLP-1R (yellow), mitochondria (magenta), and DAPI (blue) shown; white arrow points to a SNAP/FLAG-hGLP-1R-positive endosome used for CLEM analysis; size bar, 10 µm; also included CLEM overlay of aligned confocal slice with corresponding single slice of an EM tomogram (size bar, 10 µm) and magnification of EM tomogram slices (size bar, 1 µm) with indicated SNAP/FLAG-hGLP-1R-positive endosome (black arrow); images representative of n = 2 cell regions from the same biological experiment. f 3D rendering of segmentation of data from (e), with VAPB-positive ER (purple), mitochondria (orange) and SNAP/FLAG-hGLP-1R-positive endosome (yellow) shown; Created with ORS Dragonfly. g TEM micrograph of a 70 nm ultrathin section from resin embedded mouse islets transduced with SNAP/FLAG-hGLP-1R adenovirus, with SNAP/FLAG-hGLP-1R labelled with SNAP-Surface biotin + Alexa Fluor 488 Streptavidin, 5 nm colloidal gold prior to Ex-4 stimulation and fixation; ER (blue), mitochondria (yellow) and SNAP/FLAG-hGLP-1R-positive endosome (pink); inset shows magnified endosome area containing 5 nm gold particles; size bars, 100 nm; images representative of n = 3 biologically independent experiments. Data are mean ± SEM.

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