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, 11 (1), 467

Super-resolution Microscopy Compatible Fluorescent Probes Reveal Endogenous Glucagon-Like peptide-1 Receptor Distribution and Dynamics

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Super-resolution Microscopy Compatible Fluorescent Probes Reveal Endogenous Glucagon-Like peptide-1 Receptor Distribution and Dynamics

Julia Ast et al. Nat Commun.

Abstract

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Sequence and structure of LUXendin555, LUXendin645, and LUXendin651.
LUXendins are based on the antagonist Exendin4(9–39), shown in complex with GLP1R. The label can be any dye, such as TMR (top), SiR (middle), or Cy5 (bottom) to give LUXendin555, LUXendin645, and LUXendin651, respectively. The model was obtained by using the cryo-EM structure of the activated form of GLP1R in complex with a G protein (pdb: 5VAI), with the G protein and the 8 N-terminal amino acids of the ligand removed from the structure while mutating S39C and adding the respective linker. Models were obtained as representative cartoons by the in-built building capability of PyMOL (Palo Alto, CA, USA) without energy optimization. Succinimide stereochemistry is unknown and neglected for clarity.
Fig. 2
Fig. 2. LUXendin645 binding, signaling, and labeling.
a Exendin4(9–39), S39C-Exendin4(9–39), and LUXendin645 (LUX645) display similar antagonistic properties (applied at 1 µM) in HEK293-SNAP_GLP1R following 30 min GLP-1-stimulation (n = 4 independent assays). b LUXendin645 weakly activates GLP1R in the presence of the positive allosteric modulator (PAM) BETP (25 µM) (30 min stimulation in HEK293-SNAP_GLP1R) (Ex4, +ve control) (n = 4 independent assays). c LUXendin645 labels AD293-SNAP_GLP1R cells with maximal labeling at 250–500 nM (n = 4 independent assays). d LUXendin645 signal cannot be detected in YFP-AD293 cells (scale bar = 212.5 µm) (n = 3 independent assays). e Representative confocal z-stack showing LUXendin645 staining in a live islet (n = 27 islets, six animals, three separate islet preparations) (scale bar = 37.5 µm). f As for e, but two-photon z-stack (scale bar = 37.5 µm) (representative image from n = 27 islets, seven animals, three separate islet preparations). g, h 250 nM LUXendin645 internalizes GLP1R in MIN6 β-cells when agonist activity is conferred using 25 µM BETP (Ex4 and Ex9 were applied at 100 and 250 nM, respectively) (scale bar = 21 µm) (representative images from n = 12 coverslips, three independent repeats) (one-way ANOVA with Bonferroni’s test; F = 217.6, DF = 3). i, j LUXendin645 signal co-localizes with a GLP1R monoclonal antibody in islets (n = 13 islets, three separate islet preparations) and MIN6 β-cells (representative images from n = 24 coverslips, three independent repeats) (scale bar = 26 µm). k LUXendin645 improves membrane visualization compared to antibody (scale bar = 12.5 µm). Representative images are shown, with location of intensity-over-distance measures indicated in blue (n = 18 islets, five animals, three separate islet preparations). l, m LUXendin645 co-localizes with Surface 488, pre-applied to Glp1r null SNAP_hGLP1R-INS1GLP1−/− cells l. Pre-treatment with Exendin4(1–39) to internalize the GLP1R reduces LUXendin645–labeling m (scale bar = 10 µm) (representative images from n = 3 independent repeats). LUXendin645 was applied to cells at 250 nM and tissue at 50–100 nM. GLP-1 glucagon-like peptide-1; Ex9 Exendin4(9–39); S39C S39C-Exendin4(9–39); Ex4 Exendin4(1–39). Mean ± s.e.m. are shown. **P < 0.01 for all statistical tests. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. LUXendin645 is highly specific for the GLP1R.
a Schematic showing sgRNA-targeting strategy for the production of Glp1r(GE)−/− mice. The sgRNA used targeted Glp1r and the double-strand break mediated by Cas9 lies within exon1 (capital letters); intron shown in gray. b Glp1r(GE)−/ animals harbor a single-nucleotide deletion, as shown by sequencing traces. c Body weights were similar in male 8–9 weeks old Glp1r+/+, Glp1r(GE)+/, and Glp1r(GE)−/− littermates (n = 9 animals) (one-way ANOVA with Bonferroni’s test; F = 0.362, DF = 2). d The incretin-mimetic Exendin4(1–39) (Ex4; 10 nM) is unable to significantly potentiate glucose-stimulated insulin secretion in Glp1r(GE)−/ islets (n = 15 repeats, six animals for each genotype, three separate islet preparations) (between genotype comparisons: two-way ANOVA with Sidak’s test; F = 4.061, DF = 2) (within genotype comparisons: one-way ANOVA with Bonferroni’s post-hoc test; F = 14.57 (Glp1r+/+), 10.83 (Glp1r(GE)−/−); DF = 2). e Liraglutide (Lira) does not stimulate cAMP beyond vehicle (Veh) control in Glp1r(GE)−/− islets, measured using the FRET probe Epac2-camps (n = 25 islets for each genotype, three animals per genotype, two separate islet preparations). f cAMP area-under-the-curve (AUC) quantification showing absence of significant Liraglutide-stimulation in Glp1r(GE)-/- islets (n = 25 islets for each genotype, three animals per genotype, two separate islet preparations) (Kruskal–Wallis test with Dunn’s test; Kruskal–Wallis statistic = 31.78, DF = 2) (Box and Whiskers plot shows range and median) (representative images displayed above each bar; color scale shows min to max values as a ramp from blue to red). g LUXendin645 and GLP1R antibody labeling is not detectable in Glp1r(GE)−/− islets (scale bar = 40 µm) (n = 27 islets, five animals per genotype, three separate islet preparations). For all statistical tests, *P < 0.05, **P < 0.01 and NS, non-significant. In all cases, LUXendin645 was applied at 100 nM. Mean ± s.e.m. are shown. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. LUXendin645 reveals GLP1R expression in a subpopulation of α-cells.
ac LUXendin645 labeling is widespread throughout the intact islet, co-localizing predominantly with β-cells a and δ-cells b, but less so with α-cells c stained for insulin (INS), somatostatin (SST), and glucagon (GCG), respectively (n = 18 islets, seven animals, three separate islet preparations) (scale bar = 26 µm). d Following dissociation of islets into cell clusters, LUXendin645 labeling can be more accurately quantified (arrows highlight cells selected for zoom-in) (scale bar = 26 µm). e Zoom-in of d showing a LUXendin645− (left) and LUXendin645+ (right) α-cell (arrows highlight non-labeled cell membrane, which is not bounded by a β-cell) (scale bar = 26 µm). f Box-and-whiskers plot showing proportion of β-cells (INS) and α-cells (GCG) co-localized with LUXendin645 (n = 18 cell clusters, ten animals, three separate islet preparations) (box and whiskers plot shows range and median; mean is shown by a plus symbol). g Ins1CreThor;R26mT/mG dual fluorophore reporter islets express tdTomato until Cre-mediated replacement with mGFP, allowing identification of β-cells (~80% of the islet population) and non-β-cells for live imaging (scale bar = 26 µm). LUXendin645 (LUX645) highlights GLP1R expression in nearly all β-cells but relatively few non-β-cells (n = 31 islets, six animals, three separate islet preparations). h A zoom-in of the islet in g showing GLP1R expression in some non-β-cells (left) together with quantification (right) (arrows show LUXendin645-labeled non-β cells) (scale bar = 12.5 µm) (scatter dot plot shows mean ± s.e.m.). White boxes show the location of zoom-ins. In all cases, LUXendin645 was applied at 100 nM. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. LUXendin651 and LUXendin645 allow nanoscopic detection of GLP1R.
a LUXendin645 allows super-resolution snapshots of MIN6 β-cells using widefield microscopy combined with super-resolution radial fluctuations (SRRF) (representative image from n = 8 images, three independent repeatss) (scale bar = 10 µm for full-field images, 2.5 µm for zoomed-in images). bd Confocal and STED snapshots of endogenous GLP1R in LUXendin651-treated MIN6 cells at FWHM = 70 ± 10 nm (mean ± s.d.; n = 15 line profiles measured on the raw data, two independent repeats). Note the presence of punctate GLP1R expression as well as aggregation/clustering in cells imaged just away from b, close to c or next to d the coverslip using STED microscopy (representative image from n = 8 images, three independent repeats) (scale bar = 2 µm for full-field images, 1 µm for zoomed-in images). e, f Representative graph showing spatial analysis of GLP1R expression patterns using the F-function e and G-function f, which show distribution (red line) vs. a random model (black line; 95% confidence interval shown) (n = 6 from three independent repeats). g Approximately 1 in 4 MIN6 β-cells possess highly concentrated GLP1R clusters. h, i LUXendin651 allows GLP1R to be imaged in living MIN6 cells using SRRF h and STED i (representative image from n = 6 and 18 images, three independent repeats for SRRF and STED, respectively) (scale bar = 10 µm for full-field SRRF image, 2.5 µm for the zoomed-in image) (scale bar = 2 µm for STED images). White boxes show the location of zoom-ins. The following compound concentrations were used: 100 nM LUXendin645 (SRRF) and 100–400 nM LUXendin651 (STED). Mean ± s.e.m. are shown. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. LUXendin645 and LUXendin651 allow single molecule GLP1R imaging.
a Representative single molecule microscopy images showing tracking of LUXendin645- and LUXendin651-labeled GLP1R at or close to the membrane (scale bar = 3 µm). b Mean square displacement (MSD) analysis showing different GLP1R diffusion modes (representative trajectories are displayed) (scale bar = 1 µm). c GLP1R molecules with diffusion coefficient D < 0.01 are classed as immobile (left), whilst those with D > 0.01 are further divided according to their anomalous diffusion exponent (α), which defines the type of motion followed (confined, normal, or directed) (right) (pooled data from n = 16 cells, 5057–8612 trajectories, six independent repeats). LUXendin645 and LUXendin651 were used at 100 pM. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. LUXendin645 highlights GLP1R-expressing neurons in the brain.
a LUXendin645 labeling is detected in the the median eminence (ME), arcuate nucleus (ARC), area postrema (AP)/nucleus tractus solitaris (NTS), and choroid plexus (CP), in close association with GLP1-producing neurons, identified using GLU-YFP reporter animals (3V, third ventricle) (representative images from n = 4 animals) (scale bar = 106 µm). b Z-projection of an image stack (~30 µm) showing direct contacts between LUXendin645-labelled and GLP1-producing (GLU-YFP) neurons in the ARC (representative image from n = 4 animals) (scale bar = 20 µm). c, d LUXendin645 labeling co-localizes with GLP1R+ neurons in the AP/NTS c and ARC d, identified using GLP1RCre;LSL-GCaMP3 reporter animals (representative image from n = 4 animals) (scale bar = 61 µm). e Super-resolution imaging using Airyscan shows that LUXendin645 labeling is restricted to the membrane of the cell body and dendrites of GLP1R+ neurons (arrows show cell body and dendrite from left to right, respectively) (representative images from n = 4 animals) (scale bar = 9 µm). f GLP1R form nanodomains in ARC and AP neurons, as well as ependymal cells of the CP (confocal image is shown on the left for comparison) (representative images from n = 8 animals) (scale bar = 9 µm). g Mapping of LUXendin645 distribution in cleared brains shows labelling of the ARC, AP/NTS, CP, lateral ventricles (LV), fourth ventricle (4V), subfornical organ (SFO), and organum vasculosum of the lamina terminalis (OVLT) (representative images from n = 4 animals) (scale bar = 1 mm). Note that, due to suspension of the brain, the coronal section is slightly offset in the dorsal–ventral plane; hence, the SFO appears above the ARC. In all cases, LUXendin645 was injected subcutaneously at 100 pmol/g.
Fig. 8
Fig. 8. LUXendin645 labels human ESC-derived β-like cells.
a LUXendin645 (LUX645) labels β-like cells in intact spheroids, which were differentiated and cultured for 21 days. No signal is detected in undifferentiated human ES cells (day 0) or unlabelled β-like cells (-LUX645) (representative images from n = 6 spheroids) (scale bar = 100 µm). b GLP1R gene expression in day 0 undifferentiated cells, day 21 differentiated β-like cells, and human islets (n = 3 donors). c LUXendin645 labelling is localized to strongly insulin (INS)-positive but not strongly glucagon (GCG)-positive areas (representative images from n = 5–6 spheroids) (scale bar = 50 µm). d LUXendin645 (LUX) overlaps more with INS vs. GCG, as calculated using Manders’ M1 co-efficient (n = 5–6 spheroids) (unpaired Student’s t-test). e Day 21 spheroid sections (5 µm) showing expression of INS and NKX6-1, confirming a differentiated phenotype (representative images from n = 4 spheroids) (scale bar = 100 µm). f FACS plots of day 21 β-like cells with and without LUXendin645 (LUX645) incubation. LUXendin645+ (LUX+) and LUXendin645− (LUX−) cells were sorted for qPCR. g GLP1R, NKX6-1, INS, and GCG gene expression in sorted cells (n = 4 spheroids) (connecting bars indicate LUX+ and LUX− populations in the same samples) (paired Student’s t-test). LUXendin645 was applied at 100 nM. Mean ± s.e.m. are shown. *P < 0.05, **P < 0.01 for all statistical tests. Source data are provided as a Source Data file.
Fig. 9
Fig. 9. LUXendin555 allows in vivo labeling of islets.
ac LUXendin555 labels YFP-AD293_SNAP-GLP1R a but not YFP-AD293 b controls with max labeling at 600 nM c (n = 3 independent assays) (×10 scale bar = 213 µm; ×100 scale bar = 21 µm). d Surface GLP1R expression is similar in LUXendin555− (LUX555), LUXendin645− (LUX645), and 250 nM Exendin4(9–39)-treated islets (100 nM Ex4, +ve control) (representative images shown above each bar) (one-way ANOVA with Bonferroni’s test; F = 173.3, DF = 3) (n = 12 islets, seven animals, three separate islet preparations) (scale bar = 17 µm). e LUXendin555 behaves as an antagonist in HEK-SNAP_GLP1R cells using HTRF-based assays (n = 4 independent assays in duplicate). f LUXendin555 displays agonist activity in CHO-K1-SNAP_GLP1R cells, as assessed using luciferase-based detection (GLO) (n = 3 independent assays) (positive allosteric modulation was achieved using 25 µM BETP). g Schematic depicting the two-photon imaging set up for visualization of the intact pancreas in mice. h Representative image showing that LUXendin555 (100 pmol/g, IV) labels cell membranes in an islet surrounded by the vasculature in vivo (n = 3 animals) (scale bar = 50 µm). LUXendin645 and LUXendin555 were applied to cells/islets at 100 and 250 nM, respectively. GLP-1 glucagon-like peptide-1; Ex9 Exendin4(9–39); Ex4 Exendin4(1–39). Mean ± s.e.m. are shown. **P < 0.01 for all statistical tests. Source data are provided as a Source Data file.

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