Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

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.


Statistics
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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. cAMP and TIRF imaging data were acquired using Metamorph (Molecular Devices). Immunohistochemistry and live cell images were collected using Metamorph (Molecular Devices), Zen 2012 (Zeiss) and Leica Application Suite X (Leica).
LUXendins are freely available for academic use. Glp1r(GE)-/-animals are subject to a Material Transfer Agreement. The source data underlying Figs 2a-c, g, k, 3c-f, 4f and h, 5e-g, 6c, 8b and d, and 9c-f and Supplementary Fig 1 are provided

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Laboratory animals
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Field-collected samples
The measurement unit is animal, batch of islets, well of cells or spheroid. Experiments were repeated independently at least three times, usually with technical replicates. Islet data are reported from at least two separate isolation procedures.
No data were excluded unless the cells displayed a clear non-physiological state (i.e. impaired viability).
All findings were replicated independently three times, usually with different batches of compounds and across different investigators.
Samples and animals were allocated to treatment groups in a randomized manner to ensure that all states were represented in the different experiment arms.
Data were acquired using imaging setups that performed the measurement independently of the observer.
Antibodies were validated using knockout tissue (GLP1R) or known cell-type specific localizations (INS, GLU and SST), further confirmed using reporter animals.