Applications of Fluorescent Probes in Confocal Imaging of GPCRs: From Live to Fixed Cells

Applications of Fluorescent Probes in Confocal Imaging of GPCRs: From Live to Fixed Cells

GPCRs are present across cell membranes transmitting signals from the outside into the cells, making them an essential target in biomedical research. Studying their dynamics in both a spatial and temporal manner is key to understanding how they work in this signaling pathways, and one of the best tools to do so is fluorescence microscopy. To make the most out of this imaging technique, fluorescent probes must have high specificity, minimal phototoxicity and preferably be compatible with both live and fixes cell imaging.

How Fluorescent Probes Enable High-Resolution Confocal Imaging of GPCRs

To study the dynamics of GPCRs, the imaging method must be spatially precise and have real-time monitoring capabilities. This is why fluorescent tags, both attached to the protein or to pharmacophores, are so interesting. They retain the functional integrity of the GPCR while enabling selective excitation and collection of fluorescence from a specific focal plane, leading to better resolution and lower background noise.

This technique can acquire three-dimensional image stacks, facilitating the reconstruction of GPCR distribution in tissue-like environment, in both a quantitative and qualitative manner.

Figure 1. PerkinElmer High Content Analysis System Operetta CLS used by a Celtarys Research coworker. Analysis program Harmony 4.9 (PhenoLOGIC). A High Content Screening focused microscope that has a confocal-like mode as well.

Optimizing Fluorescent Probe Selection for GPCR Imaging

Probe selection is a key step to obtaining good and significative results. The biological context (live or fixed cells), the spatial temporal resolution and other experimental components should all be considered. For example, in live-cell imaging, probes should not be toxic and should be resistant to photobleaching. In the case of fluorescent ligands, where a fluorescent tag is attached to a pharmacophore, the receptor also keeps its original structure without any modification, also maintaining its physiological activity.

Ligand-directed labelling strategies are a newly developed way to attach a fluorescent tag to a GPCR in a covalent manner. This preserves functionality while being compatible with washing procedures.

Another strategy is the use of self-labelling tags such as SNAP-tag and HaloTag. In this strategy the GPCR is modified genetically to fuse an engineered enzyme. This enzyme has been modified so a small molecule will covalently bind to it, which can in turn be attached to a fluorescent tag. This provides flexibility in labelling the GPCRs but may interfere with functional activity of the receptors. SNAP-tag has been successfully used while preserving activity. They can be combined with other ligands and are very interesting in TR-FRET (Time-Resolved Förster Energy Resonance Transfer) assays.

Exploring the Applications of Confocal Fluorescence Microscopy in GPCR Research

In GPCR research one of the best used of confocal microscopy is the study of receptor trafficking, localization and signaling dynamics. This method is very precise at determining these parameters thanks to capturing fluorescence from a single focal plane. It can even study the GPCR distribution within endocytic vesicles and across subcellular compartments.

If the experiment is done on live cells, tracking real-time receptor internalization becomes possible. Studying ligand-induced clustering and evaluating protein-protein interactions becomes possible if using FRET.

On the other hand, in fixed cells studies, it facilitates the structural context necessary to map receptor localization in defined states (pharmacological stimulation, genetic modification, disease models…).

Figure 2.Left panel, confocal microscopy of living cells incubated for 1h at 37ºC with CELT-327. Right panel, the fluorescent signal is displaced when, after incubating with CELT-327, cells are incubated with an excess of the A2B/A3 antagonist, MRS1220. Fluorescence Microscopy validation performed in the ONCOMET laboratory (Health Research Institute of Santiago de Compostela).

At Celtarys, our GPCR fluorescent ligands are designed with a variety of photophysical properties to meet your needs for confocal imaging. Come check out our products or contact us for more information!

References

Fessl T, Majellaro M, Bondar A. Microscopy and spectroscopy approaches to study GPCR structure and function. Br J Pharmacol. 2023 Dec 12. doi: 10.1111/bph.16297. 

Jang W, Senarath K, Feinberg G, Lu S, Lambert NA. Visualization of endogenous G proteins on endosomes and other organelles. eLife. 2024 Nov 8; 13:RP97033. doi: 10.7554/eLife.97033.3

Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub MA, Bazin H, Tinel N, Durroux T, Prézeau L, Trinquet E, Pin JP. Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat Methods. 2008 Jun;5(6):561-7. doi: 10.1038/nmeth.1213. 

Navarro G, Sotelo E, Raïch I, Loza MI, Brea J, Majellaro M. A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery. Molecules. 2023 Dec 15;28(24):8107. doi: 10.3390/molecules28248107.