10.1 Labelling approaches
Fluorescent ligands label GPCRs and simultaneously ensure a defined receptor conformation (Figure 4a). A unique advantage of fluorescent ligands is the elimination of direct covalent labelling of signalling system components. Therefore, fluorescent ligands are suitable for imaging endogenous systems and the dynamics of GPCR activation and activity regulation. Fluorescent GPCR ligands span a range of applications from SPT (Isbilir et al., 2020) to high-throughput screening (Tahk et al., 2023) and are promising tools for the study of endogenous systems (Barbazan et al., 2022). However, this comes with the limitation that fluorescent ligands can only visualise the proteins to which they bind.
The ligand-directed fluorescent labelling approach relies on a GPCR ligand as a carrier for a fluorophore that is covalently attached to the receptor upon ligand binding and remains attached after ligand dissociation (Figure 4b) (Stoddart et al., 2020). This approach allows the labelling of endogenous GPCRs while maintaining their ability to be activated and inactivated. Ligand-directed labelling can be used in live animals (Arttamangkul et al., 2019). Ligand-directed labelling requires the presence of suitable amino acids for labelling on the receptor surface and is often limited by the existence of a suitable ligand conjugated with the appropriate fluorophore. This technique can be combined with other labelling schemes to create a RET pair for studies of GPCR signalling with FRET techniques.
Direct labelling is used mainly for in vitro experiments with applications such as smFRET and FCS and is usually based on the labelling of protein cysteines or introducing unnatural amino acids with fluorescent dyes (Figure 4c) (Mihaila et al., 2022). This approach ensures the smallest possible label size and allows for colour multiplexing. However, it has very limited compatibility with the imaging of live samples and often suffers from non-specific labelling and different labelling efficiencies causing artefacts in multicolour experiments.
FPs are workhorses of optical microscopy that are compatible with multiple imaging techniques and serve a variety of purposes (Figure 4f). FPs can be split into parts and restored using bimolecular fluorescence complementation, undergo photoswitching and photoactivation required for SMLM techniques, serve as RET donors and acceptors, can be used with 2P excitation in deep tissues, or even assist GPCR purification (reviewed in (Kim et al., 2022; Ravotto et al., 2020). The limitations of using FPs include their relatively large size, which limits localisation precision for SRN techniques and often interferes with signalling, irreversibility of labelling, and fast bleaching.
Self-labelling systems , including SNAP-tag, CLIP-tag (Gautier et al., 2008), and HaloTag (Los et al., 2008), are based on a genetically encoded modification of the molecule of interest with an enzyme which catalyses its covalent labelling with a reactive fluorescent dye (Figure 4d). These versatile systems enable specific labelling with the possibility of multiplexing and flexible experimental design, due to the existence of a variety of suitable dyes of different colours with distinct membrane permeability, some of which can be photoactivated. Self-labelling systems allow the study of multiple processes using the same labelled signalling protein. Common applications of these systems include SPT which takes advantage of the labelling specificity, photostability, and brightness of available dyes (Asher et al., 2021; Sungkaworn et al., 2017). The limitations of self-labelling systems involve mainly their large size, which is comparable to that of an FP.
Protein conjugation systems , including ALFA-tag (Gotzke et al., 2019), SpyTag (Alam et al., 2019) and binder-tag (Liu et al., 2021), rely on the target protein modification with a small peptide (7-14 amino acids) and binding of the universal fluorescently labelled small antibody-like protein. Irreversible (ALFA-tag, SpyTag) or reversible (binder-tag) conjugation allows imaging of signalling with only small direct modifications of the studied protein (Figure 4g). Moreover, in the case of binder-tag technology, the binding itself depends on protein conformation and is indicative of signalling activity. Protein conjugation systems are very promising but include relatively large labels and require delivery of exogenous fluorescently labelled proteins for labelling the experimental system.
Quantum dots and nanodiamonds provide an alternative to classical fluorescence dyes due to their outstanding brightness and photostability (Figure 4e) (Sotoma et al., 2016) and can be combined with self-labelling systems (Komatsuzaki et al., 2015). They are optically superior to fluorescent dyes but suffer from their large size (2-50 nm) and complicated intracellular delivery for applications in SPT and emerging SRN techniques.