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.