9. Imaging of endogenous signalling systems
The reliable study of physiological processes requires minimal
perturbations of endogenous systems. However, achieving this goal
remains a challenging task. Major (a thousand-fold (Cho et al., 2022))
differences in the expression of GPCR cascade components make it very
difficult to find a “one-size-fits-all” microscopy technique for the
study of endogenous systems. However, the advent of versatile
CRISPR-based gene editing has initiated a new era of GPCR studies using
endogenous protein expression levels. CRISPR technology allows the
modification of endogenously expressed proteins with genetically encoded
fluorescent and luminescent probes. The most prominent examples of
CRISPR applications in GPCR research include the Open Cell project,
which has produced and made available an outstanding amount of imaging
data on the localisation and expression levels of endogenous signalling
components (Cho et al., 2022), and the removal of selected endogenous G
protein subunits (Alvarez-Curto et al., 2016) and GRKs (Drube et al.,
2022) from HEK293 cells which has enabled the study of signalling in a
background-free setting. Careful choice of the reporting optical probes
is required for the study of endogenous systems. Small (subnanometer)
probes, with high labelling specificity that do not alter the signalling
properties of the studied system, can be delivered to the site of
labelling, and are compatible with in vivo imaging will be most
useful for the study of endogenous systems. Examples of such probes
include fluorescent ligands (Rosier et al., 2021), ligand-directed
fluorescent labelling probes (Stoddart et al., 2020), or probes for
direct protein labelling with click chemistry of unnatural amino acids
(Arsic et al., 2022; Mihaila et al., 2022). The development of CRISPR in
combination with genetic code expansion and optimised optical probes
will provide major insights into endogenous signalling systems in the
future.
Label-free techniques are a separate group of methods suitable for the
study of endogenous systems. For example, surface plasmon resonance and
dynamic mass redistribution (Figure 2l) have been used to establish the
role of PDZ-ligands in GPCR signalling (Camp et al., 2016) and detect
angiotensin 1 receptor activity in a label-free fashion (Drube et al.,
2022). Atomic force microscopy (AFM), which does not truly belong among
optical approaches, finds its uses in GPCR research. AFM scans the
sample surface with a fine probe to determine its 3D landscape and the
interaction forces between molecules. It has been used to determine the
ligand-binding free energy of PAR1 (Alsteens et al., 2015) and detect
GPCR oligomerisation through receptor unfolding (Dague et al., 2022).
Further development of label-free technologies will expand their areas
of application to provide holistic insights into endogenous cell
signalling.