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.