Figure legends
Figure 1. Spatiotemporal characteristics of the GPCR signalling cascade. (a) Temporal dynamics of individual signalling stages. Individual signalling stages range in their duration from nanoseconds (or even femtoseconds if initial steps of rhodopsin signalling are considered) to tens of minutes. (b) Spatial properties of signalling steps. Spatial properties of the signalling cascade range from Angstrom movements, which accompany conformational changes, to tens of centimetres, which characterise neuronal signalling and hormonal distribution.
Figure 2. Microscopy and spectroscopy approaches for the study of GPCR signalling. (a) STED is an SRN technique that uses a donut beam to quench all fluorophores outside a small central spot. (b) PALM and STORM use weak light intensity combined with photoactivation of fluorescent proteins or blinking of dyes to acquire resolution enhancement by imaging individual molecules. (c) DNA-PAINT uses transient binding of fluorescent oligonucleotides to reproduce the blinking behaviour required for image reconstruction from multiple frames with individual fluorescent molecules. (d) MINFLUX scans the area looking for a central minimum of fluorophore excitation corresponding to its precise position. (e) SIM projects patterns of light on a sample allowing image reconstruction with improved resolution. (f) Expansion microscopy is based on the creation of a swellable polymer network with a sample and expansion of it to enable enhanced resolution of fine sample details. (g) SmFRET allows accurate measurements of protein conformational changes and protein-protein interactions at the single-molecule level and high temporal resolution. (h) FLIM allows accurate detection of fluorescence lifetimes to filter out background signals and achieve intensity-independent reproducible measurements. (i) SPT acquires images of sparse samples and uses image processing to accurately determine the subpixel localization of individual molecules and perform their tracking with high temporal resolution. (j) FCS utilises fluorescence fluctuations to obtain information on sample dynamics with a very high temporal resolution. (k) Polarisation microscopy gains insights into protein conformation and interactions by taking advantage of the fact that light absorption in many fluorophores in constrained systems, such as cell membranes, depends on light polarisation. (l) DMR is a label-free technique that uses waveguides to detect minute changes in the distribution of cellular mass that accompany signal transduction. (m) Bioluminescence imaging uses a chemical reaction catalysed by luciferases, which is accompanied by the emission of light, to detect signalling without using excitation light and with a minimal nonspecific background. (n) 2P microscopy allows deep tissue imaging with low scatter and low autofluorescence by using two photons of infrared light instead of a single visible light photon to excite fluorescent molecules. (o) LSFM uses a sheet of light to fast scan large sample areas with minimal photodamage.
Figure 3. Spatiotemporal resolution of optical microscopy and spectroscopy techniques. (a) Temporal resolution limits of microscopy and spectroscopy techniques. (b) Spatial resolution of imaging techniques.
Figure 4. GPCR labelling strategies and examples of biosensors. (a) Fluorescent ligands simultaneously modulate GPCR activity and fluorescently label the receptors. (b) Ligand-directed labelling is based on receptor binding by a ligand bearing a fluorescent label that becomes covalently attached to the receptor. Afterwards, the ligand can be washed out, leaving the labelled GPCR. (c) Direct fluorescent labelling is based on the direct binding of a fluorescent dye to protein cysteines or unnatural amino acids. (d) Self-labelling systems (e.g., SNAP-tag, HaloTag) autocatalyse their specific labelling with fluorescent dyes. (e) Quantum dots and nanodiamonds can label specific reactive groups attached to proteins of interest and provide extraordinary brightness and photostability. (f) Fluorescent proteins are a major group of commonly used genetically encoded labels. Photoswitchable and photoactivatable fluorescent proteins are extensively used in SRN techniques. (g) Protein conjugation labelling systems (e.g., ALFA-tag, SpyTag) specifically bind a small amino acid sequence in the protein of interest. (h) Nanobodies are small single-chain antibodies that bind GPCRs in a specific conformation. (i) MiniG proteins are nanobody-like surrogates based on the GTPase domain of Gα subunits that irreversibly bind to activated GPCRs. (j) GPCR-based cAMP nanorulers allow for the detection of cAMP production with high spatial precision. (k) OptoGPCRs contain extracellular parts of rhodopsin and intracellular parts of other GPCRs so that they can be activated by light and produce a signal from those GPCRs intracellularly. (l) Circularly permuted fluorescent protein-based (cpFP) neurotransmitter sensors allow the quantitative detection of neurotransmitter concentration in the vicinity of a cell using GFP fluorophore sensitivity to the environment.