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.