Introduction
The GPCR signalling cascade is a major pathway of cellular signal
transduction and receptors play a key role in it. GPCR signalling is
responsible for a multitude of physiological processes including
eyesight, heartbeat, kidney excretion, the action of synapses, and many
others. The malfunctioning of GPCR signalling is a cause of multiple
diseases, including schizophrenia, heart failure, and multiple forms of
cancer. Therefore, the GPCR pathway is a major drug target.
Specifically, GPCRs that perceive extracellular stimuli are the primary
targets of pharmacological compounds. Accurate and precise understanding
of the spatiotemporal signalling properties of the GPCR cascade is of
utmost importance for the development of better treatments for
GPCR-related diseases.
Microscopy and spectroscopy techniques provide insights into multiple
aspects of cellular signalling at a wide range of spatial and temporal
scales. Individual stages of the GPCR signalling cascade vary greatly
depending on the nature of the ligand, GPCR itself, expression levels,
the cellular context, presence, and abundance of regulatory proteins, G
proteins, and effector types (Figure 1). Signalling events span
timescales from femtoseconds (Schoenlein et al., 1991) for the initial
steps in rhodopsin activation to tens of minutes for gene expression
regulation and the endosomal “second wave” of signalling (Tsvetanova
et al., 2015). The spatial scale of signalling events spans the range
from ångstroms for molecular conformational changes through micrometres
for redistribution of signalling molecules in the cell to millimetres
and more for the transport of hormones and neurotransmitters within the
body. Therefore, techniques for the study of GPCR signalling span the
entire range from femtosecond spectroscopy and the tracking of
individual molecules to long-term light sheet imaging of whole living
objects. For the sake of clarity, we will focus on optical microscopy
and spectroscopy techniques with high spatial and temporal resolution,
primarily spanning the spatial range from nanometres to micrometres and
the temporal range from microseconds to seconds.
Imaging GPCR signalling in high resolution
When considering a high resolution, we need to distinguish between high
spatial and high temporal resolution and techniques which excel in one
or the other of these domains or provide a good combination of both
(Figures 2 and 3, Supporting Table 1). The high spatial resolution
required for GPCR signalling ranges from ångstroms to a few tens of
nanometres (Figure 1b) while high temporal resolution ranges from
microseconds to a few hundred milliseconds (Figure 1a) if we omit the
initial photophysical events in rhodopsin activation. The group of
techniques that primarily excel in spatial resolution can be broadly
referred to as super-resolution nanoscopy. Imaging techniques which
provide the best temporal resolution are mostly based on resonance
energy transfer (RET), single-particle tracking (SPT), or fluorescence
fluctuations. Recently, several highly promising techniques enabling a
combination of high spatial and temporal resolution have emerged.
Super-resolution nanoscopy (SRN)
The spatial resolution of a conventional fluorescence microscope is
restricted by the diffraction limit, which is usually 200-300 nm in the
lateral direction and 500 nm in the axial direction. However, several
techniques use different optical phenomena to break the diffraction
limit (Figures 2 and 3). At present, the most often used are stimulated
emission depletion microscopy (STED), photoactivated localisation
microscopy (PALM), stochastic optical reconstruction microscopy (STORM),
structured illumination microscopy (SIM), DNA point accumulation in
nanoscale topography (DNA-PAINT), and super-resolution radial
fluctuations (SRRF). However, several highly promising techniques
enabling the achievement of subnanometre resolution have recently
emerged, including minimising fluorescence fluxes nanoscopy (MINFLUX),
minimal STED (MINSTED), single-molecule localization by raster scanning
a minimum of light (RASTMIN), resolution enhancement by sequential
imaging (RESI), and expansion microscopy.