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.