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G protein-coupled receptors (GPCRs), G proteins, and their signaling associates are major signal transducers that control the majority of cellular signaling and regulate key biological functions including immune, neurological, cardiovascular, and metabolic processes. These pathways are targeted by over one-third of drugs on the market; however, the current understanding of their function is limited and primarily derived from cell-destructive approaches providing an ensemble of static, multi-cell information about the status and composition of molecules. Spatiotemporal behavior of molecules involved is crucial to understanding in vivo cell behaviors both in health and disease, and the advent of genetically encoded fluorescence proteins and small fluorophore-based biosensors has facilitated the mapping of dynamic signaling in cells with subcellular acuity. Since we and others have developed optogenetic methods to regulate GPCR-G protein signaling in single cells and subcellular regions using dedicated wavelengths, the desire to develop and adopt optogenetically amenable assays to measure signaling has motivated us to take a broader look at the available optical tools and approaches compatible with measuring single-cell and subcellular GPCR-G protein signaling. Here we review such key optical approaches enabling the examination of GPCR, G protein, secondary messenger, and downstream molecules such as kinase and lipid signaling in living cells. The methods reviewed employ both fluorescence and bioluminescence detection. We not only further elaborate the underlying principles of these sensors but also discuss the experimental criteria and limitations to be considered during their use in single-cell and subcellular signal mapping.

Optical approaches for single-cell and subcellular analysis of GPCR–G protein signaling