Developing, optimizing and using FRET-based biosensors to elucidate G protein signaling

The aim of the research presented in this thesis was to gain a better understanding of how intracellular signaling by GPCRs is regulated. We used fluorescence microscopy to study GPCR signaling in live cells. In order to study protein-protein interactions involved in GPCR signaling, a high spatiotemporal resolution is required. This because these interactions are often short-lived and occur at <10nm proximities. To enable visualization of protein-protein interactions at resolutions beyond the limits of conventional microscopy, the technique Förster Resonance Energy Transfer (FRET) is exploited, applied in fluorescent biosensors. FRET is energy transfer between two fluorophores. FRET occurs if the fluorophores exist in close proximity (