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 (<10nm). The fluorescent biosensors consist of two fluorescent proteins (FPs), called the FRET pair, and a sensing domain. The sensing domain should be specific for a certain biological event involved in GPCR signaling. Upon occurrence of the biological event, the sensing domain interacts, dissociates or changes conformation leading to a change in distance or orientation between the FRET pair, altering the efficiency of energy transfer. Thereby the FRET pair indirectly reports the occurrence of the specific biological event. It is of high importance that these biosensors report biological events truthfully. Therefore a substantial part of my research was directed towards the development and optimization of biosensors and the employed fluorescent proteins that constitute the FRET pair. Another part of my research was directed towards application of developed and optimized biosensors to gain more insight into the regulation of GPCR signaling.