Fluorescent biosensors are novel pharmacological tools that allow real-time visualization of protein activations in cancerous cells, allowing biologists to visualize the dynamic pathways of proteins. The biosensor measures protein activity through genetically encoded Förster resonance energy transfer (FRET). RhoG GTPase is a signaling G protein that plays a crucial role in cellular processes and when disrupted can contribute to cancer progression. However, a fully optimized and characterized RhoG FRET biosensor currently does not exist. Therefore, the objective of this study was to validate the behavior of this biosensor through the co-expression with protein inhibitors, mutant biosensor expression, and cell stimulation. Increasing concentrations of Rho guanine nucleotide dissociation inhibitor (RhoGDI), and RabGDI were co-transfected with the RhoG biosensor, and the FRET emission from was then determined. Results displayed a significant decrease of the FRET/donor protein ratio as RhoGDI concentration increased (p<0.005). Additionally, active and inactive mutant RhoG biosensors were examined in live-cell imaging to determine the total dynamic range of the signal modulation. The results showed a 40% decrease (p<0.0004) in FRET/donor ratio between the mutants. Lastly, cellular growth factor stimulation was used to examine the biosensor’s response to cell stimulation, however additional research is required to further validate results. Future research can examine how pharmaceutical drugs affect RhoG proteins using this biosensor, establishing a foundation for cancer therapies.

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