Novel Genetically Encoded Biosensors for Functional Imaging of Cell Signaling by Polarization Microscopy

Genetically encoded fluorescent biosensors convert specific biomolecular events into optically detectable signals. By revealing biochemical processes in situ, they have revolutionized cell biology. However, imaging molecular processes often requires modifying the proteins involved, and many molecular processes are still to be imaged. Here we present a novel, widely applicable design of genetically encoded biosensors that notably expand the observation possibilities, by taking advantage of a hitherto overlooked detection principle: directionality of optical properties of fluorescent proteins. The probes, which we term FLIPs, offer an extremely simple design, high sensitivity, multiplexing capability, ratiometric readout and resilience to bleaching artifacts, without requiring any modifications to the probe targets. We demonstrate their performance on real-time single-cell imaging of activation of G protein-coupled receptors (GPCRs), G proteins, arrestins, small GTPases, as well as receptor tyrosine kinases, even at endogenous expression levels. We also identify a new, pronounced, endocytosis-associated conformational change in a GPCR–β-arrestin complex. By demonstrating a novel detection principle and allowing many more cellular processes to be visualized, FLIPs are likely to inspire numerous future developments and insights.