FLIPs: Genetically encoded molecular biosensors for functional imaging of cell signaling by linear dichroism microscopy

Genetically encoded fluorescent biosensors convert specific biomolecular events into optically detectable signals. However, imaging biomolecular processes often requires modifying the proteins involved, and many molecular processes are still to be imaged. Here, we present a biosensor design that uses a hitherto overlooked detection principle: directionality of optical properties of fluorescent proteins. The biosensors (termed FLIPs) offer an extremely simple design, high sensitivity, multiplexing capability, ratiometric readout, and other advantages, without requiring modifications to their targets. We demonstrate the sensor performance by real-time imaging activity of G protein-coupled receptors (GPCRs), G proteins, arrestins, and other membrane-associated proteins, as well as by identifying a previously undescribed, pronounced, endocytosis-associated conformational change in a GPCR-β-arrestin complex. In combination with an original tri-scanning linear dichroism confocal microscope, FLIPs allow unparalleled imaging of activity of nonmodified, endogenously expressed G proteins. Thus, FLIPs establish a powerful molecular platform for imaging cell signaling, allowing numerous future developments and insights.