Direct multiplex imaging and optogenetics of RhoGTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge due to spectral overlap of available genetically-encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein miRFP720 and the fully NIR Förster Resonance Energy Transfer (FRET) pair miRFP670-miRFP720 that enables design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use for multiplexed imaging and light control of RhoGTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and then concurrently used LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified the antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK, showed that Rac1 activity and GDI binding depend exquisitely on the spatiotemporal coordination between these two molecules, and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.