Rap1 biosensor. (A) Schematic of the Rap1 biosensor showing that FRET occurs when GTP-loaded Rap1 binds the RBD, bringing mCerulean3 (blue) and YPet (yellow) together. (B) Representative Western blot showing separation of both components of the biosensor, expressed using tandem viral 2A sequences. (C) Measuring the FRET response of the biosensor by titrating the RBD component with a constant concentration of constitutively active (G12V), dominant-negative (S17N), and WT Rap1. Results are from 12 (WT and S17N) or 13 (G12V) replicates of the full titration. Error is the SEM. There is no detectable FRET for the S17N mutant. This together with high FRET efficiency yielded a sensitive biosensor. All plate assays were performed with LinXe cells. (D) The biosensor reported activation when expressed with CalDAG-GEF1 and inactivation when coexpressed with Rap1GAP1. Results from n = 5 distinct replicates are shown. Error is the SEM. (E) FRET was greatly reduced by introducing either a K48A or K48A/H49A mutations into the RalGDS affinity reagent to abrogate binding (box and whisker plot, n = 12). (F) Individual frames of Rap1 biosensor activity in HUVECs with (right panel) and without (left panel) stimulation by a small molecule activator of Epac, a Rap1 GEF (8-CPT-cAMP). Scale bars, 10 µm. (G) HUVECs expressing the biosensor were stimulated with 8-CPT-cAMP (shown in gray), and the mean cell ratio was normalized to 1 at the first time point (n = 6 cells, error is the SEM).