Multiplexed imaging for probing RAS-RAF interactions in living cells.

GTP-bound RAS interacts with its protein effectors in response to extracellular stimuli, leading to chemical inputs for downstream pathways. Significant progress has been made in measuring these reversible protein-protein interactions (PPIs) in various cell-free environments. Yet, acquiring high sensitivity in heterogeneous solutions remains challenging. Here, using an intermolecular fluorescence resonance energy transfer (FRET) biosensing approach, we develop a method to visualize and localize HRAS-CRAF interactions in living cells. We demonstrate that the EGFR activation and the HRAS-CRAF complex formation can be concurrently probed in a single cell. This biosensing strategy discriminates EGF-stimulated HRAS-CRAF interactions at the cell and organelle membranes. In addition, we provide quantitative FRET measurements for assessing these transient PPIs in a cell-free environment. Finally, we prove the utility of this approach by showing that an EGFR-binding compound is a potent inhibitor of HRAS-CRAF interactions. The outcomes of this work form a fundamental basis for further explorations of the spatiotemporal dynamics of various signaling networks.