Multiplexed imaging for probing RAS-RAF interactions in living cells

Mohammad Ahmad, Liviu Movileanu

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


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.

Original languageEnglish (US)
Article number184173
JournalBiochimica et Biophysica Acta - Biomembranes
Issue number6
StatePublished - Aug 2023


  • Binding affinity
  • Cell signaling
  • EGFR
  • GTPase
  • Intracellular measurements
  • Multicolor microscopy
  • Protein engineering
  • Protein-protein interactions

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology


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