Single‐Molecule FRET‐Tracking of InlB‐Activated MET Receptors in Living Cells

Abstract The activation of transmembrane receptors through the binding of external ligands initiates information transfer across the cell membrane. Understanding these processes requires observations in living cells. Given the heterogeneity and lack of synchronization of such events, single‐molecule experiments are required to resolve distinct sub‐populations. Here, single‐molecule FRET microscopy and single‐particle tracking are combined to track the ligand‐induced dimerization and activation of the MET receptor tyrosine kinase in the plasma membrane of living cells. First, using fluorophore‐labeled variants of the MET ligand internalin B (InlB), the lifetime of a ligand‐activated dimeric (MET:InlB) ₂ receptor complex is determined to be ≈1 s. Next, diffusion coefficients of monomeric and dimeric MET:InlB complexes are extracted from single‐molecule FRET trajectories, revealing an ≈1.6‐fold slower diffusion of the dimeric receptor compared to the monomeric receptor, accompanied by spatially confined motion. The combination of single‐molecule FRET and single‐particle tracking provides essential biophysical parameters of membrane receptor activation in living cells.