Single-molecule FRET-tracking of InlB-activated MET receptors in living cells

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 demands for single-molecule experiments that can resolve distinct sub-populations. Here, we combine single-molecule FRET microscopy and single-particle tracking 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), we determined the lifetime of a ligand-activated dimeric (MET:InlB) ₂ receptor complex to around 1 second. Next, we determined the diffusion coefficient from monomeric and dimeric MET:InlB complexes from single-molecule FRET trajectories and found an about 1.6-fold slower diffusion of the dimeric receptor as compared to the monomeric receptor, accompanied by spatially restricted motion. The combination of single-molecule FRET and single-particle tracking delivers essential biophysical parameters of membrane receptor activation in living cells.