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

## TL;DR

This study uses advanced imaging techniques to track how a specific receptor activates in living cells when bound to a ligand.

## Contribution

The study introduces a combined approach of single-molecule FRET and tracking to observe MET receptor dimerization and mobility in real time.

## Key findings

- The lifetime of a ligand-activated dimeric MET receptor complex is approximately 1 second.
- Dimeric MET complexes diffuse 1.6 times slower than monomeric ones and show spatially confined motion.

## 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.

Single‐molecule FRET and single‐particle tracking reveal how ligand binding drives dimerization and activation of the MET receptor tyrosine kinase in living cells. Single‐molecule FRET reports on the lifetime of the (MET:InlB)2 complex. Distinct diffusion coefficients and modes of monomeric and dimeric receptor complexes uncover nanometer‐scale mobility changes.