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

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Transmembrane receptor activation by external ligands is crucial for cellular information transfer.
• Studying these dynamic processes in living cells requires high-resolution single-molecule techniques due to cellular heterogeneity.

Purpose of the Study:

• To investigate the ligand-induced dimerization and activation of the MET receptor tyrosine kinase in real-time within living cells.
• To determine the biophysical parameters governing MET receptor activation using advanced single-molecule methods.

Main Methods:

• Combined single-molecule Förster Resonance Energy Transfer (smFRET) microscopy and single-particle tracking (SPT).
• Utilized fluorophore-labeled internalin B (InlB) as the MET ligand.
• Analyzed smFRET trajectories to extract diffusion coefficients and complex lifetimes.

Main Results:

• Determined the lifetime of the ligand-activated dimeric MET:InlB receptor complex to be approximately 1 second.
• Extracted diffusion coefficients showing dimeric MET:InlB complexes diffuse ~1.6 times slower than monomeric receptors.
• Observed spatially confined motion for dimeric receptor complexes.

Conclusions:

• The combined smFRET and SPT approach provides critical biophysical insights into membrane receptor activation dynamics.
• Quantified the changes in diffusion and complex stability associated with MET receptor dimerization and activation.
• Offers a powerful methodology for studying other receptor-ligand interactions in living cells.