Long-Term Single-Molecule Tracking in Living Cells using Weak-Affinity Protein Labeling.

Area of Science:

• Cellular dynamics
• Biophysics
• Molecular imaging

Background:

• Single-particle tracking (SPT) is crucial for observing cellular protein dynamics.
• Permanent labeling in SPT is limited by photobleaching, hindering long-term studies.
• Developing new labeling strategies is essential for extended live-cell imaging.

Purpose of the Study:

• To introduce exchangeable HaloTag Ligands (xHTLs) as a novel labeling approach for live-cell SPT.
• To overcome photobleaching limitations and enable extended observation times.
• To investigate the dynamics and activation of membrane proteins, such as the epidermal growth factor receptor (EGFR), in living cells.

Main Methods:

• Utilized exchangeable HaloTag Ligands (xHTLs) for labeling proteins in live cells.
• Performed single-particle tracking (SPT) to monitor protein mobility over extended periods (up to 30 minutes).
• Applied the xHTL/HaloTag7 system to study EGFR activation kinetics and ER-luminal protein diffusion.

Main Results:

• Achieved observation times of up to 30 minutes, significantly reducing photobleaching effects.
• Generated spatial maps of EGFR diffusion, revealing non-uniform mobility and localized 'hot spots' of activation.
• Measured ER-luminal protein diffusion coefficients that correlated with the endoplasmic reticulum (ER) nanostructure.

Conclusions:

• Exchangeable HaloTag Ligands (xHTLs) provide a powerful and generalizable strategy for extended live-cell SPT.
• This approach facilitates the study of protein dynamics and functional contexts in single living cells.
• The method allows for detailed investigation of membrane protein activation and intracellular transport mechanisms.