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

Catapano, Claudia; Dietz, Marina S.; Kompa, Julian; Jang, Soohyen; Freund, Petra; Johnsson, Kai; Heilemann, Mike

Single‐particle tracking (SPT) has become a powerful tool to monitor the dynamics of membrane proteins in living cells. However, permanent labeling strategies for SPT suffer from photobleaching as a major limitation, restricting observation times, and obstructing the study of long‐term cellular processes within single living cells. Here, we use exchangeable HaloTag Ligands (xHTLs) as an easy‐to‐apply labeling approach for live‐cell SPT and demonstrate extended observation times of individual living cells of up to 30 minutes. Using the xHTL/HaloTag7 labeling system, we measure the ligand‐induced activation kinetics of the epidermal growth factor receptor (EGFR) in single living cells. We generate spatial maps of receptor diffusion in cells, report non‐uniform distributions of receptor mobility, and the formation of spatially confined 'hot spots' of EGFR activation. Furthermore, we measured the mobility of an ER‐luminal protein in living cells and found diffusion coefficients that correlated with the ER nano‐structure. This approach represents a general strategy to monitor protein mobility in a functional context and for extended observation times in single living cells.

MEMBRANE proteins; EPIDERMAL growth factor receptors; CELL receptors; PROTEIN receptors; DIFFUSION coefficients

Angewandte Chemie, 2025, Vol 137, Issue 1, p1

0044-8249

Academic Journal

10.1002/ange.202413117