Abstract
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.