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bioRxiv · 10.1101/2024.07.11.603077

Long-term single-molecule tracking in living cells using weak-affinity protein labeling

Catapano, C.·Dietz, M. S.·Kompa, J.·Jang, S.·Freund, P.·Johnsson, K.·Heilemann, M.
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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 live cells of up to 30 minutes. Using the xHTL/HT7 labeling system, we measure the ligand-induced activation kinetics of the epidermal growth factor receptor (EGFR) in single living cells. Furthermore, we generate spatial maps of receptor diffusion in cells, report non-uniform distributions of receptor activation, and the formation of spatially confined hot spots of EGFR activation. This approach represents a general strategy to monitor protein dynamics in a functional context and for extended observation times in single living cells.

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Catapano, C., Dietz, M. S., Kompa, J., Jang, S., Freund, P., Johnsson, K., Heilemann, M.. 2024-07-13. Long-term single-molecule tracking in living cells using weak-affinity protein labeling. https://doi.org/10.1101/2024.07.11.603077

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