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

Angew Chem Int Ed Engl

Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.

Published: November 2024

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.

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Source
http://dx.doi.org/10.1002/anie.202413117DOI Listing

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