Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions

Niederauer, Christian; Nguyen, Chikim; Wang-Henderson, Miles; Stein, Jeremy C.; Strauss, Sebastian; Cumberworth, Alexander; Stehr, Florian; Jungmann, Ralf; Schwille, Petra; Ganzinger, Kristina A.

doi:10.1038/s41467-023-40065-8

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

Catapano,

Dietz,

Kompa

et al. 2024

Angewandte Chemie

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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/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 activation, and the formation of spatially confined ‘hot spots’ of EGFR activation. Furthermore, we measured the mobility of the ER‐luminal protein calreticulin in living cells and found diffusion coefficients that correlated with the ER nano‐structure. 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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Long‐Term Single‐Molecule Tracking in Living Cells using Weak‐Affinity Protein Labeling

Catapano,

Dietz,

Kompa

et al. 2024

Angew Chem Int Ed

0

View full text Add to dashboard Cite

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/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 activation, and the formation of spatially confined ‘hot spots’ of EGFR activation. Furthermore, we measured the mobility of the ER‐luminal protein calreticulin in living cells and found diffusion coefficients that correlated with the ER nano‐structure. 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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Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions

Niederauer

¹

,

Nguyen

²

,

Wang-Henderson

³

et al. 2023

Nat Commun

Self Cite

9

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DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) has recently significantly enhanced observation times in in vitro SPT experiments by overcoming the constraints of fluorophore photobleaching. However, with the reported implementation, only a single target can be imaged and the technique cannot be applied straight to live cell imaging. Here we report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-color DNA-PAINT-SPT for quantifying protein dimerization and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

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Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions

Cited by 9 publications

References 49 publications

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

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

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

Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions

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