FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells

Srinjan Basu,Steven F Lee,Olga Tkachenko,Brian Hendrich,B Christoffer Lagerholm,Lawrence E Bates,Christian Eggeling,Ernest D Laue,Lisa-Maria Needham,David Lando,Julie Cramard,Wayne Boucher,Yi Lei Tan,Edward J R Taylor,Kai J Wohlfahrt,Devina Shah,Dave Klenerman

doi:10.1038/s41467-018-04486-0

Abstract

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.

Highlights

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells
To investigate whether Förster resonance energy transfer (FRET) can be exploited to modulate the properties of existing PM fluorophores, we explored a number of potential donor–acceptor pairs
We explored a number of potential FRET pairs such as PA-EGFP-mCherry and PS-CFP2-YPet, but these were unsuccessful due to problems of poor acceptor photostability, folding inefficiencies, tendencies to multimerize, high levels of pre-conversion, and limited donor brightness for live-cell single-molecule tracking

Summary

Introduction

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. We were able to modify the excited-state kinetics of the donor PM fluorophore and selectively tune photophysical properties such as the fluorescence lifetime[23] and photostability—by providing additional energetic pathways for return to the ground state instead of photobleaching This allowed us to retain the properties of specific PM fluorophores for single-molecule imaging and to exploit a known approach for stabilizing dye molecules to improve the photostability and photon budget of two PM fluorophores, photo-convertible mEos3.215 and photo-activatable dye PAJF54917. We used this approach to track proteins and complexes for substantially longer in live mammalian cells, by either labeling a protein with both the PM donor fluorophore and the acceptor dye, or by labeling one protein with the PM donor and its interaction partner with the acceptor dye

Methods

Results

Conclusion