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

Srinjan Basu, Lisa-Maria Needham, David Lando, Edward J. R. Taylor, Kai J. Wohlfahrt, Devina Shah, Wayne Boucher, Yi Lei Tan, Lawrence E. Bates, Olga Tkachenko, Julie Cramard, B. Christoffer Lagerholm, Christian Eggeling, Brian Hendrich, Dave Klenerman (), Steven F. Lee () and Ernest D. Laue ()
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Srinjan Basu: University of Cambridge
Lisa-Maria Needham: University of Cambridge
David Lando: University of Cambridge
Edward J. R. Taylor: University of Cambridge
Kai J. Wohlfahrt: University of Cambridge
Devina Shah: University of Cambridge
Wayne Boucher: University of Cambridge
Yi Lei Tan: University of Cambridge
Lawrence E. Bates: University of Cambridge
Olga Tkachenko: University of Cambridge
Julie Cramard: Wellcome Trust – MRC Stem Cell Institute, University of Cambridge
B. Christoffer Lagerholm: Weatherall Institute of Molecular Medicine, University of Oxford
Christian Eggeling: Weatherall Institute of Molecular Medicine, University of Oxford
Brian Hendrich: University of Cambridge
Dave Klenerman: University of Cambridge
Steven F. Lee: University of Cambridge
Ernest D. Laue: University of Cambridge

Nature Communications, 2018, vol. 9, issue 1, 1-14

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-04486-0

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