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

Author

Listed:
  • 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
    Wellcome Trust – MRC Stem Cell Institute, University of Cambridge)

  • Dave Klenerman

    (University of Cambridge)

  • Steven F. Lee

    (University of Cambridge)

  • Ernest D. Laue

    (University of Cambridge)

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.

Suggested Citation

  • 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. Christoffe, 2018. "FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells," Nature Communications, Nature, vol. 9(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-04486-0
    DOI: 10.1038/s41467-018-04486-0
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