Journal article
FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells
- 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.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
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(Preview, Version of record, pdf, 3.2MB, Terms of use)
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- Publisher copy:
- 10.1038/s41467-018-04486-0
Authors
- Publisher:
- Nature Publishing Group
- Journal:
- Nature Communications More from this journal
- Volume:
- 9
- Issue:
- 1
- Pages:
- 2520
- Publication date:
- 2018-06-28
- Acceptance date:
- 2018-04-27
- DOI:
- EISSN:
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2041-1723
- ISSN:
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2041-1723
- Pmid:
-
29955052
- Language:
-
English
- Keywords:
- Pubs id:
-
pubs:867703
- UUID:
-
uuid:1dac116a-c4c7-4329-9404-901dcf132b9a
- Local pid:
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pubs:867703
- Source identifiers:
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867703
- Deposit date:
-
2018-08-06
Terms of use
- Copyright holder:
- Eggeling et al
- Copyright date:
- 2018
- Notes:
- © The Author(s) 2018. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
- Licence:
- CC Attribution (CC BY)
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