Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments

Martin, K. J., McGhee, E. J., Schwarz, J. P., Drysdale, M., Brachmann, S. M., Stucke, V., Sansom, O. J. and Anderson, K. I. (2018) Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments. PLoS ONE, 13(1), e0183585. (doi: 10.1371/journal.pone.0183585) (PMID:29293509) (PMCID:PMC5749721)

Abstract

FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells. Most biosensors designed to date have been predicated on fluorescent protein pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET. Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last decade. Lifetime imaging systems have become increasingly accessible and user-friendly, and there is an entire field of biology dedicated to refining and adapting different characteristics of existing and novel fluorescent proteins. This growing pool of fluorescent proteins includes the long-lifetime green and cyan fluorescent proteins Clover and mTurquoise2, the red variant mRuby2, and the dark acceptor sREACh. Here, we have tested these donors and acceptors in appropriate combinations against the standard or recommended norms (EGFP and mTFP as donors, mCherry and either Ypet or Venus as acceptors) to determine if they could provide more reliable, reproducible and quantifiable FLIM-FRET data to improve on the dynamic range compared to other donors and breadth of application of biosensor technologies. These tests were performed for comparison on both a wide-field, frequency domain system and a multiphoton, TCSPC time domain FLIM system. Clover proved to be an excellent donor with extended dynamic range in combination with mCherry on both platforms, while mRuby2 showed a high degree of variability and poor FRET efficiencies in all cases. mTFP-Venus was the most consistent cyan-yellow pair between the two FLIM methodologies, but mTurquoise2 has better dynamic range and transfers energy consistently over time to the dark acceptor sRCh. Combination of mTFP-sRCh with Clover-mCherry would allow the simultaneous use of two FLIM-FRET biosensors within one sample by eliminating the crosstalk between the yellow acceptor and green donor emissions.

• Item Type: Articles
• Additional Information: EJM and MD were funded by Cancer Research UK (http://www.cancerresearchuk.org/, core grant C596/A17196). KJM, SMB, VS, OJS, and KIA were funded by Novartis Pharma AG (http://www.novartis.com).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Drysdale, Professor Martin and Schwarz, Ms Juliana and Anderson, Professor Kurt and McGhee, Dr Ewan and Sansom, Professor Owen

Creator Roles

McGhee, E. J.: Formal analysis, Investigation
Schwarz, J. P.: Investigation, Resources
Drysdale, M.: Conceptualization, Funding acquisition
Sansom, O. J.: Conceptualization, Funding acquisition
Anderson, K. I.: Conceptualization, Funding acquisition, Supervision, Writing – review and editing

• Authors: Martin, K. J., McGhee, E. J., Schwarz, J. P., Drysdale, M., Brachmann, S. M., Stucke, V., Sansom, O. J., and Anderson, K. I.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• ISSN (Online): 1932-6203
• Copyright Holders: Copyright © 2018 Martin et al.
• First Published: First published in PLoS ONE 13(1): e0183585
• Publisher Policy: Reproduced under a Creative Commons License