Dataset Information

Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments.

ABSTRACT: Receptor-like kinases (RLK) and receptor-like proteins (RLP) often interact in a combinatorial manner depending on tissue identity, membrane domains, or endo- and exogenous cues, and the same RLKs or RLPs can generate different signaling outputs depending on the composition of the receptor complexes they are involved in. Investigation of their interaction partners in a spatial and dynamic way is therefore of prime interest to understand their functions. This is, however, limited by the technical complexity of assessing it in endogenous conditions. A solution to close this gap is to determine protein interaction directly in the relevant tissues at endogenous expression levels using Förster resonance energy transfer (FRET). The ideal fluorophore pair for FRET must, however, fulfil specific requirements: (a) The emission and excitation spectra of the donor and acceptor, respectively, must overlap; (b) they should not interfere with proper folding, activity, or localization of the fusion proteins; (c) they should be sufficiently photostable in plant cells. Furthermore, the donor must yield sufficient photon counts at near-endogenous protein expression levels. Although many fluorescent proteins were reported to be suitable for FRET experiments, only a handful were already described for applications in plants. Herein, we compare a range of fluorophores, assess their usability to study RLK interactions by FRET-based fluorescence lifetime imaging (FLIM) and explore their differences in FRET efficiency. Our analysis will help to select the optimal fluorophore pair for diverse FRET applications.

SUBMITTER: Denay G

PROVIDER: S-EPMC6898725 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments.

Denay Grégoire G Schultz Patrick P Hänsch Sebastian S Weidtkamp-Peters Stefanie S Simon Rüdiger R

Plant direct 20191206 12

Receptor-like kinases (RLK) and receptor-like proteins (RLP) often interact in a combinatorial manner depending on tissue identity, membrane domains, or endo- and exogenous cues, and the same RLKs or RLPs can generate different signaling outputs depending on the composition of the receptor complexes they are involved in. Investigation of their interaction partners in a spatial and dynamic way is therefore of prime interest to understand their functions. This is, however, limited by the technical ...[more]

PMID: 31844834

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Project description: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.

Dataset Information

Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments.

Publications

Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments.

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