Superior robustness of ExEm‐spFRET to IIem‐spFRET method in live‐cell FRET measurement

Zhang, C.; Du, Mingde; Wang, L.; Mai, Zihao; Chen, T.

doi:10.1111/jmi.12755

Cited by 7 publications

(7 citation statements)

References 20 publications

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“…Fluorescence resonance energy transfer (FRET) analysis was performed on a user‐built microscope–spectrometer (MSM) platform that consisted of a widefield fluorescence microscope (Axio Observer, Carl Zeiss, Oberkochen, Germany) with a ×40/1.3 NA oil objective and a miniature fibre optic spectrometer (QE65 Pro, Ocean Optics, FL, USA). FRET efficiency was quantified according to the IIem‐spFRET measurement method (Zhang et al ., 2016; Lin et al ., 2018). At least 15 individual protoplasts were used for FRET efficiency quantification and statistical analysis.…”

Section: Methodsmentioning

confidence: 99%

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A plant‐unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth

Liu

Zeng

et al. 2021

New Phytologist

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During evolution, land plants generated unique proteins that participate in endosomal sorting and multivesicular endosome (MVE) biogenesis, many of them with specific phosphoinositide-binding capabilities. Nonetheless, the function of most plant phosphoinositide-binding proteins in endosomal trafficking remains elusive.Here, we analysed several Arabidopsis mutants lacking predicted phosphoinositide-binding proteins and first identified fyve4-1 as a mutant with a hypersensitive response to high-boron conditions and defects in degradative vacuolar sorting of membrane proteins such as the borate exporter BOR1-GFP.FYVE4 encodes a plant-unique, FYVE domain-containing protein that interacts with SNF7, a core component of ESCRT-III (Endosomal Sorting Complex Required for Transport III). FYVE4 affects the membrane association of the late-acting ESCRT components SNF7 and VPS4, and modulates the formation of intraluminal vesicles (ILVs) inside MVEs. The critical function of FYVE4 in the ESCRT pathway was further demonstrated by the strong genetic interactions with SNF7B and LIP5. Although the fyve4-1, snf7b and lip5 single mutants were viable, the fyve4-1 snf7b and fyve4-1 lip5 double mutants were seedling lethal, with strong defects in MVE biogenesis and vacuolar sorting of ubiquitinated membrane proteins.Taken together, we identified FYVE4 as a novel plant endosomal regulator, which functions in ESCRTing pathway to regulate MVE biogenesis.

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Section: Methodsmentioning

confidence: 99%

“…4c). When performing FRET analysis based on the IIem-spFRET method (Zhang et al, 2016;Lin et al, 2018), both SNF7A and SNF7B showed interactions with FYVE4 on these puncta (Fig. 4d).…”

Section: Fyve4 Associates With the Escrt-iii Complexmentioning

confidence: 99%

A plant‐unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth

Liu

Zeng

et al. 2021

New Phytologist

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“…All raw scans used for spectral FRET are available in S2 Fig. Once the contributing weight of donor and acceptor was determined, FRET efficiency was estimated using the following equation[27]: where E is the FRET efficiency, W A and W D are the component weights of the donor only and acceptor only emissions calculated through linear unmixing, and QY A and QY D is the quantum yield of the acceptor and donor respectively.…”

Section: Methodsmentioning

confidence: 99%

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

McCullock

MacLean

Kammermeier

2019

Preprint

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19 Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to 20 profile intra-and inter-molecular interactions. Through fusion of genetically encoded 21 fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, 22 receptor activation, and protein translocation among other biophysical phenomena.23 Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have 24 been developed. These proteins offer much improved physical properties compared to 25 previous generations of monomeric red FPs that should help facilitate more general 26 adoption of Green/Red FRET. Here we assess the ability of these two proteins, along 27 with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP 28 mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging 29 Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a 30 stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We 31 then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP 32 tandem construct. With these constructs we found that mScarlet-I and mCherry are able 33 to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, 34 mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these 35 same assays. We explore possible explanations for this poor performance but are 36 unable to definitively determine the cause, all though protein maturation seems to play a 37 role. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry 38 and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with mScarlet-I out 39 performing mCherry due it's higher brightness. 40 41 Introduction 42 Genetically encoded Fluorescent Proteins (FPs) have advanced basic and translational 43 biology immensely. Starting with the cloning of the Aequorea victoria green FP[1], a 44 massive and continual effort to expand the number of available FPs with a different 45 physical and spectral properties began. Currently, there is an enormous variety of FPs 46 at all parts of the visible spectrum, and even some parts of the ultraviolet and infrared 47 spectrums. These new proteins were either cloned from other organisms[2-4], or48 developed through evolution of already identified FPs [4][5][6][7][8][9][10][11]. This ever expanding 49 catalog of FPs has been reviewed by others [12][13][14], and new efforts and archives have 50 been created to organize this information, such as the FPbase database [15]. 51As the catalogue of FPs has expanded, so have the potential uses. Of particular 52 note is the use of FPs as biosensors which can measure signaling events, cell 53 metabolites, pH, voltage and more [16]. Many of these biosensors employ Förster 54 Resonance Energy Transfer (FRET) as part of their reporting mechanism, producing a 55 change in acceptor emission upon donor excitation when the quantity of interest 56 changes. FRET is also orientation and distance dep...

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“…For each of the acceptors, custom acceptor only scans were created based on experimentally collected data and their reported purified spectrum (as discussed in S1 Fig). All raw scans used for spectral FRET are available in S1 Fig. Once the contributing weight of donor and acceptor was determined, FRET efficiency was estimated using the following equation [27]:…”

Section: Spectral Fretmentioning

confidence: 99%

Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

2020

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Fö rster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra-and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance and determine mRuby3's protein maturation properties are a major contributor. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with the brighter mScarlet-I out performing mCherry in intensiometric studies, but mCherry out performing mScarlet-I in instances where consistent efficiency in a population is critical.

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Superior robustness of ExEm‐spFRET to IIem‐spFRET method in live‐cell FRET measurement

Cited by 7 publications

References 20 publications

A plant‐unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth

A plant‐unique ESCRT component, FYVE4, regulates multivesicular endosome biogenesis and plant growth

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

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