A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice

Yi, Bumjun; Kang, Bok Eum; Lee, Sungmoo; Braubach, Sophie; Baker, Bradley J.

doi:10.1038/s41598-018-33297-y

Cited by 21 publications

(31 citation statements)

References 32 publications

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“…However, the resulting GEVI Ilom shows similar performance as GEVIs based on tandem dimer tdTomato described here. In addition, similar to our results introduction of charged outward oriented residues on the FP surface failed to increase the dynamic range of Ilom sensitivity 45 .…”

Section: Discussionsupporting

confidence: 91%

“…Unlike with the GFP-based GEVIs, we showed that combinatorial effect of mutations of these residues lowers the voltage sensitivity compared to GEVI based on native FPs. In a recent study, the potential of dimerization of the FP for GEVI development was explored using red-shifted dimer FP dTomato 45 . However, the resulting GEVI Ilom shows similar performance as GEVIs based on tandem dimer tdTomato described here.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, mutagenesis of the residues surrounding the chromophore affected the direction of the voltage response in the GFP-based GEVIs (depolarization dependent increase vs. decrease in fluorescence intensity) 19 . Recently, this approach was implemented on the red-shifted GEVI Ilom which is based on the dimer FP dTomato 45 . The introduction of charged residues at the surface of the β barrel of dTomato failed to improve the voltage sensitivity.…”

Section: Functional Characterization Of Potential Gevis-mentioning

confidence: 99%

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Different categories of fluorescent proteins result in GEVIs with similar characteristics

Platisa

Zhou

Pieribone

2020

Preprint

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The latest generation of genetically encoded voltage indicators (GEVIs) is significantly advancing our ability to study electrical activity from large numbers of identified neurons. The further refinement of the technology will contribute to our understanding of behavior-evoked information perception, transfer and processing on a cellular level across brain regions. The development of GEVIs relies on synthetic biology which includes rational and random modifications of indicator sequence. One strategy in GEVI design is based on creating chimeras between voltage sensitive protein domains (VSDs) and fluorescent proteins (FPs). However, in this design scenario, the mechanistic details of voltage-induced fluorescence change that would inform rational design and improvements of GEVIs are still largely missing. Here we preformed a systematic study of how nature of the FP and altering the insertion site affects the characteristics of Ciona intestinalis voltage-sensitive phosphatase-based GEVIs. Surprisingly, we found that regardless of vast difference in phylogenesis, biochemical properties, fluorophore structure, sequence and excitation/emission spectra between FPs, the resulting GEVIs exhibit virtually identical decrease in fluorescence intensity in response to depolarization. These results stand in strong contrast to studies demonstrating that small numbers of targeted mutations in the FP sequence cause dramatic changes in both signal size and polarity.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Functional Characterization Of Potential Gevis-mentioning

confidence: 99%

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Different categories of fluorescent proteins result in GEVIs with similar characteristics

Platisa

Zhou

Pieribone

2020

Preprint

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“…Tracking membrane potential of cells, especially neurons, using fluorescence methods is of high interest and is an active field of research (change of membrane voltage causes change of fluorescence efficiency) [1][2][3][4][5][6][7][8][9][10]. To determine membrane voltage, a variety of voltage sensitive dyes [11][12][13], genetically encoded calcium indicators (GECI) [4,14,15], and genetically encoded voltage indicators (GEVI) based on voltage sensing domains (VSD, composed of four trans-membrane helices and fused fluorescent proteins) [16][17][18][19][20] and on microbial rhodopsins (composed of seven trans-membrane α-helices with covalently bound retinal, using the intrinsic fluorescence of retinal [9,10,[21][22][23] or the modified fluorescence from attached fluorescent proteins [19,23,24] or dyes [12]) are in use. Often, Förster-type resonance energy transfer (FRET) is involved in dye or fluorescent protein connection to VSDs and rhodopins [12,25].…”

Section: Introductionmentioning

confidence: 99%

Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

Penzkofer

Silapetere

Hegemann

2019

IJMS

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The retinal photocycle dynamics of the fluorescent voltage sensor QuasAr1 (Archaerhodopsin 3 P60S-T80S-D95H-D106H-F161V mutant from Halorubrum sodomense) in pH 8 Tris buffer was studied. The samples were photoexcited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_580 (absorption maximum at λmax ≈ 580 nm), and the retinal Schiff base photoisomerization and protonation state changes were followed by absorption spectra recordings during light exposure and after light exposure. Ret_580 turned out to be composed of two protonated retinal Schiff base isomers, namely Ret_580I and Ret_580II. Photoexcitation of Ret_580I resulted in barrier-involved isomerization to Ret_540 (quantum yield ≈ 0.056) and subsequent retinal proton release leading to Ret_410 deprotonated retinal Schiff base (RSB). In the dark, Ret_410 partially recovered to Ret_580I and partially stabilized to irreversible Ret_400 due to apoprotein restructuring (Ret_410 lifetime ≈ 2 h). Photoexcitation of Ret_580II resulted in barrier-involved isomerization to Ret_640 (quantum yield ≈ 0.00135) and subsequent deprotonation to Ret_370 (RSB). In the dark, Ret_370 partially recovered to Ret_580II and partially stabilized to irreversible Ret_350 due to apoprotein restructuring (Ret_370 lifetime ≈ 10 h). Photocycle schemes and reaction coordinate diagrams for Ret_580I and Ret_580II were developed and photocyle parameters were determined.

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“…Though the construct showed some weak signals (see SI) it did not show a large change in the light emission as observed in the FRP-VSD-eluxAB-YPet. Dimerization of the acceptor fluorescent protein causes voltage dependent FRET in fluorescent GEVIs [67]. We ruled this mechanism using a dark mutant of YPet that showed voltage dependent bioluminescent signal but no acceptor resonance energy transfer.…”

Section: Discussionmentioning

confidence: 99%

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Srinivasan

Griffin

Joshi

et al. 2019

Preprint

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1.AbstractGenetically encoded reporters have greatly increased our understanding of biology, especially in neuroscience. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach we have engineered Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential. Unlike other luciferase-luciferin bioluminescent systems, AMBER encodes the genes to express both the luciferase and luciferin. AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When AMBER is co-expressed with the luciferin producing genes it reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods we show that AMBER modulates its enzymatic activity as a function of the membrane potential. AMBER shows several-fold increase in the luminescent (ΔL/L) signal upon switching from the off to on state when the cell is depolarized. In vivo expression of AMBER in C. elegans allowed detecting pharyngeal pumping action and mechanosensory neural activity from multiple worms simultaneously. AMBER reports neural activity of multiple animals at the same time and can be used in social behavior assays to elucidate the role of membrane potential underlying behavior.2.Significance StatementThere have been many exciting advances in the development of genetically encoded voltage indicators to monitor intracelluar voltage changes. Most sensors employ fluorescence, which requires external light, potentially causing photobleaching or overheating. Consequently, there has been interest in developing luminescence reporters. However, they require addition of an exogenous substrate to produce light intracellularly. Here, we engineered a genetically encoded bioluminescent voltage indicator, AMBER, which unlike other bioluminescent activity indicators, does not require addition of an exogenous substrate. AMBER allows a large differential signal, a high signal-to-noise ratio, and causes minimal metabolic demand on cells. We used AMBER to record voltage activity in freely-moving C. elegans, demonstrating that AMBER is a important new tool for monitoring neuronal activity during social behavior.

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A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice

Cited by 21 publications

References 32 publications

Different categories of fluorescent proteins result in GEVIs with similar characteristics

Different categories of fluorescent proteins result in GEVIs with similar characteristics

Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

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