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

Abstract A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. Fo...

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Autores principales:	Bumjun Yi, Bok Eum Kang, Sungmoo Lee, Sophie Braubach, Bradley J. Baker
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Genetically Encoded Voltage Indicators (GEVI) Voltage-sensing Domain (VSD) tdTomato Small Fractional Change Acute Brain Slices Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6c4f6b7bf7454043ad72e21299734f5c
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spelling	oai:doaj.org-article:6c4f6b7bf7454043ad72e21299734f5c2021-12-02T15:09:05ZA dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice10.1038/s41598-018-33297-y2045-2322https://doaj.org/article/6c4f6b7bf7454043ad72e21299734f5c2018-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-33297-yhttps://doaj.org/toc/2045-2322Abstract A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. For red-shifted GEVI development, tdTomato was split fusing a single dTomato chromophore to the voltage sensing domain. Optimization of the amino acid length and charge composition of the linker region between the voltage sensing domain and the fluorescent protein resulted in a probe that is an order of magnitude brighter than FlicR1 at a resting potential of −70 mV and exhibits a ten-fold larger change in fluorescence (ΔF) upon 100 mV depolarization of the plasma membrane in HEK 293 cells. Unlike ArcLight, the introduction of charged residues to the exterior of dTomato did not substantially improve the dynamic range of the optical signal. As a result, this new GEVI, Ilmol, yields a 3-fold improvement in the signal-to-noise ratio compared to FlicR1 despite a smaller fractional change in fluorescence of 4% per 100 mV depolarization of the plasma membrane. Ilmol expresses well in neurons resolving action potentials in neuronal cultures and reporting population signals in mouse hippocampal acute brain slice recordings. Ilmol is the brightest red-shifted GEVI to date enabling imaging with 160-fold less light than Archon1 for primary neuron recordings (50 mW/cm2 versus 8 W/cm2) and 600-fold less light than QuasAr2 for mouse brain slice recordings (500 mW/cm2 versus 300 W/cm2). This new GEVI uses a distinct mechanism from other approaches, opening an alternate engineering path to improve sensitivity and speed.Bumjun YiBok Eum KangSungmoo LeeSophie BraubachBradley J. BakerNature PortfolioarticleGenetically Encoded Voltage Indicators (GEVI)Voltage-sensing Domain (VSD)tdTomatoSmall Fractional ChangeAcute Brain SlicesMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-14 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Genetically Encoded Voltage Indicators (GEVI) Voltage-sensing Domain (VSD) tdTomato Small Fractional Change Acute Brain Slices Medicine R Science Q
spellingShingle	Genetically Encoded Voltage Indicators (GEVI) Voltage-sensing Domain (VSD) tdTomato Small Fractional Change Acute Brain Slices Medicine R Science Q Bumjun Yi Bok Eum Kang Sungmoo Lee Sophie Braubach Bradley J. Baker A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
description	Abstract A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. For red-shifted GEVI development, tdTomato was split fusing a single dTomato chromophore to the voltage sensing domain. Optimization of the amino acid length and charge composition of the linker region between the voltage sensing domain and the fluorescent protein resulted in a probe that is an order of magnitude brighter than FlicR1 at a resting potential of −70 mV and exhibits a ten-fold larger change in fluorescence (ΔF) upon 100 mV depolarization of the plasma membrane in HEK 293 cells. Unlike ArcLight, the introduction of charged residues to the exterior of dTomato did not substantially improve the dynamic range of the optical signal. As a result, this new GEVI, Ilmol, yields a 3-fold improvement in the signal-to-noise ratio compared to FlicR1 despite a smaller fractional change in fluorescence of 4% per 100 mV depolarization of the plasma membrane. Ilmol expresses well in neurons resolving action potentials in neuronal cultures and reporting population signals in mouse hippocampal acute brain slice recordings. Ilmol is the brightest red-shifted GEVI to date enabling imaging with 160-fold less light than Archon1 for primary neuron recordings (50 mW/cm2 versus 8 W/cm2) and 600-fold less light than QuasAr2 for mouse brain slice recordings (500 mW/cm2 versus 300 W/cm2). This new GEVI uses a distinct mechanism from other approaches, opening an alternate engineering path to improve sensitivity and speed.
format	article
author	Bumjun Yi Bok Eum Kang Sungmoo Lee Sophie Braubach Bradley J. Baker
author_facet	Bumjun Yi Bok Eum Kang Sungmoo Lee Sophie Braubach Bradley J. Baker
author_sort	Bumjun Yi
title	A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
title_short	A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
title_full	A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
title_fullStr	A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
title_full_unstemmed	A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
title_sort	dimeric fluorescent protein yields a bright, red-shifted gevi capable of population signals in brain slice
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/6c4f6b7bf7454043ad72e21299734f5c
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A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice

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