Expression and testing in plants of ArcLight, a genetically–encoded voltage indicator used in neuroscience researchReport as inadecuate




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BMC Plant Biology

, 15:245

Biochemistry and physiology

Abstract

BackgroundIt is increasingly appreciated that electrical controls acting at the cellular and supra-cellular levels influence development and initiate rapid responses to environmental cues. An emerging method for non-invasive optical imaging of electrical activity at cell membranes uses genetically-encoded voltage indicators GEVIs. Developed by neuroscientists to chart neuronal circuits in animals, GEVIs comprise a fluorescent protein that is fused to a voltage-sensing domain. One well-known GEVI, ArcLight, undergoes strong shifts in fluorescence intensity in response to voltage changes in mammalian cells. ArcLight consists of super-ecliptic SE pHluorin pH -sensitive fluor escent protein with an A227D substitution, which confers voltage sensitivity in neurons, fused to the v oltage-s ensing d omain of the voltage-sensing phosphatase of C ionai ntestinalis Ci-VSD. In an ongoing effort to adapt tools of optical electrophysiology for plants, we describe here the expression and testing of ArcLight and various derivatives in different membranes of root cells in Arabidopsis thaliana.

ResultsTransgenic constructs were designed to express ArcLight and various derivatives targeted to the plasma membrane and nuclear membranes of Arabidopsis root cells. In transgenic seedlings, changes in fluorescence intensity of these reporter proteins following extracellular ATP eATP application were monitored using a fluorescence microscope equipped with a high speed camera. Coordinate reductions in fluorescence intensity of ArcLight and Ci-VSD-containing derivatives were observed at both the plasma membrane and nuclear membranes following eATP treatments. However, similar responses were observed for derivatives lacking the Ci-VSD. The dispensability of the Ci-VSD suggests that in plants, where H ions contribute substantially to electrical activities, the voltage-sensing ability of ArcLight is subordinate to the pH sensitivity of its SEpHluorin base. The transient reduction of ArcLight fluorescence triggered by eATP most likely reflects changes in pH and not membrane voltage.

ConclusionsThe pH sensitivity of ArcLight precludes its use as a direct sensor of membrane voltage in plants. Nevertheless, ArcLight and derivatives situated in the plasma membrane and nuclear membranes may offer robust, fluorescence intensity-based pH indicators for monitoring concurrent changes in pH at these discrete membrane systems. Such tools will assist analyses of pH as a signal and-or messenger at the cell surface and the nuclear periphery in living plants.

KeywordsArcLight Electrical signalling Genetically-encoded voltage indicator pH-sensitive indicator Super ecliptic pHluorin AbbreviationsArcLightSEpHluorinA227D fused to Ci-VSD

Case12Calcium sensor 12

CBL1CALCINEURIN B-LIKE PROTEIN 1. The Arabidopsis CBL1 protein At4g17615 contains a myristolated glycine and a palmitolated cysteine, which tether the fluorescent fusion protein to the cytoplasmic surface of the plasma membrane

Ci-VSDvoltage sensing domain of Ciona intestinalis voltage-sensing phosphatase

eATPextracellular ATP

FRETFörster resonance energy transfer

GEVIgenetically-encoded voltage indicator

GSTglutathione S-transferase

ITMVinduced transmembrane voltage

mCitrinemonomeric citrine

RANGAP1RAN GTPASE ACTIVATING PROTEIN 1

SEpHluorinsuper-eclipticSE pHluorin pH -sensitive fluor escent protein

SEpHluorinA227Dsuper-eclipticSE pHluorin pH -sensitive fluor escent protein containing an A227D substitution that confers voltage sensitivity in neurons

SUN2SAD1-UNC-84 DOMAIN PROTEIN 2. The Arabidopsis SUN2 protein At3g10730 has one transmembrane domain that can localize SUN2-fusion proteins at the inner nuclear membrane surface

WPPThe WPP sequence contains a TrpW-ProP-Pro motif consists of amino acids 28–131 of Arabidopsis RANGAP1 At3g63130 that is sufficient for targeting fusion proteins to the outer nuclear membrane

Electronic supplementary materialThe online version of this article doi:10.1186-s12870-015-0633-z contains supplementary material, which is available to authorized users.

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Author: Antonius J.M. Matzke - Marjori Matzke

Source: https://link.springer.com/



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