Measuring Membrane Voltage with Fluorescent Proteins

Patti, Jordan; Isacoff, Ehud Y.

doi:10.1101/pdb.top075804

Cited by 6 publications

(10 citation statements)

References 37 publications

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“…Together with classical methods for monitoring membrane potentials such as microelectrodes and patch clamp, a new generation of electrophysiological tools is being developed based on the concept of light-based or optical electrophysiology [ 4 , 5 ]. An important group of these new tools consists of genetically-encoded, protein-based voltage indicators [ 6 – 8 ].…”

Section: Introductionmentioning

confidence: 99%

“…Genetically-encoded voltage indicators (GEVIs) are composed of a fusion between a fluorescent protein (reporter) and a voltage-sensing domain (detector) [ 8 ]. GEVIs have been developed by neurobiologists over the last two decades as a non-invasive method to optically monitor changes in transmembrane potential in single and multiple neurons and other cell types [ 6 – 9 ].…”

Section: Introductionmentioning

confidence: 99%

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

Matzke

2015

BMC Plant Biol

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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 fluorescent protein) with an A227D substitution, which confers voltage sensitivity in neurons, fused to the voltage-sensing domain of the voltage-sensing phosphatase of Cionaintestinalis (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.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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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Matzke

2015

BMC Plant Biol

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“…B. Na + , K + und Mg 2+ ) zu konzentrieren, könnte weniger intensiv verfolgten Bereichen größere Aufmerksamkeit gewidmet werden. Auf fluoreszierenden Proteinen basierende Sensoren konnten auf einfache Weise an der Membran angebracht werden und erwiesen sich als leistungsstarker Ansatz zur Bestimmung der Membranspannung und anderer Vorgänge , Anders als MiTAFPs und LyTAFPs, die sich frei innerhalb der Organellen ausbreiten, sind die meisten beschriebenen MemTAFPs in der Lipiddoppelschicht verankert, was zu einer lokalen Akkumulation von Fluorophoren führen könnte und deren optische Eigenschaften beeinflusst.…”

Section: Auf Membranen Zielende Aktivierbare Fluoreszenzsonden (Memtunclassified

Wahrnehmung der chemischen Prozesse in einzelnen Organellen mit niedermolekularen Fluoreszenzsonden

et al. 2016

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Ohne genau durchdachte Fluoreszenzmarker/‐sonden kann auch das fortschrittlichste hochauflösende Mikroskopieverfahren keinen Einblick in subzelluläre Matrizes geben. Die Entwicklung der Biologie wurde in zunehmendem Maße durch Fortschritte im Bereich der Chemie vorangetrieben. Ein prominentes Beispiel hierfür sind niedermolekulare Fluoreszenzsonden, die nicht nur die Bildgebung auf zellulärer Ebene ermöglichen, sondern auch subzelluläre Abbildungen. Die meisten chemischen/biologischen Ereignisse finden innerhalb von Zellorganellen statt, sodass diese Substrukturen zunehmend mithilfe von Fluoreszenzverfahren untersucht wurden. Dieser Aufsatz fasst die vorhandenen Fluoreszenzsonden zusammen, die auf chemische/biologische Geschehnisse innerhalb einzelner Organellen zielen. Darüber hinaus werden Strategien zur Verankerung an den Organellen vorgestellt, um das Design neuer Fluoreszenzsonden anzuregen. Abschließend werden die Zukunftsaussichten für weitere Entwicklungen in der chemischen Biologie diskutiert.

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“…Recently, researchers have been able to record activity of the brain of a zebrafish embryo in 80% of its 100,000 neurons [4]. There is an ever increasing set of tools to image the brain with various fluorescent reporters [5]. The rapid improvements of optogenetics methods for shutting down or switching on neurons by shining laser light already allows stimulating up to 1000 neurons (see Refs.…”

Section: Introductionmentioning

confidence: 99%

Design of the first neuronal connectomics challenge: From imaging to connectivity

Guyon¹,

Battaglia²,

Alice

et al. 2014

2014 International Joint Conference on Neural Networks (IJCNN)

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We are organizing a challenge to reverse engineer the structure of neuronal networks from patterns of activity recorded with calcium fluorescence imaging. Unraveling the brain structure at the neuronal level at a large scale is an important step in brain science, with many ramifications in the comprehension of animal and human intelligence and learning capabilities, as well as understanding and curing neuronal diseases and injuries. However, uncovering the anatomy of the brain by disentangling the neural wiring with its very fine and intertwined dendrites and axons, making both local and far reaching synapses, is a very arduous task: traditional methods of axonal tracing are tedious, difficult, and time consuming. This challenge proposes to approach the problem from a different angle, by reconstructing the effective connectivity of a neuronal network from observations of neuronal activity of thousands of neurons, which can be obtained with state-of-the-art fluorescence calcium imaging. To evaluate the effectiveness of proposed algorithms, we will use data obtained with a realistic simulator of real neurons for which we have ground truth of the neuronal connections. We produced simulated calcium imaging data, taking into account a model of fluorescence and light scattering. The task of the participants is to reconstruct a network of 1000 neurons from time series of neuronal activities obtained with this model. This challenge is part of the official selection of the WCCI 2014 competition program.

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Measuring Membrane Voltage with Fluorescent Proteins

Cited by 6 publications

References 37 publications

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

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

Wahrnehmung der chemischen Prozesse in einzelnen Organellen mit niedermolekularen Fluoreszenzsonden

Design of the first neuronal connectomics challenge: From imaging to connectivity

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