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 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.
Highlights
It is increasingly appreciated that electrical controls acting at the cellular and supra-cellular levels influence development and initiate rapid responses to environmental cues
The fluorescent proteins tested include: classic ArcLight (Fig. 1a), which - in the absence of any other membrane targeting sequence - is directed to the plasma membrane by the Ci-VSD (Fig. 1g, sector A); ArcLight joined at the N-terminus to the WPP domain of Arabidopsis RAN GTPASE ACTIVATING PROTEIN 1 (RANGAP1) (Fig. 1b) [22, 23], which promotes targeting to the outer nuclear membrane (Fig. 1g, sector B); and ArcLight fused at the N-terminus to the Arabidopsis SAD1/UNC-84 DOMAIN PROTEIN 2 (SUN2), which contains one transmembrane domain (Fig. 1c) and is able to target the protein to the inner nuclear membrane [24] (Fig. 1g, sector C)
We tested the importance of the transmembrane Ci-VSD in voltage-sensing by replacing it with either an Arabidopsis CALCINEURIN B-LIKE PROTEIN 1 (CBL1) plasma membrane targeting peptide [25] at the Nterminus (Fig. 1d), which situates the fluorescent reporter at the cytoplasmic surface of the plasma membrane (Fig. 1g, sector D); an N-terminal WPP domain (Fig. 1e), which places the fluorescent reporter at the cytoplasmic surface of the outer nuclear membrane (Fig. 1g, sector E); or an N-terminal fusion to inner nuclear membrane protein SUN2 (Fig. 1f ), which positions the fluorescent reporter in the perinuclear space (Fig. 1g, sector F)
Summary
It 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). 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 [6,7,8,9]. In monochromatic GEVIs, a transmembrane voltage-sensing domain is fused to a single fluorescent protein that reacts to a voltage change by showing alterations in fluorescence intensity. This has been proposed to result when membrane depolarization triggers movement of the voltage-sensing domain, resulting in deformation of the linked fluorescent protein in a manner that reduces fluorescence intensity [8]
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