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

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.

Single‐cell damage elicits regional, nematode‐restricting ethylene responses in roots

“…Stress stimuli such as PAMPs, salt, or mechanical damage have been shown to induce a Ca 2+ wave that travels through the root for long distances (Steinhorst & Kudla, ; Choi et al , ; Gilroy et al , ). A number of genetically encoded live‐imaging probes for intracellular calcium are available (Albrecht et al , ; Pandey et al , ; Monshausen et al , ; Matzke & Matzke, ), and we chose the widely used, intensometric R‐GECO1 (Keinath et al , ) sensor as well as the intensity‐based concentration sensor Case12 (Matzke & Matzke, ) in order to test whether we could observe a local Ca 2+ wave upon single‐cell laser ablation. We performed ablation of cortical cells in R‐GECO lines (Fig D–I and Movie EV1).…”

“…Transgenic Arabidopsis thaliana (L.) Heynh. lines have been described elsewhere: Col ; rboh A (GABI_397C02) , rboh F (At1G64060) , rboh D (SALK_070610) (Lee et al , ) ; rboh DF (Torres et al , ) ; ein2‐1 (Guzmán & Ecker, ); ein3‐1 (Chao et al , ); pepr1‐1 pepr2‐1 (Yamaguchi et al , ); glr3.3 3.1, glr3.3 3.6 (Nguyen et al , ); aos (Park et al , ); coi1‐34 (Acosta et al , ); LOX6::LOX6‐GUS (Gasperini et al , ,b); 35S::JAS9‐Venus (Larrieu et al , ); R‐GECO1 (Keinath et al , ); Case12 (Matzke & Matzke, ); and pPR1::NLS3xVenus (Poncini et al , ). Generation of: pACS6::NLS3xVenus and pPR4::NLS3xVenus ; pJAZ10::NLS3xVenus , pAOS::NLS3xVenus .…”

Voltage imaging using transgenic mouse lines expressing the GEVI ArcLight in two olfactory cell types

“…While ArcLight has relatively slow kinetics (~10ms fast time constant), which limits its ability to detect high-frequency spiking activity, it is extremely bright, sensitive, and has been shown to respond to neuronal activity in vivo. This includes in vivo application in Drosophila, worm, mouse, and even plants [10,11,17,18,[37][38][39]. Previous imaging studies using ArcLight in mice used either in utero electroporation or AAV viral vectors under the control of different promoters (hSyn and CAG).…”

Plant mechanosensitive ion channels: an ocean of possibilities

“…Integration of localized extracellular ion flux measurements with genetically encoded ion or voltage biosensors may allow the study of MS channel function in some cellular contexts, such as pollen tubes [55]. To date, the genetically encoded sensors for transmembrane voltage used extensively in animal systems to monitor ion channel activity in vivo [56] do not yet function well in plants [57]. …”