Optical silencing of C. elegans cells with light-driven proton pumps

Okazaki, Ayako; Takahashi, Megumi; Toyoda, Naoya; Takagi, Shin

doi:10.1016/j.ymeth.2014.02.030

Cited by 9 publications

(11 citation statements)

References 41 publications

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“…Optogenetic Control of Caenorhabditis elegans-Transgenic C. elegans worms expressing AR3 or TR were generated by microinjection of DNA into the distal arms of gonads of N2 hermaphrodites as described previously (12)(13)(14). The plasmid myo-3p::TR::gfp, aex-3p::AR3::gfp or aex-3p::TR::gfp was injected with pRF4 (rol-6d), a transgenic marker conferring the roller phenotype, to create these three lines.…”

Section: Methodsmentioning

confidence: 99%

“…They found the archaeal AR3 to be the most powerful among the seven proton pumps tested (36). Here, to investigate the availability of TR as a silencer of excitable cells, we first expressed TR as a fusion construct with green fluorescent protein (GFP) in body wall muscle cells of C. elegans, which drive locomotion of worms, under the control of a promoter of the myosin heavy chain gene myo-3 (12)(13)(14). Then the locomotion of the worms was measured with and without green light (550 nm), which overlaps the absorption spectra of both TR (530 nm) and AR3 (552 nm).…”

Section: Applicability Of Tr As An Optogenetic Toolmentioning

confidence: 99%

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X-ray Crystallographic Structure of Thermophilic Rhodopsin

Tsukamoto

Mizutani

Hasegawa

et al. 2016

Journal of Biological Chemistry

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Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.

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Section: Methodsmentioning

confidence: 99%

Section: Applicability Of Tr As An Optogenetic Toolmentioning

confidence: 99%

X-ray Crystallographic Structure of Thermophilic Rhodopsin

Tsukamoto

Mizutani

Hasegawa

et al. 2016

Journal of Biological Chemistry

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“…The method used to measure animal body length was described previously [ 51 ]. The body length of ST311 unc-54; ncEx3031(myo-3p :: Arch :: gfp , rol-6d) was measured using the ImageJ public domain software and compared with that at 0.5 s before and after applying light illumination.…”

Section: Methodsmentioning

confidence: 99%

Optical silencing of body wall muscles induces pumping inhibition in Caenorhabditis elegans

Takahashi

Takagi

2017

PLoS Genet

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Feeding, a vital behavior in animals, is modulated depending on internal and external factors. In the nematode Caenorhabditis elegans, the feeding organ called the pharynx ingests food by pumping driven by the pharyngeal muscles. Here we report that optical silencing of the body wall muscles, which drive the locomotory movement of worms, affects pumping. In worms expressing the Arch proton pump or the ACR2 anion channel in the body wall muscle cells, the pumping rate decreases after activation of Arch or ACR2 with light illumination, and recovers gradually after terminating illumination. Pumping was similarly inhibited by illumination in locomotion-defective mutants carrying Arch, suggesting that perturbation of locomotory movement is not critical for pumping inhibition. Analysis of mutants and cell ablation experiments showed that the signals mediating the pumping inhibition response triggered by activation of Arch with weak light are transferred mainly through two pathways: one involving gap junction-dependent mechanisms through pharyngeal I1 neurons, which mediate fast signals, and the other involving dense-core vesicle-dependent mechanisms, which mediate slow signals. Activation of Arch with strong light inhibited pumping strongly in a manner that does not rely on either gap junction-dependent or dense-core vesicle-dependent mechanisms. Our study revealed a new aspect of the neural and neuroendocrine controls of pumping initiated from the body wall muscles.

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“…blue-light-sensitive ion channel Channelrhodopsin-2 (ChR2)) [19] or inhibitory microbial opsins (e.g. blue-light-powered proton pump (Mac) from Leptosphaeria maculans ) [20-22]. To stimulate single neurons, we take advantage of the fact that pharynx is internal and use polystyrene beads to immobilize worms [23].…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Optogenetic Stimulation of Individual Pharyngeal Neurons and Monitoring of Feeding Behavior in Intact C. elegans

Trojanowski

Fang-Yen

2015

Methods in Molecular Biology

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Summary Optogenetic approaches have proven powerful for examining the role of neural circuits in generating behaviors, especially in systems where electrophysiological manipulation is not possible. Here we describe a method for optogenetically manipulating single pharyngeal neurons in intact C. elegans while monitoring pharyngeal behavior. This approach provides bidirectional and dynamic control of pharyngeal neural activity simultaneously with a behavioral readout, and has allowed us to test hypotheses about the roles of individual pharyngeal neurons in regulating feeding behavior.

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Optical silencing of C. elegans cells with light-driven proton pumps

Cited by 9 publications

References 41 publications

X-ray Crystallographic Structure of Thermophilic Rhodopsin

X-ray Crystallographic Structure of Thermophilic Rhodopsin

Optical silencing of body wall muscles induces pumping inhibition in Caenorhabditis elegans

Simultaneous Optogenetic Stimulation of Individual Pharyngeal Neurons and Monitoring of Feeding Behavior in Intact C. elegans

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