A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons

Liu, Shuzhang; Lin, Chenjian; Xu, Yongxian; Luo, Hui‐Xin; Peng, Lirong; Zeng, Xiangmei; Zheng, Huangtao; Chen, Peng R.; Zou, Peng

doi:10.1038/s41557-021-00641-1

Cited by 61 publications

(50 citation statements)

References 47 publications

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“…Furthermore, Arch-and Mac-based positive-going eFRET GEVIs have been developed by modifying the natural proton transport pathway within microbial rhodopsins (Abdelfattah et al, 2020). In addition, other hybrid eFRET indicators, HVI-Cy3 and HVI-Cy5, were reported (Liu et al, 2021). In these constructs, the fluorophore was directly linked to a small peptide (1.6 kDa) inserted at the extracellular loop of the rhodopsin, resulting in high FRET efficiency.…”

Section: Efret-based Voltage Indicatorsmentioning

confidence: 99%

Imaging Voltage with Microbial Rhodopsins

Zhang

Yokoyama

Sakamoto

2021

Front. Mol. Biosci.

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Membrane potential is the critical parameter that reflects the excitability of a neuron, and it is usually measured by electrophysiological recordings with electrodes. However, this is an invasive approach that is constrained by the problems of lacking spatial resolution and genetic specificity. Recently, the development of a variety of fluorescent probes has made it possible to measure the activity of individual cells with high spatiotemporal resolution. The adaptation of this technique to image electrical activity in neurons has become an informative method to study neural circuits. Genetically encoded voltage indicators (GEVIs) can be used with superior performance to accurately target specific genetic populations and reveal neuronal dynamics on a millisecond scale. Microbial rhodopsins are commonly used as optogenetic actuators to manipulate neuronal activities and to explore the circuit mechanisms of brain function, but they also can be used as fluorescent voltage indicators. In this review, we summarize recent advances in the design and the application of rhodopsin-based GEVIs.

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Section: Efret-based Voltage Indicatorsmentioning

confidence: 99%

Imaging Voltage with Microbial Rhodopsins

Zhang

Yokoyama

Sakamoto

2021

Front. Mol. Biosci.

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“…They used the enzyme mediated probe incorporation method to specifically bind the trans-cyclooctene part to the mutants of Acetabularia acetabulum rhodopsin II (Ace2), which was subsequently derivatized with tetrazine-conjugated organic fluorophores via the inverse-electron-demand Diels–Alder cycloaddition (IEDDA) reaction. The resulting HVI had a dye protein structure and exhibited a strong electrochromic effect, which can be used for an all-optical electrophysiological study of cultured neurons [ 89 ].…”

Section: Protein Labeling and Modificationsmentioning

confidence: 99%

Recent Advances about the Applications of Click Reaction in Chemical Proteomics

Yao

Huang

2021

Molecules

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Despite significant advances in biological and analytical approaches, a comprehensive portrait of the proteome and its dynamic interactions and modifications remains a challenging goal. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to elucidate protein composition, distribution, and relevant physiological and pharmacological functions. Click chemistry focuses on the development of new combinatorial chemical methods for carbon heteroatom bond (C-X-C) synthesis, which have been utilized extensively in the field of chemical proteomics. Click reactions have various advantages including high yield, harmless by-products, and simple reaction conditions, upon which the molecular diversity can be easily and effectively obtained. This paper reviews the application of click chemistry in proteomics from four aspects: (1) activity-based protein profiling, (2) enzyme-inhibitors screening, (3) protein labeling and modifications, and (4) hybrid monolithic column in proteomic analysis.

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“…Therefore, there is strong interest in developing hybrid systems in which voltage-sensitive dyes are directed to cells of interest, either via expression of exogenous enzymes ( Ng and Fromherz, 2011 ; Liu et al, 2017 ; Sundukova et al, 2019 ) or via targeting of native ligands ( Fiala et al, 2020 ). Other strategies involve targeting synthetic fluorophores to genetically-encoded voltage-sensitive proteins, whether opsins ( Abdelfattah et al, 2019 ; Liu et al, 2021 ) or voltage-sensing domains ( Deo et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter

et al. 2021

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We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

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A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons

Cited by 61 publications

References 47 publications

Imaging Voltage with Microbial Rhodopsins

Imaging Voltage with Microbial Rhodopsins

Recent Advances about the Applications of Click Reaction in Chemical Proteomics

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter

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