All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

Hochbaum, Daniel; Zhao, Yongxin; Farhi, Samouil L.; Klapoetke, Nathan C.; Werley, Christopher A.; Kapoor, Vikrant; Zou, Peng; Kralj, Joel M.; Maclaurin, Dougal; Smedemark-Margulies, Niklas; Saulnier, Jessica L.; Boulting, Gabriella L.; Straub, Christoph; Cho, Yong Ku; Melkonian, Michael; Wong, Gane Ka‐Shu; Harrison, David; Murthy, Venkatesh N.; Sabatini, Bernardo L.; Boyden, Edward S.; Campbell, Robert E.; Cohen, Adam E.

doi:10.1038/nmeth.3000

Cited by 716 publications

(985 citation statements)

References 66 publications

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“…S6A). While our manuscript was in review, an Arch variant containing five mutations (P60S, T80S, D95H, D106H, and F161V) was reported with quantum yield of 8 × 10 −3 (1.5-fold less than Arch-7) (27). None of these individual mutations were identified in our evolution.…”

Section: Comparison Of Arch Variants With Other Fluorescent Proteinsmentioning

confidence: 76%

Directed evolution of a far-red fluorescent rhodopsin

McIsaac¹,

Engqvist²,

Wannier

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

115

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Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with livecell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm).optogenetics | opsins | bioelectricity | voltage sensor | near-infrared

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Section: Comparison Of Arch Variants With Other Fluorescent Proteinsmentioning

confidence: 76%

Directed evolution of a far-red fluorescent rhodopsin

McIsaac¹,

Engqvist²,

Wannier

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

115

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“…The combination of engineering and biology has allowed the precise quantification of contractile force in hPSC‐CMs (Ribeiro et al , 2015a), although it has been demonstrated that substrate stiffness plays a major role in the quantification of absolute force values (Feinberg et al , 2012; Ribeiro et al , 2015b). More recently, integrated technologies capable of simultaneously quantifying multiple parameters are being implemented with the view to providing reliable, high‐throughput tools for early preclinical stages of drug development (Hochbaum et al , 2014; Kijlstra et al , 2015; Rast et al , 2015; Song et al , 2015; Dempsey et al , 2016; Klimas et al , 2016). …”

Section: Assays and Readoutsmentioning

confidence: 99%

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

Sala

Bellin

Mummery

2016

British J Pharmacology

107

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Cardiotoxicity is a severe side effect of drugs that induce structural or electrophysiological changes in heart muscle cells. As a result, the heart undergoes failure and potentially lethal arrhythmias. It is still a major reason for drug failure in preclinical and clinical phases of drug discovery. Current methods for predicting cardiotoxicity are based on guidelines that combine electrophysiological analysis of cell lines expressing ion channels ectopically in vitro with animal models and clinical trials. Although no new cases of drugs linked to lethal arrhythmias have been reported since the introduction of these guidelines in 2005, their limited predictive power likely means that potentially valuable drugs may not reach clinical practice. Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) are now emerging as potentially more predictive alternatives, particularly for the early phases of preclinical research. However, these cells are phenotypically immature and culture and assay methods not standardized, which could be a hurdle to the development of predictive computational models and their implementation into the drug discovery pipeline, in contrast to the ambitions of the comprehensive pro‐arrhythmia in vitro assay (CiPA) initiative. Here, we review present and future preclinical cardiotoxicity screening and suggest possible hPSC‐CM‐based strategies that may help to move the field forward. Coordinated efforts by basic scientists, companies and hPSC banks to standardize experimental conditions for generating reliable and reproducible safety indices will be helpful not only for cardiotoxicity prediction but also for precision medicine.Linked ArticlesThis article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc

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“…Small-molecule voltage sensors in particular are plagued by trade-offs in speed and sensitivity. More recently, genetically encoded fluorescent voltage sensors also show promise in terms of both speed and sensitivity (17)(18)(19)(20), but current methods are similarly limited by low one-and two-photon brightness (21).…”

mentioning

confidence: 99%

Voltage-sensitive rhodol with enhanced two-photon brightness

Kulkarni

Kramer

Pourmandi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using twophoton (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue. Neuronal membrane potential dynamics drive neurotransmitter release and are therefore responsible for the unique physiology associated with neurons at cellular, circuit, and organismal levels. Despite the central importance of proper neuronal firing to human health, an integrated understanding of neuronal activity in the context of larger brain circuits remains elusive, due in part to a lack of methods for interrogating membrane potential dynamics with sufficient spatial and temporal resolution.Traditional methods for monitoring membrane potential rely heavily on the use of invasive electrodes, through one of two methods. The first method, patch-clamp electrophysiology, uses a single electrode to make contact with or puncture a cell to record changes in membrane potential, sacrificing throughput and spatial resolution to achieve a comprehensive description of a single cellular electrophysiological profile. A second method uses multielectrode arrays (MEAs), in which patterned arrays of electrodes introduced to cells or tissues report on electrical changes. Spatial resolution of MEAs depends on the number and positioning of the electrodes within the array. Although throughput is improved relative to patch-clamp electrophysiology, the extracellularly recorded signals are typically less sensitive than whole-cell methods and can be an amalgamation of several cells, making deconvolution of recorded signals and precise correlation to specific cells difficult or impossible. Addi...

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All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

Cited by 716 publications

References 66 publications

Directed evolution of a far-red fluorescent rhodopsin

Directed evolution of a far-red fluorescent rhodopsin

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

Voltage-sensitive rhodol with enhanced two-photon brightness

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