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The Advances and Applications of Optogenetics

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A special issue of Applied Sciences (ISSN 2076-3417). This special issue belongs to the section "Applied Biosciences and Bioengineering".

Deadline for manuscript submissions: closed (31 December 2019) | Viewed by 41723

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Special Issue Editors

Dr. Elena G. Govorunova

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Guest Editor

Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, TX 77065, USA
Interests: channelrhodopsins; optogenetics; neuroscience; cardiology
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Oleg A. Sineshchekov

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Guest Editor

Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, TX, USA
Interests: microbial rhodopsins; photocycle; photocurrent; optogenetics; phototaxis; photosensing; flagellate algae

Special Issue Information

Dear Colleagues,

When Karl Deisseroth coined the word “optogenetics” in 2006, he had in mind using genetically-encoded actuators to control the membrane potential and, thus, cellular excitability with light. Recruiting microbial rhodopsins to this end has yielded a revolution in neuroscience, led to clinical trials to cure blindness and is considered for the treatment of many psychiatric and neurological disorders. Other light-sensitive protein domains, mostly of plant origin, have been harnessed for photoregulation of gene expression, protein activity, oligomerization and trafficking. These efforts have been complemented by engineering proteins to bind photoswitchable ligands. Finally, an array of photosensors responsive to physiological changes in the membrane voltage or intracellular concentrations of specific ions has been created, leading to the possibility of all-optical interrogation of cellular activity. This Special Issue focuses on recent advances in the ever-broadening and exciting field of optogenetics that are expected to boost both fundamental research and clinical practice.

Dr. Elena G. Govorunova
Prof. Dr. Oleg A. Sineshchekov
Guest Editors

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Keywords

optogenetics
photoswitching
photocontrol
all-optical electrophysiology
microbial rhodopsins
ion channels
LOV domains
phytochromes
membrane potential
gene expression
oligomerization
intracellular trafficking
protein-protein interaction
signaling

Published Papers (10 papers)

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Editorial

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4 pages, 163 KiB

Open AccessEditorial

Editorial on Special Issue “The Advances and Applications of Optogenetics”

by Elena G. Govorunova and Oleg A. Sineshchekov

Appl. Sci. 2020, 10(18), 6563; https://doi.org/10.3390/app10186563 - 20 Sep 2020

Viewed by 1634

Abstract

This Special Issue provides an update for the rapidly developing technology known as “optogenetics” that is the use of genetically encoded light-sensitive molecular elements (usually derived from lower organisms) to control or report various physiological and biochemical processes within the cell. Two ongoing clinical trials use optogenetic tools for vision restoration, and optogenetic strategies have been suggested as novel therapies for several neurological, psychiatric and cardiac disorders. The Special Issue comprises two reviews and seven experimental papers on different types of light-sensitive modules widely used in optogenetic studies. These papers demonstrate the efficiency and versatility of optogenetics and are expected to be equally relevant for advanced users and beginners who only consider using optogenetic tools in their research. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

Research

Jump to: Editorial, Review

15 pages, 2024 KiB

Open AccessArticle

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

by David Ehrenberg, Nils Krause, Mattia Saita, Christian Bamann, Rajiv K. Kar, Kirsten Hoffmann, Dorothea Heinrich, Igor Schapiro, Joachim Heberle and Ramona Schlesinger

Appl. Sci. 2019, 9(22), 4905; https://doi.org/10.3390/app9224905 - 15 Nov 2019

Cited by 4 | Viewed by 2863

Abstract

Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm⁻¹, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P₂³⁸⁰ state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P₃⁵³⁰ intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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28 pages, 3501 KiB

Open AccessArticle

Modular Diversity of the BLUF Proteins and Their Potential for the Development of Diverse Optogenetic Tools

by Manish Singh Kaushik, Ramandeep Sharma, Sindhu Kandoth Veetil, Sandeep Kumar Srivastava and Suneel Kateriya

Appl. Sci. 2019, 9(18), 3924; https://doi.org/10.3390/app9183924 - 19 Sep 2019

Cited by 5 | Viewed by 3908

Abstract

Organisms can respond to varying light conditions using a wide range of sensory photoreceptors. These photoreceptors can be standalone proteins or represent a module in multidomain proteins, where one or more modules sense light as an input signal which is converted into an output response via structural rearrangements in these receptors. The output signals are utilized downstream by effector proteins or multiprotein clusters to modulate their activity, which could further affect specific interactions, gene regulation or enzymatic catalysis. The blue-light using flavin (BLUF) photosensory module is an autonomous unit that is naturally distributed among functionally distinct proteins. In this study, we identified 34 BLUF photoreceptors of prokaryotic and eukaryotic origin from available bioinformatics sequence databases. Interestingly, our analysis shows diverse BLUF-effector arrangements with a functional association that was previously unknown or thought to be rare among the BLUF class of sensory proteins, such as endonucleases, tet repressor family (tetR), regulators of G-protein signaling, GAL4 transcription family and several other previously unidentified effectors, such as RhoGEF, Phosphatidyl-Ethanolamine Binding protein (PBP), ankyrin and leucine-rich repeats. Interaction studies and the indexing of BLUF domains further show the diversity of BLUF-effector combinations. These diverse modular architectures highlight how the organism’s behaviour, cellular processes, and distinct cellular outputs are regulated by integrating BLUF sensing modules in combination with a plethora of diverse signatures. Our analysis highlights the modular diversity of BLUF containing proteins and opens the possibility of creating a rational design of novel functional chimeras using a BLUF architecture with relevant cellular effectors. Thus, the BLUF domain could be a potential candidate for the development of powerful novel optogenetic tools for its application in modulating diverse cell signaling. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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10 pages, 2795 KiB

Open AccessArticle

Light Stimulation Parameters Determine Neuron Dynamic Characteristics

by Alexander Erofeev, Evgenii Gerasimov, Anastasia Lavrova, Anastasia Bolshakova, Eugene Postnikov, Ilya Bezprozvanny and Olga L. Vlasova

Appl. Sci. 2019, 9(18), 3673; https://doi.org/10.3390/app9183673 - 05 Sep 2019

Cited by 13 | Viewed by 3880

Abstract

Optogenetics is a recently developed technique that is widely used to study neuronal function. In optogenetic experiments, neurons encode opsins (channelrhodopsins, halorhodopsins or their derivatives) by means of viruses, plasmids or genetic modification (transgenic lines). Channelrhodopsin are light activated ion channels. Their expression in neurons allows light-dependent control of neuronal activity. The duration and frequency of light stimulation in optogenetic experiments is critical for stable, robust and reproducible experiments. In this study, we performed systematic analyses of these parameters using primary cultures of hippocampal neurons transfected with channelrhodopsin-2 (ChR2). The main goal of this work was to identify the optimal parameters of light stimulation that would result in stable neuronal activity during a repeated light pulse train. We demonstrated that the dependency of the photocurrent on the light pulse duration is described by a right-skewed bell-shaped curve, while the dependence on the stimulus intensity is close to linear. We established that a duration between 10–30 ms of stimulation was the minimal time necessary to achieve a full response. Obtained results will be useful in planning and interpretation of optogenetic experiments. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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13 pages, 1816 KiB

Open AccessArticle

Ion Channel Properties of a Cation Channelrhodopsin, Gt_CCR4

by Shunta Shigemura, Shoko Hososhima, Hideki Kandori and Satoshi P. Tsunoda

Appl. Sci. 2019, 9(17), 3440; https://doi.org/10.3390/app9173440 - 21 Aug 2019

Cited by 16 | Viewed by 3500

Abstract

We previously reported a cation channelrhodopsin, Gt_CCR4, which is one of the 44 types of microbial rhodopsins from a cryptophyte flagellate, Guillardia theta. Due to the modest homology of amino acid sequences with a chlorophyte channelrhodopsin such as Cr_ChR2 from Chlamydomonas reinhardtii, it has been proposed that a family of cryptophyte channelrhodopsin, including Gt_CCR4, has a distinct molecular mechanism for channel gating and ion permeation. In this study, we compared the photocurrent properties, cation selectivity and kinetics between well-known Cr_ChR2 and Gt_CCR4 by a conventional path clamp method. Large and stable light-induced cation conduction by Gt_CCR4 at the maximum absorbing wavelength (530 nm) was observed with only small inactivation (15%), whereas the photocurrent of Cr_ChR2 exhibited significant inactivation (50%) and desensitization. The light sensitivity of Gt_CCR4 was higher (EC₅₀ = 0.13 mW/mm²) than that of Cr_ChR2 (EC₅₀ = 0.80 mW/mm²) while the channel open life time (photocycle speed) was in the same range as that of Cr_ChR2 (25~30 ms for Gt_CCR4 and 10~15 ms for Cr_ChR2). This observation implies that Gt_CCR4 enables optical neuronal spiking with weak light in high temporal resolution when applied in neuroscience. Furthermore, we demonstrated high Na⁺ selectivity of Gt_CCR4 in which the selectivity ratio for Na⁺ was 37-fold larger than that for Cr_ChR2, which primarily conducts H⁺. On the other hand, Gt_CCR4 conducted almost no H⁺ and no Ca²⁺ under physiological conditions. These results suggest that ion selectivity in Gt_CCR4 is distinct from that in Cr_ChR2. In addition, a unique red-absorbing and stable intermediate in the photocycle was observed, indicating a photochromic property of Gt_CCR4. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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17 pages, 2199 KiB

Open AccessArticle

Channelrhodopsin-2 Function is Modulated by Residual Hydrophobic Mismatch with the Surrounding Lipid Environment

by Ryan Richards, Sayan Mondal, Harel Weinstein and Robert E. Dempski

Appl. Sci. 2019, 9(13), 2674; https://doi.org/10.3390/app9132674 - 30 Jun 2019

Cited by 1 | Viewed by 3624

Abstract

Channelrhodopsin-2 (ChR2) is a light-gated ion channel that conducts cations of multiple valencies down the electrochemical gradient. This light-gated property has made ChR2 a popular tool in the field of optogenetics, allowing for the spatial and temporal control of excitable cells with light. A central aspect of protein function is the interaction with the surrounding lipid environment. To further explore these membrane-protein interactions, we demonstrate the role of residual hydrophobic mismatch (RHM) as a mechanistically important component of ChR2 function. We combined computational and functional experiments to understand how RHM between the lipid environment and ChR2 alters the structural and biophysical properties of the channel. Analysis of our results revealed significant RHM at the intracellular/lipid interface of ChR2 from a triad of residues. The resulting energy penalty is substantial and can be lowered via mutagenesis to evaluate the functional effects of this change in lipid-protein interaction energy. The experimental measurement of channel stability, conductance and selectivity resulting from the reduction of the RHM energy penalty showed changes in progressive H⁺ permeability, kinetics and open-state stability, suggesting how the modulation of ChR2 by the surrounding lipid membrane can play an important biological role and contribute to the design of targeted optogenetic constructs for specific cell types. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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11 pages, 1395 KiB

Open AccessArticle

Engineering Optogenetic Control of Endogenous p53 Protein Levels

by Pierre Wehler and Barbara Di Ventura

Appl. Sci. 2019, 9(10), 2095; https://doi.org/10.3390/app9102095 - 21 May 2019

Cited by 4 | Viewed by 3625

Abstract

The transcription factor p53 is a stress sensor that turns specific sets of genes on to allow the cell to respond to the stress depending on its severity and type. p53 is classified as tumor suppressor because its function is to maintain genome integrity promoting cell cycle arrest, apoptosis, or senescence to avoid proliferation of cells with damaged DNA. While in many human cancers the p53 gene is itself mutated, there are some in which the dysfunction of the p53 pathway is caused by the overexpression of negative regulators of p53, such as Mdm2, that keep it at low levels at all times. Here we develop an optogenetic approach to control endogenous p53 levels with blue light. Specifically, we control the nuclear localization of the Mmd2-binding PMI peptide using the light-inducible export system LEXY. In the dark, the PMI-LEXY fusion is nuclear and binds to Mdm2, consenting to p53 to accumulate and transcribe the target gene p21. Blue light exposure leads to the export of the PMI-LEXY fusion into the cytosol, thereby Mdm2 is able to degrade p53 as in the absence of the peptide. This approach may be useful to study the effect of localized p53 activation within a tissue or organ. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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11 pages, 4709 KiB

Open AccessFeature PaperArticle

Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

by Xiaodong Duan, Georg Nagel and Shiqiang Gao

Appl. Sci. 2019, 9(4), 664; https://doi.org/10.3390/app9040664 - 15 Feb 2019

Cited by 20 | Viewed by 5361

Abstract

(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca²⁺ conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na⁺ and Ca²⁺ conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na⁺ and Ca²⁺ conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na⁺ selectivity to H⁺ than XXM. PsChR D139H also showed a strongly enhanced Ca²⁺ conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na⁺ selectivity and Ca²⁺ conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca²⁺ manipulation. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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Review

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15 pages, 796 KiB

Open AccessReview

Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics

by Shai Kellner and Shai Berlin

Appl. Sci. 2020, 10(3), 805; https://doi.org/10.3390/app10030805 - 23 Jan 2020

Cited by 20 | Viewed by 5113

Abstract

Synthetic optogenetics is an emerging optical technique that enables users to photocontrol molecules, proteins, and cells in vitro and in vivo. This is achieved by use of synthetic chromophores—denoted photoswitches—that undergo light-dependent changes (e.g., isomerization), which are meticulously designed to interact with unique cellular targets, notably proteins. Following light illumination, the changes adopted by photoswitches are harnessed to affect the function of nearby proteins. In most instances, photoswitches absorb visible light, wavelengths of poor tissue penetration, and excessive scatter. These shortcomings impede their use in vivo. To overcome these challenges, photoswitches of red-shifted absorbance have been developed. Notably, this shift in absorbance also increases their compatibility with two-photon excitation (2PE) methods. Here, we provide an overview of recent efforts devoted towards optimizing azobenzene-based photoswitches for 2PE and their current applications. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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26 pages, 2559 KiB

Open AccessReview

Advances in Engineering and Application of Optogenetic Indicators for Neuroscience

by Kiryl D. Piatkevich, Mitchell H. Murdock and Fedor V. Subach

Appl. Sci. 2019, 9(3), 562; https://doi.org/10.3390/app9030562 - 08 Feb 2019

Cited by 23 | Viewed by 7343 | Correction

Abstract

Our ability to investigate the brain is limited by available technologies that can record biological processes in vivo with suitable spatiotemporal resolution. Advances in optogenetics now enable optical recording and perturbation of central physiological processes within the intact brains of model organisms. By monitoring key signaling molecules noninvasively, we can better appreciate how information is processed and integrated within intact circuits. In this review, we describe recent efforts engineering genetically-encoded fluorescence indicators to monitor neuronal activity. We summarize recent advances of sensors for calcium, potassium, voltage, and select neurotransmitters, focusing on their molecular design, properties, and current limitations. We also highlight impressive applications of these sensors in neuroscience research. We adopt the view that advances in sensor engineering will yield enduring insights on systems neuroscience. Neuroscientists are eager to adopt suitable tools for imaging neural activity in vivo, making this a golden age for engineering optogenetic indicators. Full article

(This article belongs to the Special Issue The Advances and Applications of Optogenetics)

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