Optogenetic Tools Research Articles

Activation of the periaqueductal gray controls respiratory output through a distributed brain network

IntroductionThe periaqueductal gray (PAG) has been previously established to play a key role in producing the vital changes in respiration occurring in response to threat. However, it is not fully understood how PAG activation alters the ongoing respiratory output, nor it is understood which pathways mediate these effects, as several regions have been previously identified to influence respiratory activity.MethodsWe used optogenetic tools in conjunction with EMG recordings of inspiratory and expiratory musculature to determine how PAG activation on short (250 ms) and longer (10–15 s) timescales alters respiratory muscle activity. Through cFOS mapping, we also identified key downstream brain regions which were likely modulated by PAG activation including the preBötzinger Complex (preBötC) and the lateral parafacial area (pFL). We then stimulated PAG terminals in those regions to determine whether their activity can account for the observed effects of PAG stimulation.ResultsDirectly stimulating the PAG resulted in prominent changes to all recorded muscle activities and reset the breathing rhythm in either a phase-independent or phase-dependent manner. In contrast, stimulating PAG terminals in either preBötC or pFL with long or shorter timescale stimuli could not completely replicate the effects of direct PAG stimulation and also did not produce any respiratory reset.ConclusionsOur results show that the effects of PAG activity on respiration are not mediated solely by PAG inputs to either the preBötC or pFL and more likely involve integration across a larger network of brainstem areas.

Optogenetics in medicine: innovations and therapeutic applications.

Optogenetics, an innovative approach integrating photonics and genetic engineering, enables precise control over molecular and cellular processes, opening up exciting new opportunities for precision-guided medicine. In this review, we highlight recent advances in optogenetic tools and their applications across a range of medical conditions, including vision restoration in retinitis pigmentosa via light-activated ion channels, precise immune response modulation in cancer immunotherapy, and blood glucose management in diabetes through controllable drug release. Optogenetics also plays a critical role in bioelectronic medicine, enabling seamless communication between electronic systems and biological tissues to enhance therapeutic precision. Finally, we discuss the challenges and potential transition of optogenetics from experimental models to clinical therapies, emphasizing its immense potential to transform future medical treatments.

Engineering hydrogen bonding at tyrosine-201 in the orange carotenoid protein using halogenated analogues.

The Orange Carotenoid Protein (OCP) is a unique water-soluble photoactive protein that plays a critical role in regulating the balance between light harvesting and photoprotective responses in cyanobacteria. The challenge in understanding OCP´s photoactivation mechanism stems from the heterogeneity of the initial configurations of its embedded ketocarotenoid, which in the dark-adapted state can form up to two hydrogen bonds to critical amino acids in the protein's C-terminal domain, and the extremely low quantum yield of primary photoproduct formation. While a series of experiments involving point mutations within these contacts helped us to identify these challenges, they did not resolve them. To overcome this, we shifted from classical mutagenesis to the translational introduction of non-canonical amino acid residues into the OCP structure. In this work, we demonstrate that replacing a single meta-hydrogen in tyrosine-201 with a halogen atom (chlorine, bromine, or iodine) leads to targeted modifications in the keto-carotenoid-protein matrix interaction network, both in the dark-adapted state and upon photoactivation. We found that such atomic substitutions allow us to effectively weaken key hydrogen bonds without disrupting protein folding, thereby increasing the yield of OCP photoactivation products. Such genetically encoded chemical modification of individual atoms and their systematic in situ variation in complex protein structures establishes a foundation for transforming OCP into a practical tool for optogenetics and other applications.

Backbone resonance assignments of PhoCl, a photocleavable protein.

PhoCl is a photocleavable protein engineered from a green-to-red photoconvertible fluorescent protein by circular permutation, and has been used in various optogenetic applications including precise control of protein localization and activity in cells. Upon violet light illumination, PhoCl undergoes a β-elimination reaction to be cleaved at the chromophore, resulting in spontaneous dissociation into a large empty barrel and a small C-terminal peptide. However, the structural determinants and the mechanism of the PhoCl photocleavage remain elusive, hindering the further development of more robust photocleavable optogenetic tools. Here, we report the backbone resonance assignments of PhoCl as a basis for studying the violet-light-induced self-cleavage mechanism of PhoCl.

Suppression of epileptic seizures by transcranial activation of K+-selective channelrhodopsin

Optogenetics is a valuable tool for studying the mechanisms of neurological diseases and is now being developed for therapeutic applications. In rodents and macaques, improved channelrhodopsins have been applied to achieve transcranial optogenetic stimulation. While transcranial photoexcitation of neurons has been achieved, noninvasive optogenetic inhibition for treating hyperexcitability-induced neurological disorders has remained elusive. There is a critical need for effective inhibitory optogenetic tools that are highly light-sensitive and capable of suppressing neuronal activity in deep brain tissue. In this study, we developed a highly sensitive moderately K+-selective channelrhodopsin (HcKCR1-hs) by molecular engineering of the recently discovered Hyphochytrium catenoides kalium (potassium) channelrhodopsin 1. Transcranial activation of HcKCR1-hs significantly prolongs the time to the first seizure, increases survival, and decreases seizure activity in several status epilepticus mouse models. Our approach for transcranial optogenetic inhibition of neural hyperactivity may be adapted for cell type-specific neuromodulation in both basic and preclinical settings.

Genetically encoded bioluminescent glucose indicator for biological research

Glucose is an essential energy source in living cells and is involved in various phenomena. To understand the roles of glucose, measuring cellular glucose levels is important. Here, we developed a bioluminescent glucose indicator called LOTUS-Glc. Unlike fluorescence, bioluminescence doesn’t require excitation light when imaging. Using LOTUS-Glc, we demonstrated drug effect evaluation, concurrent use with the optogenetic tool in HEK293T cells, and the measurement of light-dependent glucose fluctuations in plant-derived protoplasts. LOTUS-Glc would be a useful tool for understanding the roles of glucose in living organisms.

Live imaging of paracrine signaling: Advances in visualization and tracking techniques.

Live imaging techniques have revolutionized our understanding of paracrine signaling, a crucial form of cell-to-cell communication in biological processes. This review examines recent advances in visualizing and tracking paracrine factors through four key stages: secretion from producing cells, diffusion through extracellular space, binding to target cells, and activation of intracellular signaling within target cells. Paracrine factor secretion can be directly visualized by fluorescent protein tagging to ligand, or indirectly by visualizing the cleavage of the transmembrane pro-ligands or plasma membrane fusion of endosomes comprising the paracrine factors. Diffusion of paracrine factors has been studied using techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence decay after photoactivation (FDAP), and single-molecule tracking. Binding of paracrine factors to target cells has been visualized through various biosensors, including GPCR-activation-based (GRAB) sensors and Förster resonance energy transfer (FRET) probes for receptor tyrosine kinases. Finally, activation of intracellular signaling is monitored within the target cells by biosensors for second messengers, transcription factors, and so on. In addition to the imaging tools, the review also highlights emerging optogenetic and chemogenetic tools for triggering the release of paracrine factors, which is essential for associating the paracrine factor secretion to biological outcomes during the bioimaging of paracrine factor signaling.Key words: paracrine signaling, live imaging, biosensors, optogenetics, chemogenetics.

Dye‐Based Fluorescent Organic Nanoparticles, New Promising Tools for Optogenetics (Adv. Healthcare Mater. 2/2025)

Dye‐Based Fluorescent Organic Nanoparticles, New Promising Tools for Optogenetics (Adv. Healthcare Mater. 2/2025)

Design and Use of AsLOV2-Based Optogenetic Tools for Actin Imaging.

We present protocols for using an optogenetic tool called LILAC for actin imaging. LILAC is a light-controlled version of Lifeact that uses the Avena sativa LOV2 (AsLOV2) domain. By significantly reducing Lifeact's affinity for the cytoskeleton in the dark, LILAC reduces concentration-dependent negative side effects while enabling new image processing methods. We discuss the considerations for using this probe of live-cell actin dynamics, including fluorescent protein selection, cell maintenance, microscopy protocols, and image processing. Our work highlights the potential of AsLOV2-based optogenetics for novel imaging and control tools in cell biology.

Chlorophyll-Based Optogenetics to Control Membraneless Organelles.

Membraneless organelles (MLOs) formed via protein phase separation have garnered significant attention recently due to their relevance to cellular physiology and pathology. However, there is a lack of tools available to study their behavior and control their bioactivity in complex biological systems. This chapter describes a new optogenetic tool based on water-soluble chlorophyll protein (WSCP), a redlight-induced singlet oxygen-generating protein, to control syntheticMLOs. Upon exposure to red light, WSCP generates singlet oxygen, which triggers the crosslinking of the proteins in theMLOs, resulting in their liquid-to-solid phase transition. The effective delivery of chlorophylls enables the successful reconstitution of WSCP in living cells, thusoffering a potential approach to biological regulation at the subcellular level.

Extracellular Optogenetics to Interrogate Unmodified Primary Cell Functions.

Optogenetics often requires genetic modification of the target cells to enable the expression of specific optogenetic tools, making it difficult to study primary cells in their native state. We have recently generated a fully extracellular optogenetic system for reversible light control of T cell receptor (TCR) activation on murine naïve T cells, a cell model that is very difficult to manipulate genetically. This molecular system is very versatile and can be easily modified to study different cell systems in different species. Here we describe how to produce and use this extracellular optogenetic system to manipulate primary T cell activation.

Optoribogenetic Modulation of Transcription.

Light can be used as a precise and reversible trigger for the activation of optogenetic tools with subcellular resolution. The interaction of the photoreceptor PAL and aptamer 53 was integrated into a CRISPR/dCas9 system, which can be applied for light-controlled activation of gene expression. Here, we describe a protocol for in vitro application of light-dependent overexpression using eBFP as a proof of concept. The experiment can be done in 3days, which is split into cell seeding, transfection, and evaluation by flow cytometry. The method is broadly applicable including the upregulation of endogenous genes.

In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors.

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

Illuminating the future of food microbial control: From optical tools to Optogenetic tools.

Light as an environmental signal can effectively regulate various biological processes in microbial systems. Optical and optogenetic tools are able to utilize light for precise control methods with minimal interference. Recently, research on these tools has extended to the field of microbiology. Distinguishing from existing reviews, this review narrows the scope of application into food sector, focusing on advances in optical and optogenetic tools for microbial control, including optical tools targeting pathogenic or probiotic bacteria for non-thermal sterilization, antimicrobial photodynamic therapy, or photobiomodulation, combined with nanomaterials as photosensors for food analysis. As well as using optogenetic tools for more convenient and precise control in food production processes, covering reversible induction, metabolic flux regulation, biofilm formation, and inhibition. These tools offer new solutions to goals that cannot be achieved by traditional methods, and they are still maturing to explore other uses in the food field.

Studying ER-membrane contact sites in plants using the optogenetic approach: Taking the LiMETER as an example.

The endoplasmic reticulum (ER) links to multiple organelles through membrane contact sites (MCS), which play critical roles in signal transduction, cell homeostasis and stress response. However, studying the behaviour and functions of MCS in plants is still challenging, partially due to the lack of site-specific markers. Here, we used an optogenetic reporter, LiMETER (Light-inducible Membrane-Tethered cortical ER), to study the structure and dynamics of ER-PM contact sites (EPCS) in plants. Upon blue light activation, LiMETER is recruited to the EPCS rapidly, while this process is reversible when blue light is turned off. Compared with other EPCS reporters, LiMETER specifically and reversibly labels the contact sites, causing little side-effects on the ER structure and plant development. With its help, we re-examined the formation of ER-PM connections induced by cell-intrinsic factors or extracellular stimuli. We found that EPCSs are preferably localised at ER tubules and the edge of ER cisternae, and their number increased significantly under abiotic stress conditions. The abundance of ER and PM interaction is also developmental dependent, suggesting a direct link between ER-PM interaction, ER function and cell homeostasis. Taken together, we showed that LiMETER is an improved marker for functional and microscopical studies of ER-PM interaction, demonstrating the effectiveness of optogenetic tools in future research.

Light-activated channelrhodopsins: a revolutionary toolkit for the remote control of plant signalling.

Channelrhodopsins (CHRs), originating within algae and protists, are membrane-spanning ion channel proteins that are directly activated and/or deactivated by specific wavelengths of light. Since 2005, CHRs have been deployed as genetically encoded optogenetic tools to rapidly advance understanding of neuronal networks. CHRs provide the opportunity to finely tune ion transport across membranes and regulate membrane potential. These are fundamental biochemical signals, which in plants can be translated into physiological and developmental responses such as changes in photosynthesis, growth, turgor, vascular hydraulics, phosphorylation or reactive oxygen species (ROS) status, gene expression, or even cell death. Exploration of CHR family diversity and structure-function engineering has led to the expansion of the CHR optogenetic toolbox, offering unparalleled opportunities to precisely control and understand electrical and secondary messenger signalling in higher plants. In this Tansley Insight, we provide an overview of the recent progress in the application of CHR optogenetics in higher plants and discuss their possible uses in the remote control of plant biology, illuminating a new future domain for plant research enabled through synthetic biology.

Control Intracellular Protein Condensates with Light.

Protein phase transitions are gaining traction among biologists for their wide-ranging roles in biological regulation. However, achieving precise control over these phenomena in vivo remains a formidable task. Optogenetic techniques present us with a potential means to control protein phase behavior with spatiotemporal precision. This review delves into the design of optogenetic tools, particularly those aimed at manipulating protein phase transitions in complex biological systems. We begin by discussing the pivotal roles of subcellular phase transitions in physiological and pathological processes. Subsequently, we offer a thorough examination of the evolution of optogenetic tools and their applications in regulating these protein phase behaviors. Furthermore, we highlight the tailored design of optogenetic tools for controlling protein phase transitions and the construction of synthetic condensates using these innovative techniques. In the long run, the development of optogenetic tools not only holds the potential to elucidate the roles of protein phase transitions in various physiological processes but also to antagonize pathological ones to reinstate cellular homeostasis, thus bringing about novel therapeutic strategies. The integration of optogenetic techniques into the study of protein phase transitions represents a significant step forward in our understanding and manipulation of biology at the subcellular level.

Blue Light-Induced, Dosed Protein Expression of Active BDNF in Human Cells Using the Optogenetic CRY2/CIB System.

The use of optogenetic tools offers an excellent method for spatially and temporally regulated gene and protein expression in cell therapeutic approaches. This could be useful as a concomitant therapeutic measure, especially in small body compartments such as the inner ear, for example, during cochlea implantation, to enhance neuronal cell survival and function. Here, we used the blue light activatable CRY2/CIB system to induce transcription of brain-derived neurotrophic factor (BDNF) in human cells. Transfection with three plasmids, encoding for the optogenetic system and the target, as well as illumination protocols were optimized with luciferase as a reporter to achieve the highest protein expression in human embryonic kidney cells 293. Illumination was performed either with a light-emitting diode or with a scanning laser setup. The optimized protocols were applied for the production of BDNF. We could demonstrate a 64.7-fold increase of BNDF expression upon light induction compared to the basal level. Light-induced BDNF was biologically active and enhanced survival and neurite growth of spiral ganglion neurons. The optogenetic approach can be transferred to autologous cell systems, such as bone marrow-derived mesenchymal stem cells, and thus represents the first optogenetic neurotrophic therapy for the inner ear.

Optogenetic control of Corynebacterium glutamicum gene expression.

Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.

Bacteriorhodopsin of purple membrane reverses anisotropy outside the pH range of proton pumping based on logic gate realization

The bacteriorhodopsin of purple membrane is the first discovered light-sensing protein among ion transporting microbial rhodopsins, some of which (e.g. Archaerhodopsin 3) could be broadly used as tools in optogenetics having wide potential of medical applications. Since its discovery as early as in 1971, bacteriorhodopsin has attracted wide interests in nano-biotechnology, particularly in optoelectronics devices. Therefore, the present work has been motivated due to two topics; firstly, anisotropy demand became indispensible in bioelectronics; secondly, the stationary level of electric response in bacteriorhodopsin within the pH range of proton pumping (pH 3 – pH 10) implies, in turn, raising here a question about whether the electric anisotropy is implicated for reducing (or switching off) such level beyond such pH range. Noteworthy is that the purple membrane converts to blue form upon acidification, while to reddish purple form upon alkalization. In the present study, the acidic and alkaline forms of bacteriorhodopsin have exhibited most probable state of reversal for the dielectric anisotropy around pH 2.5 and pH 10.5, respectively. This is underscored by proposing a correlation seemingly found between disassembly of the crystalline structure of bacteriorhodopsin and the reversal of dielectric anisotropy, at such acidic and alkaline reversal pH’s, in terms of the essence of the crystalline lattice. Most importantly, the results have substantiated dual frequency characteristics and logic gate-based dielectric anisotropy reversal to bacteriorhodopsin, which may implicate it for potential applications in bioelectronics.