Curated Optogenetic Publication Database

Abstract: Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over the last two years, there have been notable advances in the development of novel optogenetic switches, their utilization in elucidating intricate signaling pathways, and their progress toward practical applications in biotechnological processes, material sciences, and therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, and contemplate future directions.

Abstract: The tumor microenvironment is a barrier to breast cancer therapy. Cancer-associated fibroblast cells (CAFs) can support tumor proliferation, metastasis, and drug resistance by secreting various cytokines and growth factors. Abnormal angiogenesis provides sufficient nutrients for tumor proliferation. Considering that CAFs express the sigma receptor (which recognizes anisamide, AA), we developed a CAFs and breast cancer cells dual-targeting nano drug delivery system to transport the LightOn gene express system, a spatiotemporal controlled gene expression consisting of a light-sensitive transcription factor and a specific minimal promoter. We adopted RGD (Arg-Gly-Asp) to selectively bind to the αvβ3 integrin on activated vascular endothelial cells and tumor cells. After the LightOn system has reached the tumor site, LightOn gene express system can spatiotemporal controllably express toxic Pseudomonas exotoxin An under blue light irradiation. The LightOn gene express system, combined with multifunctional nanoparticles, achieved high targeting delivery efficiency both in vitro and in vivo. It also displayed strong tumor and CAFs inhibition, anti-angiogenesis ability and anti-metastasis ability, with good safety. Moreover, it improved survival rate, survival time, and lung metastasis rate in a mouse breast cancer model. This study proves the efficacy of combining the LightOn system with targeted multifunctional nanoparticles in tumor and anti-metastatic therapy and provides new insights into tumor microenvironment regulation.

Abstract: Optogenetics has opened new possibilities in the remote control of diverse cellular functions with high spatiotemporal precision using light. However, delivering light to optically non-transparent systems remains a challenge. Here, we describe the photoactivation of light-oxygen-voltage-sensing domains (LOV domains) with in situ generated light from a chemiluminescence reaction between luminol and H2O2. This activation is possible due to the spectral overlap between the blue chemiluminescence emission and the absorption bands of the flavin chromophore in LOV domains. All four LOV domain proteins with diverse backgrounds and structures (iLID, BcLOV4, nMagHigh/pMagHigh, and VVDHigh) were photoactivated by chemiluminescence as demonstrated using a bead aggregation assay. The photoactivation with chemiluminescence required a critical light-output below which the LOV domains reversed back to their dark state with protein characteristic kinetics. Furthermore, spatially confined chemiluminescence produced inside giant unilamellar vesicles (GUVs) was able to photoactivate proteins both on the membrane and in solution, leading to the recruitment of the corresponding proteins to the GUV membrane. Finally, we showed that reactive oxygen species produced by neutrophil like cells can be converted into sufficient chemiluminescence to recruit the photoswitchable protein BcLOV4-mCherry from solution to the cell membrane. The findings highlight the utility of chemiluminescence as an endogenous light source for optogenetic applications, offering new possibilities for studying cellular processes in optically non-transparent systems.

Abstract: RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions.

Abstract: The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies.

Abstract: Optogenetics is a cutting-edge technology that merges light control and genetics to achieve targeted control of tissue cells. Compared to traditional methods, optogenetics offers several advantages in terms of time and space precision, accuracy, and reduced damage to the research object. Currently, optogenetics is primarily used in pathway research, drug screening, gene expression regulation, and the stimulation of molecule release to treat various diseases. The selection of light-sensitive proteins is the most crucial aspect of optogenetic technology; structural changes occur or downstream channels are activated to achieve signal transmission or factor release, allowing efficient and controllable disease treatment. In this review, we examine the extensive research conducted in the field of biomedicine concerning optogenetics, including the selection of light-sensitive proteins, the study of carriers and delivery devices, and the application of disease treatment. Additionally, we offer critical insights and future implications of optogenetics in the realm of clinical medicine.

Abstract: Optogenetic approaches in transparent zebrafish models have provided numerous insights into vertebrate neurobiology. The purpose of this study was to develop methods to activate light-sensitive transgene products simultaneously throughout an entire larval zebrafish.

Abstract: Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.

Abstract: The dysregulation of protein kinases is associated with multiple diseases due to the kinases’ involvement in a variety of cell signaling pathways. Manipulating protein kinase function, by controlling the active site, is a promising therapeutic and investigative strategy to mitigate and study diseases. Kinase active sites share structural similarities making it difficult to specifically target one kinase, allosteric control allows specific regulation and study of kinase function without directly targeting the active site. Allosteric sites are distal to the active site but coupled via a dynamic network of inter-atomic interactions between residues in the protein. Establishing an allosteric control over a kinase requires understanding the allosteric wiring of the protein. Computational techniques offer effective and inexpensive mapping of the allosteric sites on a protein. Here, we discuss methods to map and regulate allosteric communications in proteins, and strategies to establish control over kinase functions in live cells and organisms. Protein molecules, or “sensors” are engineered to function as tools to control allosteric activity of the protein as these sensors have high spatiotemporal resolution and help in understanding cell phenotypes after immediate activation or inactivation of a kinase. Traditional methods used to study protein functions, such as knockout, knockdown, or mutation, cannot offer a sufficiently high spatiotemporal resolution. We discuss the modern repertoire of tools to regulate protein kinases as we enter a new era in deciphering cellular signaling and developing novel approaches to treat diseases associated with signal dysregulation.

Abstract: Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform – Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially-designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Abstract: Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.

Abstract: The increasing integration between biological and digital interfaces has led to heightened interest in utilizing biological materials to store digital data, with the most promising one involving the storage of data within defined sequences of DNA that are created by de novo DNA synthesis. However, there is a lack of methods that can obviate the need for de novo DNA synthesis, which tends to be costly and inefficient. Here, in this work, we detail a method of capturing 2-dimensional light patterns into DNA, by utilizing optogenetic circuits to record light exposure into DNA, encoding spatial locations with barcoding, and retrieving stored images via high-throughput next-generation sequencing. We demonstrate the encoding of multiple images into DNA, totaling 1152 bits, selective image retrieval, as well as robustness to drying, heat and UV. We also demonstrate successful multiplexing using multiple wavelengths of light, capturing 2 different images simultaneously using red and blue light. This work thus establishes a 'living digital camera', paving the way towards integrating biological systems with digital devices.

Abstract: Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 μm) and does not fully exploit the features of the "high spatial resolution" of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII.

Abstract: Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Abstract: Photoreceptor proteins are versatile toolbox for developing biosensors for optogenetic applications. These molecular tools get activated upon illumination of blue light, which in turn offers a non-invasive method for gaining high spatiotemporal resolution and precise control of cellular signal transduction. The Light-Oxygen-Voltage (LOV) domain family of proteins is a well-recognized system for constructing optogenetic devices. Translation of these proteins into efficient cellular sensors is possible by tuning their photochemistry lifetime. However, the bottleneck is the need for more understanding of the relationship between the protein environment and photocycle kinetics. Significantly, the effect of the local environment also modulates the electronic structure of chromophore, which perturbs the electrostatic and hydrophobic interaction within the binding site. This work highlights the critical factors hidden in the protein networks, linking with their experimental photocycle kinetics. It presents an opportunity to quantitatively examine the alternation in chromophore's equilibrium geometry and identify details which have substantial implications in designing synthetic LOV constructs with desirable photocycle efficiency.

Abstract: Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye.

Abstract: Optogenetics tools for precise temporal and spatial control of protein abundance are valuable in studying diverse complex biological processes. In the present study, we engineer a monomeric tag of stabilization upon light induction (SULI) for yeast and zebrafish based on a single light-oxygen-voltage domain from Neurospora crassa. Proteins of interest fused with SULI are stable upon light illumination but are readily degraded after transfer to dark conditions. SULI shows a high dynamic range and a high tolerance to fusion at different positions of the target protein. Further studies reveal that SULI-mediated degradation occurs through a lysine ubiquitination-independent proteasome pathway. We demonstrate the usefulness of SULI in controlling the cell cycle in yeast and regulating protein stability in zebrafish, respectively. Overall, our data indicate that SULI is a simple and robust tool to quantitatively and spatiotemporally modulate protein levels for biotechnological or biomedical applications.

Abstract: Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.

Abstract: The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

Abstract: The light-regulated Gal4-UAS system has offered new ways to control cellular activities with precise spatial and temporal resolution in zebrafish and Drosophila. However, the existing optogenetic Gal4-UAS systems suffer from having multiple protein components and a dependence on extraneous light-sensitive cofactors, which increase the technical complexity and limit the portability of these systems. To overcome these limitations, we herein describe the development of a novel optogenetic Gal4-UAS system (ltLightOn) for both zebrafish and Drosophila based on a single light-switchable transactivator, termed GAVPOLT, which dimerizes and binds to gene promoters to activate transgene expression upon blue light illumination. The ltLightOn system is independent of exogenous cofactors and exhibits a more than 2400-fold ON/OFF gene expression ratio, allowing quantitative, spatial, and temporal control of gene expression. We further demonstrate the usefulness of the ltLightOn system in regulating zebrafish embryonic development by controlling the expression of lefty1 by light. We believe that this single-component optogenetic system will be immensely useful in understanding the gene function and behavioral circuits in zebrafish and Drosophila.

Abstract: Antibiotics are a key control mechanism for synthetic biology and microbiology. Resistance genes are used to select desired cells and regulate bacterial populations, however their use to-date has been largely static. Precise spatiotemporal control of antibiotic resistance could enable a wide variety of applications that require dynamic control of susceptibility and survival. Here, we use light-inducible Cre recombinase to activate expression of drug resistance genes in Escherichia coli. We demonstrate light-activated resistance to four antibiotics: carbenicillin, kanamycin, chloramphenicol, and tetracycline. Cells exposed to blue light survive in the presence of lethal antibiotic concentrations, while those kept in the dark do not. To optimize resistance induction, we vary promoter, ribosome binding site, and enzyme variant strength using chromosome and plasmid-based constructs. We then link inducible resistance to expression of a heterologous fatty acid enzyme to increase production of octanoic acid. These optogenetic resistance tools pave the way for spatiotemporal control of cell survival.

Abstract: Individualized immunotherapy has attracted great attention due to its high specificity, effectiveness, and safety. We used an exogenous antigen to label tumor cells with MHC I molecules, which allowed neoantigen-specific T cells to recognize and kill tumor cells. A neoantigen vaccine alone cannot achieve complete tumor clearance due to a tumor immunosuppressive microenvironment. The LightOn system was developed to effectively eliminate tumor cells through the spatiotemporally controllable expression of diphtheria toxin A fragment, leading to antigen release in the tumor region. These antigens stimulated and enhanced immunological function and thus, recruited neoantigen-specific T cells to infiltrate tumor tissue. Using the nanoparticle delivery system, neoantigens produced higher delivery efficiency to lymph nodes and improved tumor targeting ability for tumor cell labelling. Good tumor inhibition and prolonged survival were achieved, while eliciting a strong immune response. The combination of a spatiotemporally controllable transgene system with tumor neoantigen labeling has great potential for tumor immunotherapy.

Abstract: The initiation of apical-basal (AB) polarity and the process of mitotic cell division are both characterised by the generation of specialised plasma membrane and cortical domains. These are generated using shared mechanisms, such as asymmetric protein accumulation, Rho GTPase signalling, cytoskeletal reorganisation, vesicle trafficking and asymmetric phosphoinositide distribution. In epithelial tissue, the coordination of AB polarity and mitosis in space and time is important both during initial epithelial development and to maintain tissue integrity and ensure appropriate cell differentiation at later stages. Whilst significant progress has been made in understanding the mechanisms underlying cell division and AB polarity, it has so far been challenging to fully unpick the complex interrelationship between polarity, signalling, morphogenesis, and cell division. However, the recent emergence of optogenetic protein localisation techniques is now allowing researchers to reversibly control protein activation, localisation and signalling with high spatiotemporal resolution. This has the potential to revolutionise our understanding of how subcellular processes such as apical-basal polarity are integrated with cell behaviours such as mitosis and how these processes impact whole tissue morphogenesis. So far, these techniques have been used to investigate processes such as cleavage furrow ingression, mitotic spindle positioning, and in vivo epithelial morphogenesis. This review describes some of the key shared mechanisms of cell division and apical-basal polarity establishment, how they are coordinated during development and how the advance of optogenetic techniques is furthering this research field.

Abstract: Cell communication is a widespread mechanism in biology, allowing the transmission of information about environmental conditions. In order to understand how cell communication modulates relevant biological processes such as survival, division, differentiation, and apoptosis, different synthetic systems based on chemical induction have been successfully developed. In this work, we coupled cell communication and optogenetics in the budding yeast Saccharomyces cerevisiae. Our approach is based on two strains connected by the light-dependent production of α-factor pheromone in one cell type, which induces gene expression in the other type. After the individual characterization of the different variants of both strains, the optogenetic intercellular system was evaluated by combining the cells under contrasting illumination conditions. Using luciferase as a reporter gene, specific co-cultures at a 1:1 ratio displayed activation of the response upon constant blue light, which was not observed for the same cell mixtures grown in darkness. Then, the system was assessed at several dark/blue-light transitions, where the response level varies depending on the moment in which illumination was delivered. Furthermore, we observed that the amplitude of response can be tuned by modifying the initial ratio between both strains. Finally, the two-population system showed higher fold inductions in comparison with autonomous strains. Altogether, these results demonstrated that external light information is propagated through a diffusible signaling molecule to modulate gene expression in a synthetic system involving microbial cells, which will pave the road for studies allowing optogenetic control of population-level dynamics.