Curated Optogenetic Publication Database

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: 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: 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.

Abstract: The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.

Abstract: The past decade has witnessed enormous progress in optogenetics, which uses photo-sensitive proteins to control signal transduction in live cells and animals. The ever-increasing amount of optogenetic tools, however, could overwhelm the selection of appropriate optogenetic strategies. In this work, we summarize recent progress in this emerging field and highlight the application of opsin-free optogenetics in studying embryonic development, focusing on new insights gained into optical induction of morphogenesis, cell polarity, cell fate determination, tissue differentiation, neuronal regeneration, synaptic plasticity, and removal of cells during development.

Abstract: In the budding yeast Saccharomyces cerevisiae, the FUN-LOV (FUNgal Light Oxygen and Voltage) optogenetic switch enables high levels of light-activated gene expression in a reversible and tunable fashion. The FUN-LOV components, under identical promoter and terminator sequences, are encoded in two different plasmids, which limits its future applications in wild and industrial yeast strains. In this work, we aim to expand the molecular versatility of the FUN-LOV switch to increase its biotechnological applications. Initially, we generated new variants of this system by replacing the promoter and terminator sequences and by cloning the system in a single plasmid (FUN-LOVSP). In a second step, we included the nourseothricin (Nat) or hygromycin (Hph) antibiotic resistances genes in the new FUN-LOVSP plasmid, generating two new variants (FUN-LOVSP-Nat and FUN-LOVSP-Hph), to allow selection after genome integration. Then, we compared the levels of light-activated expression for each FUN-LOV variants using the luciferase reporter gene in the BY4741 yeast strain. The results indicate that FUN-LOVSP-Nat and FUN-LOVSP-Hph, either episomally or genome integrated, reached higher levels of luciferase expression upon blue-light stimulation compared the original FUN-LOV system. Finally, we demonstrated the functionality of FUN-LOVSP-Hph in the 59A-EC1118 wine yeast strain, showing similar levels of reporter gene induction under blue-light respect to the laboratory strain, and with lower luciferase expression background in darkness condition. Altogether, the new FUN-LOV variants described here are functional in different yeast strains, expanding the biotechnological applications of this optogenetic tool.

Abstract: RNA is the cornerstone of biology's central dogma, which was initially thought to serve only as an intermediate between DNA and protein. Decades of research, in particular the discovery of new classes of non-coding RNAs (ncRNAs), have unveiled a plethora of activities that RNAs can fulfil besides coding for proteins, ranging from catalysis over scaffolding to regulatory functions. These ncRNAs not only play important roles in healthy individuals, but also are implicated in a wide range of diseases, including cancers, cardiovascular and neurological diseases, which have been demonstrated in several clinical studies.1 For this reason, the study of RNA metabolism, including transcription, pre-mRNA processing, mRNA export, RNA trafficking and translation, represents a crucial milestone for understanding the biology of cells and molecular pathology of disease. However, when compared to our knowledge of proteins and genomes, our understanding of RNA's diverse biological roles is significantly lacking, in part because of the transient and complex dynamics of RNA and the challenges associated with precisely manipulating RNA metabolism from synthesis to degradation. While methods so far developed have made a significant impact in shedding light on the mysteries of RNA, tools that allow precise spatiotemporal control of RNA metabolism are still urgently needed for deeper insight into the diverse physiological functions of RNA.

Abstract: Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

Abstract: The expression of genes of interest (GOI) can be initiated by providing external stimuli such as temperature shifts and light irradiation. The application of thermal or light stimuli triggers structural changes in stimuli-sensitive biomolecules within the cell, thereby inducing or repressing gene expression. Over the past two decades, several groups have reported genetic circuits that use natural or engineered stimuli-sensitive modules to manipulate gene expression. Here, we summarize versatile strategies of thermosensors and light-driven systems for the conditional expression of GOI in bacterial hosts.

Abstract: Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.

Abstract: Molecular optogenetics is a highly dynamic research field. In the past two years, the field was characterized by the development of new allosteric switches as well as the forward integration of optogenetics research towards application. Further, two areas of research have significantly gathered momentum, the use of optogenetics to control liquid-liquid phase separation as well as the application of optogenetic tools in the extracellular space. Here, we review these areas and discuss future directions.

Abstract: Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to control gene expression independently from mammalian transcriptional and/or translational machineries thus working orthogonally to endogenous regulatory mechanisms. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize, and optimize a new optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool is orthogonal to the cellular machinery for transcription and consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7-mOptoT7). In our study we exploited the T7 polymerase's viral origins to tune our system's expression level, reaching up to an almost 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate the gene expression burden when compared to another optogenetic construct. These properties make mOptoT7 a powerful new tool to use when orthogonality and viral RNA species (that lack endogenous RNA modifications) are desired.