Curated Optogenetic Publication Database

Abstract: The last two decades have witnessed the emergence of optogenetics; a field that has given researchers the ability to use light to control biological processes at high spatio-temporal and quantitative resolution, in a reversible manner with minimal side effects. Optogenetics has revolutionised the neurosciences, increased our understanding of cellular signalling and metabolic networks and resulted in variety of applications in biotechnology and biomedicine. However, implementing optogenetics in plants has been less straight forward given their dependency on light for their life cycle. Here, we highlight some of the widely used technologies in microorganisms and animal systems derived from plant photoreceptor proteins and discuss strategies recently implemented to overcome the challenges for using optogenetics in plants.

Abstract: Understanding how cells self-organize into functional higher-order structures is of great interest, both towards deciphering animal development, as well as for our ability to predictably build custom tissues to meet research and therapeutic needs. The proper organization of cells across length-scales results from interconnected and dynamic networks of molecules and cells. Optogenetic probes provide dynamic and tunable control over molecular events within cells, and thus represent a powerful approach to both dissect and control collective cell behaviors. Here we emphasize the breadth of the optogenetic toolkit and discuss how these methods have already been used to reverse-engineer the design rules of developing organisms. We also offer our perspective on the rich potential for optogenetics to power forward-engineering of tissue assembly towards the generation of bespoke tissues with user-defined properties.

Abstract: The precise control of signaling proteins is a prerequisite to decipher the complexity of the signaling network and to reveal and to study pathways involved in regulating cellular metabolism and gene expression. Optogenetic approaches play an emerging role as they enable the spatiotemporal control of signaling processes. Herein, a multichromatic system is developed by combining the blue light cryptochrome 2 system and the red/far-red light phytochrome B system. The use of three wavelengths allows the orthogonal control of the RAF/ERK and the AKT signaling pathway. Continuous exposure of cells to blue light leads to activation of AKT while simultaneous pulses of red and far-red light enable the modulation of ERK signaling in cells with constantly active AKT signaling. The optimized, orthogonal multichromatic system presented here is a valuable tool to better understand the fine grained and intricate processes involved in cell fate decisions.

Abstract: Signaling networks control the flow of information through biological systems and coordinate the chemical processes that constitute cellular life. Optogenetic actuators - genetically encoded proteins that undergo light-induced changes in activity or conformation - are useful tools for probing signaling networks over time and space. They have permitted detailed dissections of cellular proliferation, differentiation, motility, and death, and enabled the assembly of synthetic systems with applications in areas as diverse as photography, chemical synthesis, and medicine. In this review, we provide a brief introduction to optogenetic systems and describe their application to molecular-level analyses of cell signaling. Our discussion highlights important research achievements and speculates on future opportunities to exploit optogenetic systems in the study and assembly of complex biochemical networks.

Abstract: Cybergenetic systems use computer interfaces to enable feed-back controls over biological processes in real time. The complex and dynamic nature of cellular metabolism makes cybergenetics attractive for controlling engineered metabolic pathways in microbial fermentations. Cybergenetics would not only create new avenues of research into cellular metabolism, it would also enable unprecedented strategies for pathway optimization and bioreactor operation and automation. Implementation of metabolic cybergenetics, however, will require new capabilities from actuators, biosensors, and control algorithms. The recent application of optogenetics in metabolic engineering, the expanding role of genetically encoded biosensors in strain development, and continued progress in control algorithms for biological processes suggest that this technology will become available in the not so distant future.

Abstract: Inhibition of receptor tyrosine kinases (RTKs) by small molecule inhibitors and monoclonal antibodies is used to treat cancer. Conversely, activation of RTKs with their ligands, including growth factors and insulin, is used to treat diabetes and neurodegeneration. However, conventional therapies that rely on injection of RTK inhibitors or activators do not provide spatiotemporal control over RTK signaling, which results in diminished efficiency and side effects. Recently, a number of optogenetic and optochemical approaches have been developed that allow RTK inhibition or activation in cells and in vivo with light. Light irradiation can control RTK signaling non-invasively, in a dosed manner, with high spatio-temporal precision, and without the side effects of conventional treatments. Here we provide an update on the current state of the art of optogenetic and optochemical RTK technologies and the prospects of their use in translational studies and therapy.

Abstract: Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).

Abstract: The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

Abstract: A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.

Abstract: Multi-objective optimization of microbial chassis for the production of xenobiotic compounds requires the implementation of metabolic control strategies that permit dynamic distribution of cellular resources between biomass and product formation. We addressed this need in a previous study by engineering the T7 RNA polymerase to be thermally responsive. The modified polymerase is activated only after the temperature of the host cell falls below 18oC, and Escherichia coli cells that employ the protein to transcribe the heterologous lycopene biosynthetic pathway exhibit impressive improvements in productivity. We have expanded our toolbox of metabolic switches in the current study by engineering a version of the T7 RNA polymerase that drives the transition between biomass and product formation upon stimulation with red light. The engineered polymerase is expressed as two distinct polypeptide chains. Each chain comprises one of two photoactive components from Arabidopsis thaliana, phytochrome B (PhyB) and phytochrome-integrating factor 3 (PIF3), as well as the N- or C-terminus domains of both, the vacuolar ATPase subunit (VMA) intein of Saccharomyces cerevisiae and the polymerase. Red light drives photodimerization of PhyB and PIF3, which then brings together the N- and C-terminus domains of the VMA intein. Trans-splicing of the intein follows suit and produces an active form of the polymerase that subsequently transcribes any sequence that is under the control of a T7 promoter. The photodimerization also involves a third element, the cyanobacterial chromophore phycocyanobilin (PCB), which too is expressed heterologously by E. coli. We deployed this version of the T7 RNA polymerase to control the production of lycopene in E. coli and observed tight control of pathway expression. We tested a variety of expression configurations to identify one that imposes the lowest metabolic burden on the strain, and we subsequently optimized key parameters such as the source, moment and duration of photostimulation. We also identified targets for future refinement of the circuit. In summary, our work is a significant advance for the field and greatly expands on previous work by other groups that have used optogenetic circuits to control heterologous metabolism in prokaryotic hosts.

Abstract: Precise manipulation of gene expression with temporal and spatial control is essential for functional analysis and determining cell lineage relationships in complex biological systems. The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre. Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. Upon red light illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of the cofactor phycocyanobilin (PCB) to restore Cre activity. Red light exposure of zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter injected with CreLite mRNAs and PCB resulted in Cre activity as measured by the generation of multi-spectral cell labeling in several different tissues. Our data show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different developmental stages, and suggests CreLite may also be useful for temporal and spatial control of gene expression in cell culture, ex vivo organ culture, and other animal models. This article is protected by copyright. All rights reserved.

Abstract: Since the neurobiological inception of optogenetics, light-controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light-sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle-specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Physiology of Model Organisms Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Cellular Models.

Abstract: G protein-coupled receptors (GPCRs) form the largest class of membrane receptors in the mammalian genome with nearly 800 human genes encoding for unique subtypes. Accordingly, GPCR signaling is implicated in nearly all physiological processes. However, GPCRs have been difficult to study due in part to the complexity of their function which can lead to a plethora of converging or diverging downstream effects over different time and length scales. Classic techniques such as pharmacological control, genetic knockout and biochemical assays often lack the precision required to probe the functions of specific GPCR subtypes. Here we describe the rapidly growing set of optogenetic tools, ranging from methods for optical control of the receptor itself to optical sensing and manipulation of downstream effectors. These tools permit the quantitative measurements of GPCRs and their downstream signaling with high specificity and spatiotemporal precision.

Abstract: Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90 min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.

Abstract: Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.

Abstract: In order to understand the systematic regulation of the cell cycle, we need more precise tools for cell-cycle perturbation. Optogenetics is a powerful technique for precisely controlling cellular signaling at higher spatial and temporal resolution. Here, we report optogenetic tools for the rapid and reversible control of cell-cycle checkpoints with a red/far-red light photoreceptor, phytochrome B (PhyB). We established fission yeast cells producing phycocyanobilin as a chromophore of PhyB, and demonstrated light-dependent protein recruitment to the plasma membrane, nucleus, and kinetochore. Using this system, we developed optogenetic manipulation of the cell cycle in two ways: the Opto-G2/M checkpoint triggered G2/M cell cycle arrest in response to red light, and Opto-SAC induced a spindle assembly checkpoint (SAC) in response to red light and then quickly released the SAC by far-red light.

Abstract: We present here PhotoGal4, a phytochrome B-based optogenetic switch for fine-tuned spatiotemporal control of gene expression in Drosophila explants. This switch integrates the light-dependent interaction between phytochrome B and PIF6 from plants with regulatory elements from the yeast Gal4/UAS system. We found that PhotoGal4 efficiently activates and deactivates gene expression upon red- or far-red-light irradiation, respectively. In addition, this optogenetic tool reacts to different illumination conditions, allowing for fine modulation of the light-dependent response. Importantly, by simply focusing a laser beam, PhotoGal4 induces intricate patterns of expression in a customized manner. For instance, we successfully sketched personalized patterns of GFP fluorescence such as emoji-like shapes or letterform logos in Drosophila explants, which illustrates the exquisite precision and versatility of this tool. Hence, we anticipate that PhotoGal4 will expand the powerful Drosophila toolbox and will provide a new avenue to investigate intricate and complex problems in biomedical research.

Abstract: Plant phytochromes enable vital adaptations to red and far-red light. At the molecular level, these responses are mediated by light-regulated interactions between phytochromes and partner proteins, foremost the phytochrome-interacting factors (PIF). Although known for decades, quantitative analyses of these interactions have long been sparse. To address this deficit, we here studied by an integrated fluorescence-spectroscopic approach the equilibrium and kinetics of Arabidopsis thaliana phytochrome B (AtPhyB) binding to a tetramerized PIF6 variant. Several readouts consistently showed the stringently light-regulated interaction to be little affected by PIF tetramerization. Analysis of the binding kinetics allowed the determination of bimolecular association and unimolecular dissociation rate constants as a function of light. Unexpectedly, the stronger affinity of AtPhyB under red light relative to far-red light is entirely due to accelerated association rather than decelerated dissociation. The association reaction under red light is highly efficient and only threefold slower than the diffusion limit. The present findings pertain equally to the analysis of signal transduction in plants and to the biotechnological application of phytochromes.

Abstract: The zebrafish (Danio rerio) is a popular vertebrate model organism to investigate molecular mechanisms driving development and disease. Due to its transparency at embryonic and larval stages, investigations in the living organism are possible with subcellular resolution using intravital microscopy. The beneficial optical characteristics of zebrafish not only allow for passive observation, but also active manipulation of proteins and cells by light using optogenetic tools. Initially, photosensitive ion channels have been applied for neurobiological studies in zebrafish to dissect complex behaviors on a cellular level. More recently, exciting non-neural optogenetic tools have been established to control gene expression or protein localization and activity, allowing for unprecedented non-invasive and precise manipulation of various aspects of cellular physiology. Zebrafish will likely be a vertebrate model organism at the forefront of in vivo application of non-neural optogenetic tools and pioneering work has already been performed. In this review, we provide an overview of non-neuromodulatory optogenetic tools successfully applied in zebrafish to control gene expression, protein localization, cell signaling, migration and cell ablation.

Abstract: Morphogenesis of multicellular systems is governed by precise spatiotemporal regulation of biochemical reactions and mechanical forces which together with environmental conditions determine the development of complex organisms. Current efforts in the field aim at decoding the system-level principles underlying the regulation of developmental processes. Toward this goal, optogenetics, the science of regulation of protein function with light, is emerging as a powerful new tool to quantitatively perturb protein function in vivo with unprecedented precision in space and time. In this review, we provide an overview of how optogenetics is helping to address system-level questions of multicellular morphogenesis and discuss future directions.

Abstract: Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.

Abstract: T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx.

Abstract: In the field of extracellular optogenetics, photoreceptors are applied outside of cells to obtain systems with a desired functionality. Among the diverse applied photoreceptors, phytochromes are the only ones that can be actively and reversibly switched between the active and inactive photostate by the illumination with cell-compatible red and far-red light. In this protocol, we describe the production of a biotinylated variant of the photosensory domain of A. thaliana phytochrome B (PhyB-AviTag) in E. coli with a single, optimized expression plasmid. We give detailed instructions for the purification of the protein by immobilized metal affinity chromatography and the characterization of the protein in terms of purity, biotinylation, spectral photoswitching and the light-dependent interaction with its interaction partner PIF6. In comparison to previous studies applying PhyB-AviTag, the optimized expression plasmid used in this protocol simplifies the production process and shows an increased yield and purity.

Abstract: Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.

Abstract: Tissue repair is a complex process that requires effective communication and coordination between cells across multiple tissues and organ systems. Two of the initial intracellular signals that encode injury signals and initiate tissue repair responses are calcium and extracellular signal-regulated kinase (ERK). However, calcium and ERK signaling control a variety of cellular behaviors important for injury repair including cellular motility, contractility, and proliferation, as well as the activity of several different transcription factors, making it challenging to relate specific injury signals to their respective repair programs. This knowledge gap ultimately hinders the development of new wound healing therapies that could take advantage of native cellular signaling programs to more effectively repair tissue damage. The objective of this review is to highlight the roles of calcium and ERK signaling dynamics as mechanisms that link specific injury signals to specific cellular repair programs during epithelial and stromal injury repair. We detail how the signaling networks controlling calcium and ERK can now also be dissected using classical signal processing techniques with the advent of new biosensors and optogenetic signal controllers. Finally, we advocate the importance of recognizing calcium and ERK dynamics as key links between injury detection and injury repair programs that both organize and execute a coordinated tissue repair response between cells across different tissues and organs. This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Biological Mechanisms > Cell Signaling Laboratory Methods and Technologies > Imaging Models of Systems Properties and Processes > Organ, Tissue, and Physiological Models.