Curated Optogenetic Publication Database

Abstract: Upconversion-mediated optogenetics is an emerging powerful technique to remotely control and manipulate the deep-tissue protein functions and signaling pathway activation. This technique uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and through near-infrared light to indirectly activate the traditional optogenetic proteins. With the merits of high spatiotemporal resolution and minimal invasiveness, this technique enables cell-type specific manipulation of cellular activities in deep tissues as well as in living animals. In this review, we introduce the latest development of optogenetic modules and UCNPs, with emphasis on the integration of UCNPs with cellular optogenetics and their biomedical applications on the control of neural/brain activity, cancer therapy and cardiac optogenetics in vivo. Furthermore, we analyze the current developed strategies to optimize and advance the upconversion-mediated optogenetics and discuss the remaining challenges of its further applications in biomedical study and clinical translational research. STATEMENT OF SIGNIFICANCE: Optogenetics harnesses photoactivatable proteins to optically stimulate and control intracellular activities. UCNPs-mediated NIR-activatable optogenetics uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and utilizes near-infrared (NIR) light to indirectly activate the traditional optogenetic proteins. The integration of UCNPs with cellular optogenetics has showed great promise in biomedical applications in regulating neural/brain activity, cancer therapy and cardiac optogenetics in vivo. The evolution and optimization of functional UCNPs and the discovery and engineering of novel optogenetic modules would both contribute to the advance of such unique hybrid technology, which may lead to discoveries in biomedical research and provide new treatments for human diseases.

Abstract: The photochemistry of cobalamins has recently been found to have biological importance, with the discovery of bacterial photoreceptor proteins, such as CarH and AerR. CarH and AerR, are involved in the light regulation of carotenoid biosynthesis and bacteriochlorophyll biosynthesis, respectively, in bacteria. Experimental transient absorption spectroscopic studies have indicated unusual photochemical behavior of 5'-deoxy-5'-adenosylcobalamin (AdoCbl) in CarH, with excited-state charge separation between cobalt and adenosyl and possible heterolytic cleavage of the Co-adenosyl bond, as opposed to the homolytic cleavage observed in aqueous solution and in many AdoCbl-based enzymes. We employ molecular dynamics and hybrid quantum mechanical/molecular mechanical calculations to obtain a microscopic understanding of the modulation of the excited electronic states of AdoCbl by the CarH protein environment, in contrast to aqueous solution and AdoCbl-based enzymes. Our results indicate a progressive stabilization of the electronic states involving charge transfer (CT) from cobalt/corrin to adenine on changing the environment from gas phase to water to solvated CarH. The solvent exposure of the adenosyl ligand in CarH, the π-stacking interaction between a tryptophan and the adenine moiety, and the hydrogen-bonding interaction between a glutamate and the lower axial ligand of cobalt are found to contribute to the stabilization of the states involving CT to adenine. The combination of these three factors, the latter two of which can be experimentally tested via mutagenesis studies, is absent in an aqueous solvent environment and in AdoCbl-based enzymes. The favored CT from metal and/or corrin to adenine in CarH may promote heterolytic cleavage of the cobalt-adenosyl bond proposed by experimental studies. Overall, this work provides novel, to our knowledge, physical insights into the mechanism of CarH function and directions for future experimental investigations. The fundamental understanding of the mechanism of CarH functioning will serve the development of optogenetic tools based on the new class of B12-dependent photoreceptors.

Abstract: The field of optogenetics is rapidly growing in relevance and number of developed tools. Amongst other things, the optogenetic repertoire includes light-responsive ion channels and methods for gene regulation. This review will be confined to the optogenetic control of gene expression in mammalian cells as suitable models for clinical applications. Here optogenetic gene regulation might offer an excellent method for spatially and timely regulated gene and protein expression in cell therapeutic approaches. Well-known systems for gene regulation, such as the LOV-, CRY2/CIB-, PhyB/PIF-systems, as well as other, in mammalian cells not yet fully established systems will be described. Advantages and disadvantages with regard to clinical applications are outlined in detail. Among the many unanswered questions concerning the application of optogenetics, we discuss items such as the use of exogenous chromophores and their effects on the biology of the cells and methods for a gentle, but effective gene transfection method for optogenetic tools for in vivo applications. This article is protected by copyright. All rights reserved.

Abstract: Optogenetics initially used plant photoreceptors to monitor neural circuits, later it has expanded to include engineered plant photoreceptors. Recently photoreceptors from bacteria, algae and cyanobacteria have been used as an optogenetic tool. Bilin-based photoreceptors are common light-sensitive photoswitches in plants, algae, bacteria and cyanobacteria. Here we discuss the photoreceptors from cyanobacteria. Several new photoreceptors have been explored in cyanobacteria which are now proposed as cyanobacteriochrome. The domains in the cyanobacteriochrome, light-induced signaling transduction, photoconversion, are the most attractive features for the optogenetic system. The wider spectral feature of cyanobacteriochrome from UV to visible radiation makes it a light potential sensitive optogenetic tool. Besides, cyanobacterial phytochrome responses to yellow, orange and blue light have more application in optogenetics. This chapter summarizes the photoconversion, phototaxis, cell aggregation, cell signaling mediated by cyanobacteriochrome and cyanophytochrome. As there is a wide range of cyanobacteriochrome and its combination delivers a varied light-sensitive response. Besides coordination among cyanobacteriochromes in cell signaling reduces the engineering of photoreceptors for the optogenetic system.

Abstract: Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.

Abstract: Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

Abstract: Abstract not available.

Abstract: Recently, re-purposing of cyanobacterial photoreceptors as optogentic actuators enabled light-regulated protein expression in different host systems. These new bi-stable optogenetic tools enable interesting new applications, but their light-driven working mechanism remains largely elusive on a molecular level. Here, we study the optogenetic cyanobacteriochrome Am1-c0023g2 with isotope labeling and two dimensional infrared (2D-IR) spectroscopy. Isotope labeling allows us to isolate two site-specific carbonyl marker modes from the overwhelming mid-IR signal of the peptide backbone vibrations. Unlike conventional difference-FTIR spectroscopy, 2D-IR is sensitive to homogeneous and inhomogeneous broadening mechanisms of these two vibrational probes in the different photostates of the protein. We analyse the 2D-IR line shapes in the context of available structural models and find that they reflect the hydrogen-bonding environment of these two marker groups.

Abstract: Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Abstract: Optimization of engineered biological systems requires precise control over the rates and timing of gene expression. Optogenetics is used to dynamically control gene expression as an alternative to conventional chemical-based methods since it provides a more convenient interface between digital control software and microbial culture. Here, we describe the construction of a real-time optogenetics platform, which performs closed-loop control over the CcaR-CcaS two-plasmid system in Escherichia coli. We showed the first model-based design approach by constructing a nonlinear representation of the CcaR-CcaS system, tuned the model through open-loop experimentation to capture the experimental behavior, and applied the model in silico to inform the necessary changes to build a closed-loop optogenetic control system. Our system periodically induces and represses the CcaR-CcaS system while recording optical density and fluorescence using image processing techniques. We highlight the facile nature of constructing our system and how our model-based design approach will potentially be used to model other systems requiring closed-loop optogenetic control.

Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.

Abstract: The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.

Abstract: Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.

Abstract: Dynamically tunable biomaterials are of particular interest in the field of biomedical engineering because of the potential utility for shape-change materials, drug and cell delivery and tissue regeneration. Stimuli-responsive proteins formed into hydrogels are potential candidates for such systems, due to the genetic tailorability and control over structure-function relationships. Here we report the synthesis of genetically engineered Silk-Elastin-Like Protein (SELP) photoresponsive hydrogels. Polymerization of the SELPs and monomeric adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) was achieved using genetically encoded SpyTag-SpyCatcher peptide-protein pairs under mild physiological conditions. The hydrogels exhibited a partial collapse of the crosslinked molecular network with both decreased loss and storage moduli upon exposure to visible light. The materials were also evaluated for cytotoxicity and the encapsulation and release of L929 murine fibroblasts from 3D cultures. The design of these photo-responsible proteins provides new stimuli-responsive SELP-CarHC hydrogels for dynamically tunable protein-based materials.

Abstract: Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.

Abstract: Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.

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: 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: Axon regeneration constitutes a fundamental challenge for regenerative neurobiology, which necessitates the use of tailor-made biomaterials for controllable delivery of cells and biomolecules. An increasingly popular approach for creating these materials is to directly assemble engineered proteins into high-order structures, a process that often relies on sophisticated protein chemistry. Here, we present a simple approach for creating injectable, photoresponsive hydrogels via metal-directed assembly of His6-tagged proteins. The B12-dependent photoreceptor protein CarHC can complex with transition metal ions through an amino-terminal His6-tag, which can further undergo a sol-gel transition upon addition of AdoB12, leading to the formation of hydrogels with marked injectability and photodegradability. The inducible phase transitions further enabled facile encapsulation and release of cells and proteins. Injecting the Zn2+-coordinated gels decorated with leukemia inhibitory factor into injured mouse optic nerves led to prolonged cellular signaling and enhanced axon regeneration. This study illustrates a powerful strategy for designing injectable biomaterials.

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: 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: The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.Bio, a parallelised open-source platform that represents a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities, it incorporates tunable light outputs, spectrometry, and advanced automation features. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins in situ. By integrating precise measurement and actuation hardware into a single low-cost platform, Chi.Bio facilitates novel experimental methods for synthetic, systems, and evolutionary biology and broadens access to cutting-edge research capabilities.

Abstract: Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware.

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.