Curated Optogenetic Publication Database

Abstract: Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Abstract: Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spatiotemporal control of RhoA GTPase. Cytokinesis failure can lead to changes in cell fate or aneuploidy, which can be detrimental during development and/or can lead to cancer. However, our knowledge of the pathways that regulate RhoA during cytokinesis is limited, and the role of other Rho family GTPases is not clear. This is largely because the study of Rho GTPases presents unique challenges using traditional cell biological and biochemical methods, and they have pleiotropic functions making genetic studies difficult to interpret. The recent generation of optogenetic tools and biosensors that control and detect active Rho has overcome some of these challenges and is helping to elucidate the role of RhoA in cytokinesis. However, improvements are needed to reveal the role of other Rho GTPases in cytokinesis, and to identify the molecular mechanisms that control Rho activity. This review examines some of the outstanding questions in cytokinesis, and explores tools for the imaging and control of Rho GTPases.

Abstract: During embryonic development, signalling pathways orchestrate organogenesis by controlling tissue-specific gene expression programmes and differentiation. Although the molecular components of many common developmental signalling systems are known, our current understanding of how signalling inputs are translated into gene expression outputs in real-time is limited. Here we employ optogenetics to control the activation of Notch signalling during Drosophila embryogenesis with minute accuracy and follow target gene expression by quantitative live imaging. Light-induced nuclear translocation of the Notch Intracellular Domain (NICD) causes a rapid activation of target mRNA expression. However, target gene transcription gradually decays over time despite continuous photo-activation and nuclear NICD accumulation, indicating dynamic adaptation to the signalling input. Using mathematical modelling and molecular perturbations, we show that this adaptive transcriptional response fits to known motifs capable of generating near-perfect adaptation and can be best explained by state-dependent inactivation at the target cis-regulatory region. Taken together, our results reveal dynamic nuclear adaptation as a novel mechanism controlling Notch signalling output during tissue differentiation.

Abstract: Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.

Abstract: Protein-based hydrogels can mimic many aspects of native extracellular matrices (ECMs) and are promising biomedical materials that find various applications in cell proliferation, drug/cell delivery, and tissue engineering. To be adapted for different tasks, it is important that the mechanical and/or biochemical properties of protein-based hydrogels can be regulated by external stimuli. Light as a regulation stimulus is of advantage because it can be easily applied in demanded spatiotemporal manners. The noncovalent binding between the light-oxygen-voltage-sensing domain 2 (LOV2) and its binding partner ZDark1 (zdk1), named as LOVTRAP, is a light-responsive interaction. The binding affinity of LOVTRAP is much higher in dark than that under blue light irradiation. Taking advantage of these light-responsive interactions, herein we endeavored to use LOVTRAP as a crosslinking mechanism to engineer light-responsive protein hydrogels. Using LOV2-containing and zdk1-containing multifunctional protein building blocks, we successfully engineered a light-responsive protein hydrogel whose viscoelastic properties can change in response to light: in the dark, the hydrogel showed higher storage modulus; under blue light irradiation, the storage modulus decreased. Due to the noncovalent nature of the LOVTRAP, the engineered LOVTRAP protein hydrogels displayed shear-thinning and self-healing properties and served as an excellent injectable protein hydrogel. We anticipated that this new class of light-responsive protein hydrogels will broaden the scope of dynamic protein hydrogels and help develop other light-responsive protein hydrogels for biomedical applications.

Abstract: Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Abstract: Optogenetics has opened new insights into biomedical research with the ability to manipulate and control cellular activity using light in combination with genetically engineered photosensitive proteins. By stimulating with light, this method provides high spatiotemporal and high specificity resolution, which is in contrast to conventional pharmacological or electrical stimulation. Optogenetics was initially introduced to control neural activities but was gradually extended to other biomedical fields.

Abstract: Microbial co-culture fermentations can improve chemical production from complex biosynthetic pathways over monocultures by distributing enzymes across multiple strains, thereby reducing metabolic burden, overcoming endogenous regulatory mechanisms, or exploiting natural traits of different microbial species. However, stabilizing and optimizing microbial subpopulations for maximal chemical production remains a major obstacle in the field. In this study, we demonstrate that optogenetics is an effective strategy to dynamically control populations in microbial co-cultures. Using a new optogenetic circuit we call OptoTA, we regulate an endogenous toxin-antitoxin system, enabling tunability of Escherichia coli growth using only blue light. With this system we can control the population composition of co-cultures of E. coli and Saccharomyces cerevisiae. When introducing in each strain different metabolic modules of biosynthetic pathways for isobutyl acetate or naringenin, we found that the productivity of co-cultures increases by adjusting the population ratios with specific light duty cycles. This study shows the feasibility of using optogenetics to control microbial consortia populations and the advantages of using light to control their chemical production.

Abstract: Bidirectional optogenetic control of yeast gene expression has great potential for biotechnological applications. Our group has developed optogenetic inverter circuits that activate transcription using darkness, as well as amplifier circuits that reach high expression levels under limited light. However, because both types of circuits harness Gal4p and Gal80p from the galactose (GAL) regulon they cannot be used simultaneously. Here, we apply the Q System, a transcriptional activator/inhibitor system from Neurospora crassa, to build circuits in Saccharomyces cerevisiae that are inducible using quinic acid, darkness, or blue light. We develop light-repressed OptoQ-INVRT circuits that initiate darkness-triggered transcription within an hour of induction, as well as light-activated OptoQ-AMP circuits that achieve up to 39-fold induction. The Q System does not exhibit crosstalk with the GAL regulon, allowing coutilization of OptoQ-AMP circuits with previously developed OptoINVRT circuits. As a demonstration of practical applications in metabolic engineering, we show how simultaneous use of these circuits can be used to dynamically control both growth and production to improve acetoin production, as well as enable light-tunable co-production of geraniol and linalool, two terpenoids implicated in the hoppy flavor of beer. OptoQ-AMP and OptoQ-INVRT circuits enable simultaneous optogenetic signal amplification and inversion, providing powerful additions to the yeast optogenetic toolkit.

Abstract: Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. Here, we present the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, we substantially increased LEXY's efficiency and generated variants with different recovery times. iLEXY enables rapid (t1/2 < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, we phenocopy loss-of-function mutants, precisely map the Twist-sensitive embryonic stages, and investigate the effects of timed Twist depletions. Our results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.

Abstract: The ability to stimulate mammalian cells with light, brought along by optogenetic control, has significantly broadened our understanding of electrically excitable tissues. Backed by advanced (bio)materials, it has recently paved the way towards novel biosensing concepts supporting bio-analytics applications transversal to the main biomedical stream. The advancements concerning enabling biomaterials and related novel biosensing concepts involving optogenetics are reviewed with particular focus on the use of engineered cells for cell-based sensing platforms and the available toolbox (from mere actuators and reporters to novel multifunctional opto-chemogenetic tools) for optogenetic-enabled real-time cellular diagnostics and biosensor development. The key advantages of these modified cell-based biosensors concern both significantly faster (minutes instead of hours) and higher sensitivity detection of low concentrations of bioactive/toxic analytes (below the threshold concentrations in classical cellular sensors) as well as improved standardization as warranted by unified analytic platforms. These novel multimodal functional electro-optical label-free assays are reviewed among the key elements for optogenetic-based biosensing standardization. This focused review is a potential guide for materials researchers interested in biosensing based on light-responsive biomaterials and related analytic tools.

Abstract: We re-engineered a commonly-used light-sensing protein, AsLOV2, using a circular permutation strategy to allow photoswitchable control of the C-terminus of a peptide. We demonstrate that the circularly permuted AsLOV2 can be used on its own or together with the original AsLOV2 for enhanced caging. In summary, circularly permuted AsLOV2 could expand the engineering capabilities of optogenetic tools.

Abstract: Inducible biomolecular condensates play fundamental roles in cellular responses to intracellular and environmental cues. Knowledge about their composition is crucial to understand the functions that arise specifically from the assembly of condensates. This protocol combines an optogenetic and an efficient proximity labeling approach to analyze protein modifications driven by protein condensation in cultured cells. Low endogenous biotin level ensures sharp signals. For complete details on the use and execution of this protocol, please refer to Frattini et al. (2021).

Abstract: Lipids are highly dynamic molecules that, due to their hydrophobicity, are spatially confined to membrane environments. From these locations, certain privileged lipids serve as signaling molecules. For understanding the biological functions of subcellular pools of signaling lipids, induced proximity tools have been invaluable. These methods involve controlled heterodimerization, by either small-molecule or light triggers, of functional proteins. In the arena of lipid signaling, induced proximity tools can recruit lipid-metabolizing enzymes to manipulate lipid signaling and create artificial tethers between organelle membranes to control lipid trafficking pathways at membrane contact sites. Here, we review recent advances in methodology development and biological application of chemical-induced and light-induced proximity tools for manipulating lipid metabolism, trafficking, and signaling.

Abstract: Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation drives yes-associated protein (YAP) nuclear localization within minutes and consequent mechanotransduction verified by YAP-transcriptional enhanced associate domain transcriptional activity. These single-transgene tools do not require protein binding partners for dynamic membrane localization and permit spatiotemporally precise control over RhoA signaling to advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

Abstract: Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar-polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin pattern influence the multiple cell behaviors, including cell intercalation, cell shape changes, and apical cell area fluctuations, that simultaneously occur during morphogenesis. Here, we developed two optogenetic tools, optoGEF and optoGAP, to activate or deactivate Rho1 signaling, respectively. We used these tools to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation and analyzed the effects on contractile cell behaviors. We show that uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar-polarized pattern of myosin at cell junctions on the timescale of 3-5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation but have distinct effects on cell area fluctuations and cell packings that are linked with changes in the medial and junctional myosin pools. These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar-polarized myosin pattern in the germband during axis elongation. Moreover, our results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles not only in organizing the cell rearrangements that are known to directly contribute to axis elongation but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.

Abstract: With the rise of new tools, from controlled genetic manipulations and optogenetics to improved microscopy, it is now possible to make clear, quantitative and reproducible measurements of biological processes. The humble fruit fly Drosophila melanogaster, with its ease of genetic manipulation combined with excellent imaging accessibility, has become a major model system for performing quantitative in vivo measurements. Such measurements are driving a new wave of interest from physicists and engineers, who are developing a range of testable dynamic models of active systems to understand fundamental biological processes. The reproducibility of the early Drosophila embryo has been crucial for understanding how biological systems are robust to unavoidable noise during development. Insights from quantitative in vivo experiments in the Drosophila embryo are having an impact on our understanding of critical biological processes, such as how cells make decisions and how complex tissue shape emerges. Here, to highlight the power of using Drosophila embryogenesis for quantitative biology, I focus on three main areas: (1) formation and robustness of morphogen gradients; (2) how gene regulatory networks ensure precise boundary formation; and (3) how mechanical interactions drive packing and tissue folding. I further discuss how such data has driven advances in modelling.

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: Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus in glucose and cellulosic media at different temperatures.

Abstract: Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Although Htl has been extensively studied, the dynamics of its action are poorly understood after the initial phases of mesoderm formation and spreading. To begin to address this challenge, we have developed an optogenetic version of the FGFR Heartless in Drosophila (Opto-htl). Opto-htl enables us to activate the FGFR pathway in selective spatial (~ 35 μm section from one of the lateral sides of the embryo) and temporal domains (ranging from 40 min to 14 h) during embryogenesis. Importantly, the effects can be tuned by the intensity of light-activation, making this approach significantly more flexible than other genetic approaches. We performed controlled perturbations to the FGFR pathway to define the contribution of Htl signalling to the formation of the developing embryonic heart and somatic muscles. We find a direct correlation between Htl signalling dosage and number of Tinman-positive heart cells specified. Opto-htl activation favours the specification of Tinman positive cardioblasts and eliminates Eve-positive DA1 muscles. This effect is seen to increase progressively with increasing light intensity. Therefore, fine tuning of phenotypic responses to varied Htl signalling dosage can be achieved more conveniently than with other genetic approaches. Overall, Opto-htl is a powerful new tool for dissecting the role of FGFR signalling during development.

Abstract: Many organs and tissues have an intrinsic ability to regenerate from a dedicated, tissue-specific stem cell pool. As organisms age, the process of self-regulation or homeostasis begins to slow down with fewer stem cells available for tissue repair. Tissues become more fragile and organs less efficient. This slowdown of homeostatic processes leads to the development of cellular and neurodegenerative diseases. In this review, we highlight the recent use and future potential of optogenetic approaches to study homeostasis. Optogenetics uses photosensitive molecules and genetic engineering to modulate cellular activity in vivo, allowing precise experiments with spatiotemporal control. We look at applications of this technology for understanding the mechanisms governing homeostasis and degeneration as applied to widely used model organisms, such as Drosophila melanogaster, where other common tools are less effective or unavailable.

Abstract: Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Abstract: Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated transport of proteins in both directions, and decreasing modification slowed transport. Superresolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the nonspecific permeability of the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.

Abstract: Engineered anaerobic bacteria known as live biotherapeutic products (LBPs) have shown great advances in cancer therapy. One advantage of anaerobic bacteria as drug carrier is that it spontaneously target to tumor and persistently release anti-tumor factors. To realize effective anti-cancer therapeutics, one essential premise is to improve the controllability of treatment. Here, we designed near-infrared (NIR)-light responsive bacteria as anti-tumor agent, which is based on a blue-light responsive module and upconversion nanoparticles. The upconversion nanoparticles converted external NIR light to local blue light to noninvasively activate blue-light responsive module (EL222) in engineered LBPs. The activated LBPs then produce tumor necrosis factor α (TNFα) for precise tumor ablation. In vitro and in vivo results have proven that this engineered NIR-light-responsive bacteria could efficiently inhibit tumor growth. We anticipate that this controllable and safe bacteria-based therapy can facilitate the application of LBPs to accurately and effectively regulate diseases.

Abstract: Protein-protein interactions (PPIs) are ubiquitously involved in cellular processes such as gene expression, enzymatic catalysis, and signal transduction. To study dynamic PPIs, real-time methods such as Förster resonance energy transfer and bioluminescence resonance energy transfer can provide high temporal resolution, but they only allow PPI detection in a limited area at a time and do not permit post-PPI analysis or manipulation of the cells. Integration methods such as the yeast two-hybrid system and split protein systems integrate PPI signals over time and allow subsequent analysis, but they lose information on dynamics. To address some of these limitations, an assay named SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics) has recently been published. Similar to many existing integrators, SPARK converts PPIs into a transcriptional signal. SPARK, however, also adds blue light as a co-stimulus to achieve temporal gating; SPARK only records PPIs during light stimulation. Here, we describe the procedures for using SPARK assays to study a dynamic PPI of interest, including designing DNA constructs and optimization in HEK293T/17 cell cultures. These protocols are generally applicable to various PPI partners and can be used in different biological contexts. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Designing DNA constructs for SPARK Basic Protocol 2: Performing the SPARK assay in HEK293T/17 cell cultures Support Protocol 1: Lentivirus preparation Support Protocol 2: Immunostaining of SPARK components.