Curated Optogenetic Publication Database

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: Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Here we show that nuclear forces differentially control both passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than facilitated diffusion. Due to this differential effect, force leads to the translocation into or out of the nucleus of cargoes within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism, and engineered exogenously by introducing appropriate nuclear localization signals. Our work sets a novel framework to understand mechanically induced signalling, with potential general applicability across signalling pathways and pathophysiological scenarios.

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: 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: 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: 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: 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: 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: 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.

Abstract: Cell–cell adhesions are fundamental in regulating multicellular behavior and lie at the center of many biological processes from embryoid development to cancer development. Therefore, controlling cell–cell adhesions is fundamental to gaining insight into these phenomena and gaining tools that would help in the bioartificial construction of tissues. For addressing biological questions as well as bottom-up tissue engineering the challenge is to have multiple cell types self-assemble in parallel and organize in a desired pattern from a mixture of different cell types. Ideally, different cell types should be triggered to self-assemble with different stimuli without interfering with the other and different types of cells should sort out in a multicellular mixture into separate clusters. In this chapter, we will summarize the developments in photoregulation cell–cell adhesions using non-neuronal optogenetics. Among the concepts, we will cover is the control of homophylic and heterophilic cell–cell adhesions, the independent control of two different types with blue or red light and the self-sorting of cells into distinct structures and the importance of cell–cell adhesion dynamics. These tools will give an overview of how the spatiotemporal regulation of cell–cell adhesion gives insight into their role and how tissues can be assembled from cells as the basic building block.

Abstract: Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that cross-linked chromatin can mechanically suppress droplets' coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

Abstract: Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.

Abstract: Cells commonly communicate with each other through diffusible molecules but nonchemical communication remains elusive. While bioluminescent organisms communicate through light to find prey or attract mates, it is still under debate if signaling through light is possible at the cellular level. Here, we demonstrate that cell to cell signaling through light is possible in artificial cell communities derived from biomimetic vesicles. In our design, artificial sender cells produce an intracellular light signal, which triggers the adhesion to receiver cells. Unlike soluble molecules, the light signal propagates fast, independent of diffusion and without the need for a transporter across membranes. To obtain a predator-prey relationship, the luminescence predator cells is loaded with a secondary diffusible poison, which is transferred to the prey cell upon adhesion and leads to its lysis. This design provides a blueprint for light based intercellular communication, which can be used for programing artificial and natural cell communities.

Abstract: Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Abstract: Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.

Abstract: FG-repeat nucleoporins at the center of the nuclear pore complex (NPC) are highly modified with O-GlcNAc. In this issue, Yoo and Mitchison (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010141) use optogenetic probes to show that O-GlcNAc enhances permeability of the NPC, accelerating transport in both directions.

Abstract: Cell shape regulation is important but the mechanisms that govern shape are not fully understood, in part due to limited experimental models where cell shape changes and underlying molecular processes can be rapidly and non-invasively monitored in real time. Here, we use an optogenetic tool to activate RhoA in the middle of mononucleated macrophages to induce contraction, resulting in a side with the nucleus that retains its shape and a non-nucleated side which was unable to maintain its shape and collapsed. In cells overexpressing focal adhesion kinase (FAK), the non-nucleated side exhibited a wide flat morphology and was similar in adhesion area to the nucleated side. In cells overexpressing fascin, an actin bundling protein, the non-nucleated side assumed a spherical shape and was similar in height to the nucleated side. This effect of fascin was also observed in fibroblasts even without inducing furrow formation. Based on these results, we conclude that FAK and fascin work together to maintain cell shape by regulating adhesion area and height, respectively, in different cell types.

Abstract: The spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration.

Abstract: Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.