Curated Optogenetic Publication Database

Abstract: The enteroendocrine system plays an important role in metabolism. The gut microbiome regulates enteroendocrine in an extensive way, arousing attention in biomedicine. However, conventional strategies of enteroendocrine regulation via gut microbiome are usually non-specific or imprecise. Here, an optogenetic operated probiotics system was developed combining synthetic biology and flexible electronics to achieve in situ controllable secretion to mimic enteroendocrine. Firstly, optogenetic engineered Lactococcus lactis (L. lactis) were administrated in the intestinal tract. A wearable optogenetic device was designed to control optical signals remotely. Then, L. lactis could secrete enteroendocrine hormone according to optical signals. As an example, optogenetic L. lactis could secrete glucagon-like peptide-1(GLP-1) under the control of the wearable optogenetic device. To improve the half-life of GLP-1 in vivo, the Fc domain from immunoglobulin was fused. Treated with this strategy, blood glucose, weight and other features were relatively well controlled in rats and mice models. Furthermore, up-conversion microcapsules were introduced to increase the excitation wavelength of the optogenetic system for better penetrability. This strategy has biomedical potential in metabolic diseases therapy by mimicking enteroendocrine.

Abstract: Deciphering gene function requires the ability to control gene expression in space and time. Binary systems such as the Gal4/UAS provide a powerful means to modulate gene expression and to induce loss or gain of function. This is best exemplified in Drosophila, where the Gal4/UAS system has been critical to discover conserved mechanisms in development, physiology, neurobiology, and metabolism, to cite a few. Here we describe a transgenic light-inducible Gal4/UAS system (ShineGal4/UAS) based on Magnet photoswitches. We show that it allows efficient, rapid, and robust activation of UAS-driven transgenes in different tissues and at various developmental stages in Drosophila. Furthermore, we illustrate how ShineGal4 enables the generation of gain and loss-of-function phenotypes at animal, organ, and cellular levels. Thanks to the large repertoire of UAS-driven transgenes, ShineGal4 enriches the Drosophila genetic toolkit by allowing in vivo control of gene expression with high temporal and spatial resolutions.

Abstract: The type III secretion system (T3SS) enables direct injection of bacterial effector proteins into eukaryotic cells. We found that the dynamic cytosolic interface of the system allows Yersinia enterocolitica to suppress premature secretion at low pH, ensuring rapid activation at the site of action. Exploiting this principle, we developed a light-controlled T3SS based on optogenetic interaction switches, which provides unprecedented spatiotemporal control of protein secretion and translocation.

Abstract: Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

Abstract: Synthetic biology seeks to develop modular biocircuits that combine to produce complex, controllable behaviors. These designs are often subject to noisy fluctuations and uncertainties, and most modern synthetic biology design processes have focused to create robust components to mitigate the noise of gene expression and reduce the heterogeneity of single-cell responses. However, a deeper understanding of noise can achieve control goals that would otherwise be impossible. We explore how an "Optogenetic Maxwell Demon" could selectively amplify noise to control multiple cells using single-input-multiple-output (SIMO) feedback. Using data-constrained stochastic model simulations and theory, we show how an appropriately selected stochastic SIMO controller can drive multiple different cells to different user-specified configurations irrespective of initial conditions. We explore how controllability depends on cells' regulatory structures, the amount of information available to the controller, and the accuracy of the model used. Our results suggest that gene regulation noise, when combined with optogenetic feedback and non-linear biochemical auto-regulation, can achieve synergy to enable precise control of complex stochastic processes.

Abstract: The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood.

Abstract: Advances in protein design have brought us within reach of developing a nanoscale programming language, in which molecules serve as operands and their conformational states function as logic gates with precise input and output behaviors. Combining these nanoscale computing agents into larger molecules and molecular complexes will allow us to write and execute "code". Here, in an important step toward this goal, we report an engineered, single protein design that is allosterically regulated to function as a 'two-input logic OR gate'. Our system is based on chemo- and optogenetic regulation of focal adhesion kinase. In the engineered FAK, all of FAK domain architecture is retained and key intramolecular interactions between the kinase and the FERM domains are externally controlled through a rapamycin-inducible uniRapR module in the kinase domain and a light-inducible LOV2 module in the FERM domain. Orthogonal regulation of protein function was possible using the chemo- and optogenetic switches. We demonstrate that dynamic FAK activation profoundly increased cell multiaxial complexity in the fibrous extracellular matrix microenvironment and decreased cell motility. This work provides proof-of-principle for fine multimodal control of protein function and paves the way for construction of complex nanoscale computing agents.

Abstract: Photosensor proteins are important not only because of their biological functions but also because of their applications in optogenetics. To understand the molecular mechanism behind their biological functions and consequently seek possible applications to optogenetics, the dynamics of their intermolecular interaction (for example, association/dissociation reaction and conformational changes) upon photoexcitation need to be elucidated. Although it has been difficult to trace such reactions in the time domain using traditional spectroscopic techniques, the time-resolved diffusion method based on the transient grating technique has been demonstrated to possess a significant advantage in detecting such spectrally silent dynamics in a time-resolved manner. In this paper, the principle and studies on blue light sensor proteins, phototropins, are described. Reaction kinetics of dimerization, dissociation reactions, and conformational changes were measured, and reaction schemes were determined. This method can be employed to study protein reactions from the viewpoint of diffusion and to elucidate the reaction schemes and kinetics that cannot be detected by other spectroscopic methods.

Abstract: Optogenetics holds the promise of controlling biological processes with superb temporal and spatial resolution at minimal perturbation. Although many of the light-reactive proteins used in optogenetic systems are derived from prokaryotes, applications were largely limited to eukaryotes for a long time. In recent years, however, an increasing number of microbiologists use optogenetics as a powerful new tool to study and control key aspects of bacterial biology in a fast and often reversible manner. After a brief discussion of optogenetic principles, this review provides an overview of the rapidly growing number of optogenetic applications in bacteria, with a particular focus on studies venturing beyond transcriptional control. To guide future experiments, we highlight helpful tools, provide considerations for successful application of optogenetics in bacterial systems, and identify particular opportunities and challenges that arise when applying these approaches in bacteria.

Abstract: Cells employ intracellular signaling pathways to sense and respond to changes in their external environment. In recent years, live-cell biosensors have revealed complex pulsatile dynamics in many pathways, but studies of these signaling dynamics are limited by the necessity of live-cell imaging at high spatiotemporal resolution. Here, we describe an approach to infer pulsatile signaling dynamics from a single measurement in fixed cells using a pulse-detecting gene circuit. We computationally screened for circuits with the capability to selectively detect signaling pulses, revealing an incoherent feedforward topology that robustly performs this computation. We implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. Our "recorder of Erk activity dynamics" (READer) responds sensitively to spontaneous and stimulus-driven Erk pulses. READer circuits open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.

Abstract: Abstract not available.

Abstract: Chimeric antigen receptor (CAR) T cell-based immunotherapy, approved by the US Food and Drug Administration, has shown curative potential in patients with haematological malignancies. However, owing to the lack of control over the location and duration of the anti-tumour immune response, CAR T cell therapy still faces safety challenges arising from cytokine release syndrome and on-target, off-tumour toxicity. Herein, we present the design of light-switchable CAR (designated LiCAR) T cells that allow real-time phototunable activation of therapeutic T cells to precisely induce tumour cell killing. When coupled with imaging-guided, surgically removable upconversion nanoplates that have enhanced near-infrared-to-blue upconversion luminescence as miniature deep-tissue photon transducers, LiCAR T cells enable both spatial and temporal control over T cell-mediated anti-tumour therapeutic activity in vivo with greatly mitigated side effects. Our nano-optogenetic immunomodulation platform not only provides a unique approach to interrogate CAR-mediated anti-tumour immunity, but also sets the stage for developing precision medicine to deliver personalized anticancer therapy.

Abstract: The Cre-loxP system has been widely used for specific DNA recombination which induces gene inactivation or expression. Recently, photoactivatable-Cre (PA-Cre) proteins have been developed as a tool for spatiotemporal control of the enzymatic activity of Cre recombinase. Here, we generated transgenic mice bearing a PA-Cre gene and systematically investigated the conditions of photoactivation for the PA-Cre in embryonic stem cells (ESCs) derived from the transgenic mice and in a simple mathematical model. Cre-mediated DNA recombination was induced in 16% of the PA-Cre ESCs by 6 hr continuous illumination. We show that repetitive pulsed illumination efficiently induced DNA recombination with low light energy as efficient as continuous illumination in the ESCs (96 ± 15% of continuous illumination when pulse cycle was 2 s), which was also supported by a minimal mathematical model. DNA recombination by the PA-Cre was also successfully induced in the transgenic mouse pre-implantation embryos under the developed conditions. These results suggest that strategies based on repetitive pulsed illumination are efficient for the activation of photoactivatable Cre and, possibly other photo-switchable proteins.

Abstract: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro. ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention.

Abstract: This review adds the bilin-binding phytochromes to the Chemical Reviews thematic issue "Optogenetics and Photopharmacology". The work is structured into two parts. We first outline the photochemistry of the covalently bound tetrapyrrole chromophore and summarize relevant spectroscopic, kinetic, biochemical, and physiological properties of the different families of phytochromes. Based on this knowledge, we then describe the engineering of phytochromes to further improve these chromoproteins as photoswitches and review their employment in an ever-growing number of different optogenetic applications. Most applications rely on the light-controlled complex formation between the plant photoreceptor PhyB and phytochrome-interacting factors (PIFs) or C-terminal light-regulated domains with enzymatic functions present in many bacterial and algal phytochromes. Phytochrome-based optogenetic tools are currently implemented in bacteria, yeast, plants, and animals to achieve light control of a wide range of biological activities. These cover the regulation of gene expression, protein transport into cell organelles, and the recruitment of phytochrome- or PIF-tagged proteins to membranes and other cellular compartments. This compilation illustrates the intrinsic advantages of phytochromes compared to other photoreceptor classes, e.g., their bidirectional dual-wavelength control enabling instant ON and OFF regulation. In particular, the long wavelength range of absorption and fluorescence within the "transparent window" makes phytochromes attractive for complex applications requiring deep tissue penetration or dual-wavelength control in combination with blue and UV light-sensing photoreceptors. In addition to the wide variability of applications employing natural and engineered phytochromes, we also discuss recent progress in the development of bilin-based fluorescent proteins.

Abstract: How T lymphocytes tune their responses to different strengths of stimulation is a fundamental question in immunology. Recent work using new optogenetic, single-cell genomic, and live-imaging approaches has revealed that stimulation strength controls the rate of individual cell responses within a population. Moreover, these responses have been found to use shared molecular programs, regardless of stimulation strength. However, additional data indicate that stimulation duration or cytokine feedback can impact later gene expression phenotypes of activated cells. In-depth molecular studies have suggested mechanisms by which stimulation strength might modulate the probability of T cell activation. This emerging model allows activating T cells to achieve a wide range of population responses through probabilistic control within individual cells.

Abstract: RNAs in circulation carry sequence-specific regulatory information between cells in animal, plant, and host-pathogen systems. Double-stranded RNA (dsRNA) delivered into the extracellular space of the nematode C. elegans accumulates within the germline and reaches progeny. Here we provide evidence for spatial, temporal, and substrate specificity in the transport of dsRNA from parental circulation to progeny. Temporary loss of dsRNA transport resulted in the persistent accumulation of mRNA from a germline gene. The expression of this gene varied among siblings and even between gonad arms within one animal. Perturbing RNA regulation of the gene created new epigenetic states that lasted for many generations. Thus, one role for the transport of dsRNA into the germline in every generation is to limit heritable changes in gene expression.

Abstract: Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, we present an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, our system enables dynamic control of consortia composition in continuous cultures for extended periods. We further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. Our artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use.

Abstract: Optogenetic technologies have transformed our ability to precisely control biological processes in time and space. Yet, current eukaryotic optogenetic systems are limited by large or complex optogenetic modules, long illumination times, low tissue penetration or slow activation and deactivation kinetics. Here, we report a red/far-red light-mediated and miniaturized Δphytochrome A (ΔPhyA)-based photoswitch (REDMAP) system based on the plant photoreceptor PhyA, which rapidly binds the shuttle protein far-red elongated hypocotyl 1 (FHY1) under illumination with 660-nm light with dissociation occurring at 730 nm. We demonstrate multiple applications of REDMAP, including dynamic on/off control of the endogenous Ras/Erk mitogen-activated protein kinase (MAPK) cascade and control of epigenetic remodeling using a REDMAP-mediated CRISPR-nuclease-deactivated Cas9 (CRISPR-dCas9) (REDMAPcas) system in mice. We also demonstrate the utility of REDMAP tools for in vivo applications by activating the expression of transgenes delivered by adeno-associated viruses (AAVs) or incorporated into cells in microcapsules implanted into mice, rats and rabbits illuminated by light-emitting diodes (LEDs). Further, we controlled glucose homeostasis in type 1 diabetic (T1D) mice and rats using REDMAP to trigger insulin expression. REDMAP is a compact and sensitive tool for the precise spatiotemporal control of biological activities in animals with applications in basic biology and potentially therapy.

Abstract: Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.

Abstract: Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

Abstract: The design and implementation of synthetic circuits that operate robustly in the cellular context is fundamental for the advancement of synthetic biology. However, their practical implementation presents challenges due to low predictability of synthetic circuit design and time-intensive troubleshooting. Here, we present the Cyberloop, a testing framework to accelerate the design process and implementation of biomolecular controllers. Cellular fluorescence measurements are sent in real-time to a computer simulating candidate stochastic controllers, which in turn compute the control inputs and feed them back to the controlled cells via light stimulation. Applying this framework to yeast cells engineered with optogenetic tools, we examine and characterize different biomolecular controllers, test the impact of non-ideal circuit behaviors such as dilution on their operation, and qualitatively demonstrate improvements in controller function with certain network modifications. From this analysis, we derive conditions for desirable biomolecular controller performance, thereby avoiding pitfalls during its biological implementation.

Abstract: Regulating stem cell functions by precisely controlling the nanoscale presentation of bioactive ligands has a substantial impact on tissue engineering and regenerative medicine but remains a major challenge. Here it is shown that bioactive ligands can become mechanically "invisible" by increasing their tether lengths to the substrate beyond a critical length, providing a way to regulate mechanotransduction without changing the biochemical conditions. Building on this finding, light switchable tethers are rationally designed, whose lengths can be modulated reversibly by switching a light-responsive protein, pdDronpa, in between monomer and dimer states. This allows the regulation of the adhesion, spreading, and differentiation of stem cells by light on substrates of well-defined biochemical and physical properties. Spatiotemporal regulation of differential cell fates on the same substrate is further demonstrated, which may represent an important step toward constructing complex organoids or mini tissues by spatially defining the mechanical cues of the cellular microenvironment with light.

Abstract: Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modeling, optogenetics, and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane–associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and that coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.

Abstract: One of the major cellular mechanisms to ensure protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is typically triggered by accumulation of misfolded proteins in the ER lumen. Here we describe activation of ER stress via protein aggregation in the cell nucleus. We find in the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) activation of ER stress due to the aggregation of the diseases-causing progerin protein at the nuclear envelope. The presence of nucleoplasmic protein aggregates is sensed and signaled to the ER lumen via immobilization and clustering of theinner nuclear membrane protein SUN2, leading to activation of the Unfolded Protein Response (UPR). These results identify a nuclear trigger of ER stress and they provide insight into the molecular disease mechanisms of HGPS.