Curated Optogenetic Publication Database

Abstract: Animal development is characterized by signaling events that occur at precise locations and times within the embryo, but determining when and where such precision is needed for proper embryogenesis has been a long-standing challenge. Here we address this question for extracellular signal regulated kinase (Erk) signaling, a key developmental patterning cue. We describe an optogenetic system for activating Erk with high spatiotemporal precision in vivo. Implementing this system in Drosophila, we find that embryogenesis is remarkably robust to ectopic Erk signaling, except from 1 to 4 hr post-fertilization, when perturbing the spatial extent of Erk pathway activation leads to dramatic disruptions of patterning and morphogenesis. Later in development, the effects of ectopic signaling are buffered, at least in part, by combinatorial mechanisms. Our approach can be used to systematically probe the differential contributions of the Ras/Erk pathway and concurrent signals, leading to a more quantitative understanding of developmental signaling.

Abstract: It has become clear that biological processes are highly dynamic and heterogeneous within and among cells. Conventional analytical tools and chemical or genetic manipulations are unsuitable for dissecting the role of their spatiotemporally dynamic nature. Recently, optical control of biomolecular signaling, a technology called “optogenetics,” has gained much attention. The technique has enabled spatial and temporal regulation of specific signaling pathways both in vitro and in vivo. This review presents strategies for optogenetic systems development and application for biological research. Combinations with other technologies and future perspectives are also discussed herein. Although many optogenetic approaches are designed to modulate ion channel conductivity, we mainly examine systems that target other biomolecular reactions such as gene expression, protein translocations, and kinase or receptor signaling pathways.

Abstract: Cells receive many different environmental clues to which they must adapt accordingly. Therefore, a complex signal transduction network has evolved. Cellular signal transduction is a highly dynamic process, in which the specific outcome is a result of the exact spatial and temporal resolution of single sub-events. While conventional techniques, like chemical inducer systems, have led to a sound understanding of the architecture of signal transduction pathways, the spatiotemporal aspects were often impossible to resolve. Optogenetics, based on genetically encoded light-responsive proteins, has the potential to revolutionize manipulation of signal transduction processes. Light can be easily applied with highest precision and minimal invasiveness. This review focuses on examples of optogenetic systems which were generated and applied to manipulate non-neuronal mammalian signaling processes at various stages of signal transduction, from cell membrane through cytoplasm to nucleus. Further, the future of optogenetic signaling will be discussed.

Abstract: In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

Abstract: Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca(2+) and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.

Abstract: Optogenetics is an emerging and powerful technique that allows the control of protein activity with light. The possibility of inhibiting or stimulating protein activity with the spatial and temporal precision of a pulse of laser light is opening new frontiers for the investigation of developmental pathways and cell biological bases underlying organismal development. With this powerful technique in hand, it will be possible to address old and novel questions about how cells, tissues, and organisms form. In this review, we focus on the applications of existing optogenetic tools for addressing issues in animal morphogenesis.

Abstract: Microbial opsin-based optogenetic tools have been transformative for neuroscience. To extend optogenetic approaches to the immune system to remotely control immune responses with superior spatiotemporal precision, pioneering tools have recently been crafted to modulate lymphocyte trafficking, inflammasome activation, dendritic cell (DC) maturation, and antitumor immunity through the photoactivation of engineered chemokine receptors and calcium release-activated calcium channels. We highlight herein some conceptual design strategies for installing light sensitivities into the immune signaling network and, in parallel, we propose potential solutions for in vivo optogenetic applications in living organisms with near-infrared light-responsive upconversion nanomaterials. Moreover, to move beyond proof-of-concept into translational applications, we discuss future prospects for integrating personalized immunoengineering with optogenetics to overcome critical hurdles in cancer immunotherapy.

Abstract: A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.

Abstract: Dynamic control of gene expression can have far-reaching implications for biotechnological applications and biological discovery. Thanks to the advantages of light, optogenetics has emerged as an ideal technology for this task. Current state-of-the-art methods for optical expression control fail to combine precision with repeatability and cannot withstand changing operating culture conditions. Here, we present a novel fully automatic experimental platform for the robust and precise long-term optogenetic regulation of protein production in liquid Escherichia coli cultures. Using a computer-controlled light-responsive two-component system, we accurately track prescribed dynamic green fluorescent protein expression profiles through the application of feedback control, and show that the system adapts to global perturbations such as nutrient and temperature changes. We demonstrate the efficacy and potential utility of our approach by placing a key metabolic enzyme under optogenetic control, thus enabling dynamic regulation of the culture growth rate with potential applications in bacterial physiology studies and biotechnology.

Abstract: Optogenetics describes the use of genetically encoded photosensitive proteins to direct intended biological processes with light in recombinant and native systems. While most of these light-responsive proteins were originally discovered in photosynthetic organisms, the past few decades have been punctuated by experiments that not only commandeer but also engineer and enhance these natural tools to explore a wide variety of physiological questions. In addition, the ability to tune dynamic range and kinetic rates of optogenetic actuators is a challenging question that is heavily explored with computational methods devised to facilitate optimization of these systems. Here, we explain the basic mechanisms of a few popular photodimerizing optogenetic systems, discuss applications, compare optogenetic tools against more traditional chemical methods, and propose a simple quantitative understanding of how actuators exert their influence on targeted processes.

Abstract: Living cells respond to their environment using networks of signaling molecules that act as sensors, information processors, and actuators. These signaling systems are highly modular at both the molecular and network scales, and much evidence suggests that evolution has harnessed this modularity to rewire and generate new physiological behaviors. Conversely, we are now finding that, following nature's example, signaling modules can be recombined to form synthetic tools for monitoring, interrogating, and controlling the behavior of cells. Here we highlight recent progress in the modular design of synthetic receptors, optogenetic switches, and phospho-regulated proteins and circuits, and discuss the expanding role of combinatorial design in the engineering of cellular signaling proteins and networks.

Abstract: Plants deploy a wide array of signalling networks integrating environmental cues with growth, defence and developmental responses. The high level of complexity, redundancy and connection between several pathways hampers a comprehensive understanding of involved functional and regulatory mechanisms. The implementation of synthetic biology approaches is revolutionizing experimental biology in prokaryotes, yeasts and animal systems and can likewise contribute to a new era in plant biology. This review gives an overview on synthetic biology approaches for the development and implementation of synthetic molecular tools and techniques to interrogate, understand and control signalling events in plants, ranging from strategies for the targeted manipulation of plant genomes up to the spatiotemporally resolved control of gene expression using optogenetic approaches. We also describe strategies based on the partial reconstruction of signalling pathways in orthogonal platforms, like yeast, animal and in vitro systems. This allows a targeted analysis of individual signalling hubs devoid of interconnectivity with endogenous interacting components. Implementation of the interdisciplinary synthetic biology tools and strategies is not exempt of challenges and hardships but simultaneously most rewarding in terms of the advances in basic and applied research. As witnessed in other areas, these original theoretical-experimental avenues will lead to a breakthrough in the ability to study and comprehend plant signalling networks.

Abstract: Cells have evolved a variety of mechanisms to regulate the enormous complexity of processes taking place inside them. One mechanism consists in tightly controlling the localization of macromolecules, keeping them away from their place of action until needed. Since a large fraction of the cellular response to external stimuli is mediated by gene expression, it is not surprising that transcriptional regulators are often subject to stimulus-induced nuclear import or export. Here we review recent methods in chemical biology and optogenetics for controlling the nuclear localization of proteins of interest inside living cells. These methods allow researchers to regulate protein activity with exquisite spatiotemporal control, and open up new possibilities for studying the roles of proteins in a broad array of cellular processes and biological functions.

Abstract: We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS UirS-UirR from Synechocystis PCC6803 to E. coli. We demonstrate that the promoter of the small RNA csiR1, previously shown to be activated by inorganic carbon stress, is a UirS-UirR output. Additionally, in contrast to a recently proposed sequestration model, we show that the sensor histidine kinase UirS phosphorylates the response regulator UirR to activate PcsiR1 transcription in response to UV-violet light. Finally, we measure changes in UirS-UirR output minutes after a change in light input and exploit these rapid dynamics to program a challenging gene expression signal with high predictability. UirS-UirR is the first engineered transcriptional regulatory tool activated exclusively by UV-violet light, and the most blue shifted photoreversible transcriptional regulatory tool.

Abstract: Light-mediated control of protein-protein interactions to regulate cellular pathways is an important application of optogenetics. Here, we report an optogenetic system based on the reversible light-induced binding between the bacterial phytochrome BphP1 and its natural partner PpsR2 from Rhodopseudomonas palustris bacteria. We extensively characterized the BphP1-PpsR2 interaction both in vitro and in mammalian cells and then used this interaction to translocate target proteins to specific cellular compartments, such as the plasma membrane and the nucleus. We showed light-inducible control of cell morphology that resulted in a substantial increase of the cell area. We demonstrated light-dependent gene expression with 40-fold contrast in cultured cells, 32-fold in subcutaneous mouse tissue, and 5.7-fold in deep tissues in mice. Characteristics of the BphP1-PpsR2 optogenetic system include its sensitivity to 740- to 780-nm near-infrared light, its ability to utilize an endogenous biliverdin chromophore in eukaryotes (including mammals), and its spectral compatibility with blue-light-driven optogenetic systems.

Abstract: Plastid-to-nucleus retrograde signals emitted by dysfunctional chloroplasts impact photomorphogenic development, but the molecular link between retrograde- and photosensory-receptor signalling has remained unclear. Here, we show that the phytochrome and retrograde signalling (RS) pathways converge antagonistically to regulate the expression of the nuclear-encoded transcription factor GLK1, a key regulator of a light-induced transcriptional network central to photomorphogenesis. GLK1 gene transcription is directly repressed by PHYTOCHROME-INTERACTING FACTOR (PIF)-class bHLH transcription factors in darkness, but light-activated phytochrome reverses this activity, thereby inducing expression. Conversely, we show that retrograde signals repress this induction by a mechanism independent of PIF mediation. Collectively, our data indicate that light at moderate levels acts through the plant's nuclear-localized sensory-photoreceptor system to induce appropriate photomorphogenic development, but at excessive levels, sensed through the separate plastid-localized RS system, acts to suppress such development, thus providing a mechanism for protection against photo-oxidative damage by minimizing the tissue exposure to deleterious radiation.

Abstract: Cyanobacteriochromes (CBCRs) are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB) and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV). The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2) from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.

Abstract: Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.

Abstract: Optogenetic tools to control gene expression have many advantages over the classical chemically inducible systems, overcoming intrinsic limitations of chemical inducers such as solubility, diffusion, and cell toxicity. They offer an unmatched spatiotemporal resolution and permit quantitative and noninvasive control of the gene expression. Here we describe a protocol of a synthetic light-inducible system for the targeted control of gene expression in plants based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). The synthetic toggle switch system is in the ON state when plant protoplasts are illuminated with red light (660 nm) and can be returned to the OFF state by subsequent illumination with far-red light (760 nm). In this protocol, the implementation of a red light-inducible expression system in plants using Light-Emitting Diode (LED) illumination boxes is described, including the isolation and transient transformation of plant protoplasts from Arabidopsis thaliana and Nicotiana tabacum.

Abstract: Synthetic biology aims at manipulating biological systems by rationally designed and genetically introduced components. Efforts in photoactuator engineering resulted in microorganisms reacting to extracellular light-cues with various cellular responses. Some of them lead to the formation of macroscopically observable outputs, which can be used to generate images made of living matter. Several methods have been developed to convert colorless compounds into visible pigments by an enzymatic conversion. This has been exploited as a showcase for successful creation of an optogenetic tool; examples for basic light-controlled biological processes that have been coupled to this biophotography comprise regulation of transcription, protein stability, and second messenger synthesis. Moreover, biological reproduction of images is used as means to facilitate quantitative characterization of optogenetic switches as well as a technique to investigate complex cellular signaling circuits. Here, we will compare the different techniques for biological image generation, introduce experimental approaches, and provide future-perspectives for biophotography.

Abstract: Recent advances in the understanding of photosensing in biological systems have enabled the use of photoreceptors as novel genetic tools. Exploiting various photoreceptors that cyanobacteria possess, a green light-inducible gene expression system was previously developed for the regulation of gene expression in cyanobacteria. However, the applications of cyanobacterial photoreceptors are not limited to these bacteria but are also available for non-photosynthetic microorganisms by the coexpression of a cyanobacterial chromophore with a cyanobacteria-derived photosensing system. An Escherichia coli-derived self-aggregation system based on Antigen 43 (Ag43) has been shown to induce cell self-aggregation of various bacteria by exogenous introduction of the Ag43 gene.

Abstract: Biological phenomena, such as cellular differentiation and phagocytosis, are fundamental processes that enable cells to fulfill important physiological roles in multicellular organisms. In the field of synthetic biology, the study of these behaviors relies on the use of a broad range of molecular tools that enable the real-time manipulation and measurement of key components in the underlying signaling pathways. This Review will focus on a subset of synthetic biology tools known as bottom-up techniques, which use technologies such as optogenetics and chemically induced dimerization to reconstitute cellular behavior in cells. These techniques have been crucial not only in revealing causal relationships within signaling networks but also in identifying the minimal signaling components that are necessary for a given cellular function. We discuss studies that used these systems in a broad range of cellular and molecular phenomena, including the time-dependent modulation of protein activity in cellular proliferation and differentiation, the reconstitution of phagocytosis, the reconstitution of chemotaxis, and the regulation of actin reorganization. Finally, we discuss the potential contribution of synthetic biology to medicine.

Abstract: We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms.

Abstract: Photoreceptors are found in all kingdoms of life and mediate crucial responses to environmental challenges. Nature has evolved various types of photoresponsive protein structures with different chromophores and signaling concepts for their given purpose. The abundance of these signaling proteins as found nowadays by (meta-)genomic screens enriched the palette of optogenetic tools significantly. In addition, molecular insights into signal transduction mechanisms and design principles from biophysical studies and from structural and mechanistic comparison of homologous proteins opened seemingly unlimited possibilities for customizing the naturally occurring proteins for a given optogenetic task. Here, a brief overview on the photoreceptor concepts already established as optogenetic tools in natural or engineered form, their photochemistry and their signaling/design principles is given. Finally, so far not regarded photosensitive modules and protein architectures with potential for optogenetic application are described.

Abstract: The optogenetics techniques that have long been used in neuroscience are now giving biologists the power to probe cellular structures with unprecedented precision.