Curated Optogenetic Publication Database

Abstract: Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions but also their link to pathological aggregates.

Abstract: Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling.

Abstract: Photo-controlled or 'optogenetic' effectors interfacing with endogenous protein machinery allow the roles of endogenous proteins to be probed. There are two main approaches being used to develop optogenetic effectors: (i) caging strategies using photo-controlled conformational changes, and (ii) protein relocalization strategies using photo-controlled protein-protein interactions. Numerous specific examples of these approaches have been reported and efforts to develop general methods for photo-control of endogenous proteins are a current focus. The development of improved screening and selection methods for photo-switchable proteins would advance the field.

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: 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: 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: 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: Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein-protein interactions.

Abstract: The regulation of cellular signaling in vivo has been a challenging task owing to the lack of effective methods for tunable control of the amplitude, location, and duration of cell-signaling events at a deep-tissue level. In this issue of ACS Nano, an intriguing paper by Ambrosone et al. demonstrates that deep-tissue-penetrating near-infrared (NIR) light can be used to control the Wnt/β-catenin-signaling pathway in a single-cell organism (Hydra) by utilizing microcapsules that contain plasmonic gold nanoparticles. In parallel, in recent work, we proposed upconversion nanoparticles (UCNPs) as NIR-light-activatable "wireless" optogenetic tools, and we showed their ability to modulate cell signaling pathways in both mammalian cells and mice. We believe that these interesting NIR-light-responsive nanotechnologies will open new avenues for both basic research and clinical applications.

Abstract: Growth cones of extending axons navigate to correct targets by sensing a guidance cue gradient via membrane protein receptors. Although most signaling mechanisms have been clarified using an in vitro approach, it is still difficult to investigate the growth cone behavior in complicated extracellular environment of living animals due to the lack of tools. We develop a system for the light-dependent activation of a guidance receptor, Deleted in Colorectal Cancer (DCC), using Arabidopsis thaliana Cryptochrome 2, which oligomerizes upon blue-light absorption. Blue-light illumination transiently activates DCC via its oligomerization, which initiates downstream signaling in the illuminated subcellular region. The extending axons are attracted by illumination in cultured chick dorsal root ganglion neurons. Moreover, light-mediated navigation of the growth cones is achieved in living Caenorhabditis elegans. The photo-manipulation system is applicable to investigate the relationship between the growth cone behavior and its surrounding environment in living tissue.

Abstract: Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.

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: 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: Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.

Abstract: In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.

Abstract: Sensory photoreceptors not only control diverse adaptive responses in Nature, but as light-regulated actuators they also provide the foundation for optogenetics, the non-invasive and spatiotemporally precise manipulation of cellular events by light. Novel photoreceptors have been engineered that establish control by light over manifold biological processes previously inaccessible to optogenetic intervention. Recently, photoreceptor engineering has witnessed a rapid development, and light-regulated actuators for the perturbation of a plethora of cellular events are now available. Here, we review fundamental principles of photoreceptors and light-regulated allostery. Photoreceptors dichotomize into associating receptors that alter their oligomeric state as part of light-regulated allostery and non-associating receptors that do not. A survey of engineered photoreceptors pinpoints light-regulated association reactions and order-disorder transitions as particularly powerful and versatile design principles. Photochromic photoreceptors that are bidirectionally toggled by two light colors augur enhanced spatiotemporal resolution and use as photoactivatable fluorophores. By identifying desirable traits in engineered photoreceptors, we provide pointers for the design of future, light-regulated actuators.

Abstract: The photoreceptor cryptochrome 2 (CRY2) has become a powerful optogenetic tool that allows light-inducible manipulation of various signaling pathways and cellular processes in mammalian cells with high spatiotemporal precision and ease of application. However, it has also been shown that the behavior of CRY2 under blue light is complex, as the photoexcited CRY2 can both undergo homo-oligomerization and heterodimerization by binding to its dimerization partner CIB1. To better understand the light-induced CRY2 activities in mammalian cells, this article systematically characterizes CRY2 homo-oligomerization in different cellular compartments, as well as how CRY2 homo-oligomerization and heterodimerization activities affect each other. Quantitative analysis reveals that membrane-bound CRY2 has drastically enhanced oligomerization activity compared to that of its cytoplasmic form. While CRY2 homo-oligomerization and CRY2-CIB1 heterodimerization could happen concomitantly, the presence of certain CIB1 fusion proteins can suppress CRY2 homo-oligomerization. However, the homo-oligomerization of cytoplasmic CRY2 can be significantly intensified by its recruitment to the membrane via interaction with the membrane-bound CIB1. These results contribute to the understanding of the light-inducible CRY2-CRY2 and CRY2-CIB1 interaction systems and can be used as a guide to establish new strategies utilizing the dual optogenetic characteristics of CRY2 to probe cellular processes.

Abstract: Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artefacts. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signalling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.

Abstract: Cellular processes such as proliferation, differentiation, or migration depend on precise spatiotemporal coordination of protein activities. Correspondingly, reaching a quantitative understanding of cellular behavior requires experimental approaches that enable spatial and temporal modulation of protein activity. Recently, a variety of light-sensitive protein domains have been engineered as optogenetic actuators to spatiotemporally control protein activity. In the present review, we discuss the principle of these optical control methods and examples of their applications in modulating signaling pathways. By controlling protein activity with spatiotemporal specificity, tunable dynamics, and quantitative control, light-controllable proteins promise to accelerate our understanding of cellular and organismal biology.

Abstract: Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein-like probes, including near-infrared fluorescence, independence of oxygen, small size, and photosensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.

Abstract: Variation in signaling activity across a cell plays a crucial role in processes such as cell migration. Signaling activity specific to organelles within a cell also likely plays a key role in regulating cellular functions. To understand how such spatially confined signaling within a cell regulates cell behavior, tools that exert experimental control over subcellular signaling activity are required. Here, we discuss the advantages of using optogenetic approaches to achieve this control. We focus on a set of optical triggers that allow subcellular control over signaling through the activation of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases and downstream signaling proteins, as well as those that inhibit endogenous signaling proteins. We also discuss the specific insights with regard to signaling and cell behavior that these subcellular optogenetic approaches can provide.

Abstract: The ability to perturb living systems is essential to understand how cells sense, integrate, and exchange information, to comprehend how pathologic changes in these processes relate to disease, and to provide insights into therapeutic points of intervention. Several molecular technologies based on natural photoreceptor systems have been pioneered that allow distinct cellular signaling pathways to be modulated with light in a temporally and spatially precise manner. In this review, we describe and discuss the underlying design principles of natural photoreceptors that have emerged as fundamental for the rational design and implementation of synthetic light-controlled signaling systems. Furthermore, we examine the unique challenges that synthetic protein technologies face when applied to the study of neural dynamics at the cellular and network level.

Abstract: Biological clocks play key roles in organismal development, homeostasis and function. In recent years, much work has focused on circadian clocks, but emerging studies have highlighted the existence of ultradian oscillators - those with a much shorter periodicity than 24 h. Accumulating evidence, together with recently developed optogenetic approaches, suggests that such ultradian oscillators play important roles during cell fate decisions, and analyzing the functional links between ultradian oscillation and cell fate determination will contribute to a deeper understanding of the design principle of developing embryos. In this Review, we discuss the mechanisms of ultradian oscillatory dynamics and introduce examples of ultradian oscillators in various biological contexts. We also discuss how optogenetic technology has been used to elucidate the biological significance of ultradian oscillations.