Curated Optogenetic Publication Database

Abstract: Exploiting the optically controlled association and dissociation behavior of a photoswitchable fluorescent protein, Dronpa145N, here we demonstrate the engineering of an optically switchable reversible protein hydrogel using Dronpa145N-based protein building blocks. Our results open the possibility to optically tune the mechanical, chemical and structural properties of protein hydrogels.

Abstract: The breakthrough CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nuclease has revolutionized our ability in genome engineering. Although Cas9 is already a powerful tool for simple and efficient target endogenous gene manipulation, further engineering of Cas9 will improve the performance of Cas9, such as gene-editing efficiency and accuracy in vivo, and expand the application possibility of this Cas9 technology. The emerging inducible Cas9 methods, which can control the activity of Cas9 using an external stimulus such as chemicals and light, have the potential to provide spatiotemporal gene manipulation in user-defined cell population at a specific time and improve the accuracy of Cas9-mediated genome editing. In this review, we focus on the recent advance in inducible Cas9 technologies, especially light-inducible Cas9, and related methodologies, and also discuss future directions of this emerging tools.

Abstract: The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications.

Abstract: Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Abstract: Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.

Abstract: Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.

Abstract: Precise spatial and temporal control of cellular processes is in life sciences a highly sought-after capability. In the recent years, this goal has become progressively achievable through the field of optogenetics, which utilizes light as a non-invasive means to control genetically encoded light-responsive proteins. The latest optogenetic systems, such as those for control of subcellular localization or cellular decision-making and tissue morphogenesis provide us with insights to gain a deeper understanding of the cellular inner workings. Besides, they hold a potential for further development into biomedical applications, from in vitro optogenetics-assisted drug candidate screenings to light-controlled gene therapy and tissue engineering.

Abstract: The zebrafish ( Danio rerio) is a powerful vertebrate model to study cellular and developmental processes in vivo. The optical clarity and their amenability to genetic manipulation make zebrafish a model of choice when it comes to applying optical techniques involving genetically encoded photoresponsive protein technologies. In recent years, a number of fluorescent protein and optogenetic technologies have emerged that allow new ways to visualize, quantify, and perturb developmental dynamics. Here, we explain the principles of these new tools and describe some of their representative applications in zebrafish.

Abstract: To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.

Abstract: Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.

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: Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

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: 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: Dronpa is a novel photochromic fluorescent protein that exhibits fast response to light. The present article is the first report of the resonance and preresonance Raman spectra of Dronpa. We used the intensity and frequency of Raman bands to determine the structure of the Dronpa chromophore in two thermally stable photochromic states. The acid-base equilibrium in one photochromic state was observed by spectroscopic pH titration. The Raman spectra revealed that the chromophore in this state shows a protonation/deprotonation transition with a pKa of 5.2 ± 0.3 and maintains the cis configuration. The observed resonance Raman bands showed that the other photochromic state of the chromophore is in a trans configuration. The results demonstrate that Raman bands selectively enhanced for the chromophore yield valuable information on the molecular structure of the chromophore in photochromic fluorescent proteins after careful elimination of the fluorescence background.

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: Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events.

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: Rational design of optogenetic tools is inherently linked to the understanding of photoreceptor function. Structural analysis of elements involved in signal integration in individual sensor domains provides an initial idea of their mode of operation, but understanding how local structural rearrangements eventually affect signal transmission to output domains requires inclusion of the effector regions in the characterization. However, the dynamic nature of these assemblies renders their structural analysis challenging and therefore a combination of high- and low-resolution techniques is required to appreciate functional aspects of photoreceptors. This review focuses on the potential of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) for complementing the structural characterization of photoreceptors. In this respect, the ability of HDX-MS to provide information on conformational dynamics and the possibility to address multiple functionally relevant states in solution render this methodology ideally suitable. We highlight recent examples demonstrating the potential of HDX-MS and discuss how these results can help to improve existing optogenetic systems or guide the design of novel optogenetic tools.

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: 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: The authors discovered some errors regarding reference group labels in Table 2. The corrected table is attached. The authors regret these errors.