Curated Optogenetic Publication Database

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: 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: 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: 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: Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

Abstract: Protein switches are ubiquitous in biological signal transduction systems, enabling cells to sense and respond to a variety of molecular queues in a rapid, specific, and integrated fashion. Analogously, tailor-engineered protein switches with custom input and output functions have become invaluable research tools for reporting on distinct physiological states and actuating molecular functions in real time and in situ. Here, we analyze recent progress in constructing protein-based switches while assessing their potential in the assembly of defined signaling motifs. We anticipate such systems will ultimately pave the way towards a new generation of molecular diagnostics and facilitate the construction of artificial signaling systems that operate in parallel to the signaling machinery of a host cell for applications in synthetic biology.

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: Molecular signals are sensed by their respective receptors and information is transmitted and processed by a sophisticated intracellular network controlling various biological functions. Optogenetic tools allow the targeting of specific signaling nodes for a precise spatiotemporal control of downstream effects. These tools are based on photoreceptors such as phytochrome B (PhyB), cryptochrome 2, or light-oxygen-voltage-sensing domains that reversibly bind to specific interaction partners in a light-dependent manner. Fusions of a protein of interest to the photoreceptor or their interaction partners may enable the control of the protein function by light-mediated dimerization, a change of subcellular localization, or due to photocaging/-uncaging of effectors. In this review, we summarize the photoreceptors and the light-based mechanisms utilized for the modulation of signaling events in mammalian cells focusing on non-neuronal applications. We discuss in detail optogenetic tools and approaches applied to control signaling events mediated by second messengers, Rho GTPases and growth factor-triggered signaling cascades namely the RAS/RAF and phosphatidylinositol-3-kinase pathways. Applying the latest generation of optogenetic tools allows to control cell fate decisions such as proliferation and differentiation or to deliver therapeutic substances in a spatiotemporally controlled manner.

Abstract: The light-based control of ion channels has been transformative for the neurosciences, but the optogenetic toolkit does not stop there. An expanding number of proteins and cellular functions have been shown to be controlled by light, and the practical considerations in deciding between reversible optogenetic systems (such as systems that use light-oxygen-voltage domains, phytochrome proteins, cryptochrome proteins and the fluorescent protein Dronpa) are well defined. The field is moving beyond proof of concept to answering real biological questions, such as how cell signalling is regulated in space and time, that were difficult or impossible to address with previous tools.

Abstract: Enabling optical control over biological processes is a defining goal of the new field of optogenetics. Control of membrane voltage by natural rhodopsin family ion channels has found widespread acceptance in neuroscience, due to the fact that these natural proteins control membrane voltage without further engineering. In contrast, optical control of intracellular biological processes has been a fragmented effort, with various laboratories engineering light-responsive properties into proteins in different manners. In the present article, we review the various systems that have been developed for controlling protein functions with light based on vertebrate rhodopsins, plant photoregulatory proteins and, most recently, the photoswitchable fluorescent protein Dronpa. By allowing biology to be controlled with spatiotemporal specificity and tunable dynamics, light-controllable proteins will find applications in the understanding of cellular and organismal biology and in synthetic biology.

Abstract: Light is fundamental to life on earth. Therefore, nature has evolved a multitude of photoreceptors that sense light across all kingdoms. This natural resource provides synthetic biology with a vast pool of light-sensing components with distinct spectral properties that can be harnessed to engineer novel optogenetic tools. These devices enable control over gene expression, cell morphology and signaling pathways with superior spatiotemporal resolution and are maturing towards elaborate applications in basic research, in the production of biopharmaceuticals and in biomedicine. This article provides a summary of the recent advances in optogenetics that use light for the precise control of biological functions in mammalian cells.

Abstract: Fluorescent proteins (FPs) are widely used as optical sensors, whereas other light-absorbing domains have been used for optical control of protein localization or activity. Here, we describe light-dependent dissociation and association in a mutant of the photochromic FP Dronpa, and we used it to control protein activities with light. We created a fluorescent light-inducible protein design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains associate and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control.