Curated Optogenetic Publication Database

Abstract: Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics, is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and for the first time, combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.

Abstract: Abstract not available.

Abstract: Single-cell behaviors play essential roles during early-stage biofilms formation. In this study, we evaluated whether biofilm formation could be guided by precisely manipulating single cells behaviors. Thus, we established an illumination method to precisely manipulate the type IV pili (TFP) mediated motility and microcolony formation of Pseudomonas aeruginosa by using a combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation and adaptive microscopy. We termed this method as Adaptive Tracking Illumination (ATI). We reported that ATI enables the precise manipulation of TFP mediated motility and microcolony formation during biofilm formation by manipulating bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) levels in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms can be controlled using ATI. Thus, the established method (i.e., ATI) can markedly promote ongoing studies of biofilms.

Abstract: Cells receive diverse signaling cues from their environment that trigger cascades of biochemical reactions in a dynamic manner. Single-cell imaging technologies have revealed that not only molecular species but also dynamic patterns of signaling inputs determine the fates of signal-receiving cells; however it has been challenging to elucidate how such dynamic information is delivered and decoded in complex networks of inter-cellular and inter-molecular interactions. The recent development of optogenetic technology with photo-sensitive proteins has changed this situation; the combination of microscopy and optogenetics provides fruitful insights into the mechanism of dynamic information processing at the single-cell level. Here, we review recent efforts to visualize the flows of dynamic patterns in signaling pathways, which utilize methods integrating single-cell imaging and optogenetics.

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: Structures arising from actin-based cell membrane movements, including ruffles, lamellipodia, and filopodia, play important roles in a broad spectrum of cellular functions, such as cell motility, axon guidance in neurons, wound healing, and micropinocytosis. Previous studies investigating these cell membrane dynamics often relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies did not allow the modulation of protein activity at specific regions of cells, tissues, and organs in animals with high spatial and temporal precision. Recently, optogenetic tools for inducing cell membrane dynamics have been developed which address several of the disadvantages of previous techniques. In a recent study, we developed a powerful optogenetic tool, called the Magnet system, to change cell membrane dynamics through Tiam1 and PIP3 signal transductions with high spatial and temporal resolution. In this review, we summarize recent advances in optogenetic tools that allow us to induce actin-regulated cell membrane dynamics and unique membrane ruffles that we discovered using our Magnet system.

Abstract: Mammalian fertilization relies on sperm finding the egg and penetrating the egg vestments. All steps in a sperm's lifetime crucially rely on changes in the second messenger cAMP (cyclic adenosine monophosphate). In recent years, it has become clear that signal transduction in sperm is not a continuum, but rather organized in subcellular domains, e.g. the sperm head and the sperm flagellum, with the latter being further separated into the midpiece, principal piece, and endpiece. To understand the underlying signaling pathways controlling sperm function in more detail, experimental approaches are needed that allow to study sperm signaling with spatial and temporal precision. Here, we will give a comprehensive overview on cAMP signaling in mammalian sperm, describing the molecular players involved in these pathways and the sperm functions that are controlled by cAMP. Furthermore, we will highlight recent advances in analyzing and manipulating sperm signaling with spatio-temporal precision using light.

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: Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.

Abstract: Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make use of various sensor-response circuits, called two-component systems (TCSs). A TCS comprises a histidine protein kinase (HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two components are coupled via a phosphorylation control mechanism. In a recent study, we adopted an optogenetics approach to re-engineer the sensor HKs in Escherichia coli as a light-sensing fusion protein. We constructed a light-controllable HK by replacing the original signal-specific sensing domain of HK with the light-sensing domain of Cph1 from Cyanobacteria Synechocystis, so that HK can be investigated by red light. Here, we extended the study to other 16 HK-RR TCSs and constructed a library of light-responsible HK-Cph1 chimeras. By taking the NarX-NarL system as an example, we demonstrated the light responsiveness of the constructed chimera and investigated the frequency response of the NarXNarL system. The constructed library serves as a toolkit for future TCS study using optogenetics approach.

Abstract: Signaling pathways involving second messenger c-di-GMP regulate various aspects of bacterial physiology and behavior. We describe the use of a red light-activated diguanylate cyclase (c-di-GMP synthase) and a blue light-activated c-di-GMP phosphodiesterase (hydrolase) for manipulating intracellular c-di-GMP levels in bacterial cells. We illustrate the application of these enzymes in regulating several c-di-GMP-dependent phenotypes, i.e., motility and biofilm phenotypes in E. coli and chemotactic behavior in the alphaproteobacterium Azospirillum brasilense. We expect these light-activated enzymes to be also useful in regulating c-di-GMP-dependent processes occurring at the fast timescale, for spatial control of bacterial populations, as well as for analyzing c-di-GMP-dependent phenomena at the single-cell level.

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: Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene’s transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

Abstract: Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana. Robust gene expression and high protein levels are achieved by combining genome integrated red light-sensing components with an episomal high-copy reporter construct. The gene of interest as well as the synTALE DNA-binding domain can be easily exchanged, allowing the flexible regulation of any desired gene by targeting endogenous or heterologous promoter regions. To allow low-cost induction of gene expression for industrial fermentation processes, we engineered yeast to endogenously produce the chromophore required for the effective dimerization of PhyB and PIF3. Time course experiments demonstrate high-level induction over a period of at least 48 h.

Abstract: The Ras/Erk signaling pathway plays a central role in diverse cellular processes ranging from development to immune cell activation to neural plasticity to cancer. In recent years, this pathway has been widely studied using live-cell fluorescent biosensors, revealing complex Erk dynamics that arise in many cellular contexts. Yet despite these high-resolution tools for measurement, the field has lacked analogous tools for control over Ras/Erk signaling in live cells. Here, we provide detailed methods for one such tool based on the optical control of Ras activity, which we call "Opto-SOS." Expression of the Opto-SOS constructs can be coupled with a live-cell reporter of Erk activity to reveal highly quantitative input-to-output maps of the pathway. Detailed herein are protocols for expressing the Opto-SOS system in cultured cells, purifying the small molecule cofactor necessary for optical stimulation, imaging Erk responses using live-cell microscopy, and processing the imaging data to quantify Ras/Erk signaling dynamics.

Abstract: The dynamic behavior of the plant red/far-red light photoreceptor phytochrome B (phyB) has been elucidated in natural and synthetic systems. Red light switches phyB from the inactive Pr state to the active Pfr state, a process that is reversed by far-red light. Alongside light signals, phyB activity is constrained by thermal reversion (that is prominent in the dark) and protein-protein interactions between phyB, other phytochrome molecules, and, among others, PHYTOCHROME INTERACTING FACTORs (PIFs). Requirements for phyB-PIF association have been well studied and are central to light-regulated synthetic tools. However, it is unknown whether PIF interactions influence transitions of phyB between different conformers. Here, we show that the in vitro thermal reversion of phyB involves multiple reactions. Thermal reversion of phyB in vitro is inhibited by PIF6, and this effect is observed at all temperatures tested. We analyzed our experimental data using a mathematical model containing multiple Pfr conformers, in accordance with previous findings. Remarkably, each Pfr conformer is differentially regulated by PIF6 and temperature. As a result, we speculate that in vivo phytochrome signaling networks may require similar levels of complexity to fine-tune responses to the external environment.

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: Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades.

Abstract: Cells are bombarded by extrinsic signals that dynamically change in time and space. Such dynamic variations can exert profound effects on behaviors, including cellular signaling, organismal development, stem cell differentiation, normal tissue function, and disease processes such as cancer. Although classical genetic tools are well suited to introduce binary perturbations, new approaches have been necessary to investigate how dynamic signal variation may regulate cell behavior. This fundamental question is increasingly being addressed with optogenetics, a field focused on engineering and harnessing light-sensitive proteins to interface with cellular signaling pathways. Channelrhodopsins initially defined optogenetics; however, through recent use of light-responsive proteins with myriad spectral and functional properties, practical applications of optogenetics currently encompass cell signaling, subcellular localization, and gene regulation. Now, important questions regarding signal integration within branch points of signaling networks, asymmetric cell responses to spatially restricted signals, and effects of signal dosage versus duration can be addressed. This review summarizes emerging technologies and applications within the expanding field of optogenetics.

Abstract: Optogenetic tools use colored light to rapidly control gene expression in space and time. We designed a genetically encoded system that gives Escherichia coli the ability to distinguish between red, green, and blue (RGB) light and respond by changing gene expression. We use this system to produce 'color photographs' on bacterial culture plates by controlling pigment production and to redirect metabolic flux by expressing CRISPRi guide RNAs.

Abstract: Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.

Abstract: Optogenetic tools offer fast and reversible control of protein activity with subcellular spatial precision. In the past few years, remarkable progress has been made in engineering photoactivatable systems regulating the activity of cellular proteins. In this review, we discuss general strategies in designing and optimizing such optogenetic tools and highlight recent advances in the field, with specific focus on applications regulating protein catalytic activity.

Abstract: Abstract not available.

Abstract: Several fields in neuroscience have been revolutionized by the advent of optogenetics, a technique that offers the possibility to modulate neuronal physiology in response to light stimulation. This innovative and far-reaching tool provided unprecedented spatial and temporal resolution to explore the activity of neural circuits underlying cognition and behaviour. With an exponential growth in the discovery and synthesis of new photosensitive actuators capable of modulating neuronal networks function, other fields in biology are experiencing a similar re-evolution. Here, we review the various optogenetic toolboxes developed to influence cellular physiology as well as the diverse ways in which these can be engineered to precisely modulate intracellular signalling and transcription. We also explore the processes required to successfully express and stimulate these photo-actuators in vivo before discussing how such tools can enlighten our understanding of neuronal plasticity at the systems level.