Curated Optogenetic Publication Database

Abstract: Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.

Abstract: Despite the large diversity of the proteins involved in cellular signaling, many intracellular signaling pathways converge onto one of only dozens of small molecule second messengers. Cyclic adenosine monophosphate (cAMP), one of these second messengers, is known to regulate activity of both Protein Kinase A (PKA) and the Extracellular Regulated Kinase (ERK), among other signaling pathways. In its role as an important cellular signaling hub, intracellular cAMP concentration has long been assumed to monotonically regulate its known effectors. Using an optogenetic tool that can introduce precise amounts of cAMP in MDCKI cells, we identify genes whose expression changes biphasically with monotonically increasing cAMP levels. By examining the behavior of PKA and ERK1/2 in the same dose regime, we find that these kinases also respond biphasically to increasing cAMP levels, with opposite phases. We reveal that this behavior results from an elaborate integration by PKA of many cellular signals triggered by cAMP. In addition to the direct activation of PKA, cAMP also modulates the activity of p38 and ERK, which then converge to inhibit PKA. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, influencing the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational modeling, synthesize a set of network interconnections involving PKA and other important signaling pathways into a model that demonstrates how cells can capitalize on signal integration to create a diverse set of responses to cAMP concentration and produce complex input-output relationships.

Abstract: Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.

Abstract: Optogenetic manipulations have transformed neuroscience in recent years. While sophisticated tools now exist for controlling the firing patterns of neurons, it remains challenging to optogenetically define the plasticity state of individual synapses. A variety of synapses in the mammalian brain express presynaptic long-term potentiation (LTP) upon elevation of presynaptic cyclic adenosine monophosphate (cAMP), but the molecular expression mechanisms as well as the impact of presynaptic LTP on network activity and behavior are not fully understood. In order to establish optogenetic control of presynaptic cAMP levels and thereby presynaptic potentiation, we developed synaptoPAC, a presynaptically targeted version of the photoactivated adenylyl cyclase bPAC. In cultures of hippocampal granule cells of Wistar rats, activation of synaptoPAC with blue light increased action potential-evoked transmission, an effect not seen in hippocampal cultures of non-granule cells. In acute brain slices of C57BL/6N mice, synaptoPAC activation immediately triggered a strong presynaptic potentiation at mossy fiber synapses in CA3, but not at Schaffer collateral synapses in CA1. Following light-triggered potentiation, mossy fiber transmission decreased within 20 min, but remained enhanced still after 30 min. The optogenetic potentiation altered the short-term plasticity dynamics of release, reminiscent of presynaptic LTP. Our work establishes synaptoPAC as an optogenetic tool that enables acute light-controlled potentiation of transmitter release at specific synapses in the brain, facilitating studies of the role of presynaptic potentiation in network function and animal behavior in an unprecedented manner. Read the Editorial Highlight for this article on page 270.

Abstract: Cybergenetic systems use computer interfaces to enable feed-back controls over biological processes in real time. The complex and dynamic nature of cellular metabolism makes cybergenetics attractive for controlling engineered metabolic pathways in microbial fermentations. Cybergenetics would not only create new avenues of research into cellular metabolism, it would also enable unprecedented strategies for pathway optimization and bioreactor operation and automation. Implementation of metabolic cybergenetics, however, will require new capabilities from actuators, biosensors, and control algorithms. The recent application of optogenetics in metabolic engineering, the expanding role of genetically encoded biosensors in strain development, and continued progress in control algorithms for biological processes suggest that this technology will become available in the not so distant future.

Abstract: Since the neurobiological inception of optogenetics, light-controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light-sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle-specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Physiology of Model Organisms Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Cellular Models.

Abstract: cAMP is a crucial mediator of multiple cell signaling pathways. This cyclic nucleotide requires strict spatiotemporal control for effective function. Light-activated proteins have become a powerful tool to study signaling kinetics due to having quick on/off rates and minimal off-target effects. The photoactivated adenylyl cyclase from Beggiatoa (bPAC) produces cAMP rapidly upon stimulation with blue light. However, light delivery is not always feasible, especially in vivo. Hence, we created a luminescence-activated cyclase by fusing bPAC with nanoluciferase (nLuc) to allow chemical activation of cAMP activity. This dual-activated adenylyl cyclase can be stimulated using short bursts of light or long-term chemical activation with furimazine and other related luciferins. Together these can be used to mimic transient, chronic, and oscillating patterns of cAMP signaling. Moreover, when coupled to compartment-specific targeting domains, these reagents provide a new powerful tool for cAMP spatiotemporal dynamic studies. Here, we describe detailed methods for working with bPAC-nLuc in mammalian cells, stimulating cAMP production with light and luciferins, and measuring total cAMP accumulation.

Abstract: G protein-coupled receptors (GPCRs) form the largest class of membrane receptors in the mammalian genome with nearly 800 human genes encoding for unique subtypes. Accordingly, GPCR signaling is implicated in nearly all physiological processes. However, GPCRs have been difficult to study due in part to the complexity of their function which can lead to a plethora of converging or diverging downstream effects over different time and length scales. Classic techniques such as pharmacological control, genetic knockout and biochemical assays often lack the precision required to probe the functions of specific GPCR subtypes. Here we describe the rapidly growing set of optogenetic tools, ranging from methods for optical control of the receptor itself to optical sensing and manipulation of downstream effectors. These tools permit the quantitative measurements of GPCRs and their downstream signaling with high specificity and spatiotemporal precision.

Abstract: Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

Abstract: Yeast has been a robust platform to manufacture a broad range of biofuels, commodity chemicals, natural products and pharmaceuticals. The membrane-bound organelles in yeast provide us the means to access the specialized metabolism for various biosynthetic applications. The separation and compartmentalization of genetic and metabolic events presents us the opportunity to precisely control and program gene expression for higher order biological functions. To further advance yeast synthetic biology platform, genetically encoded biosensors and actuators haven been engineered for in vivo monitoring and controlling cellular processes with spatiotemporal resolutions. The dynamic response, sensitivity and operational range of these genetically encoded sensors are determined by the regulatory architecture, dynamic assemly and interactions of the related proteins and genetic elements. This review provides an update of the basic design principles underlying the allosteric transcription factors, GPCR and optogenetics-based sensors, aiming to precisely analyze and control yeast cellular processes for various biotechnological applications.

Abstract: cAMP is a positive regulator tightly involved in certain types of synaptic plasticity and related memory functions. However, its spatiotemporal roles at the synaptic and neural circuit levels remain elusive. Using a combination of a cAMP optogenetics approach and voltage-sensitive dye (VSD) imaging with electrophysiological recording, we define a novel capacity of postsynaptic cAMP in enabling dentate gyrus long-term potentiation (LTP) and depolarization in acutely prepared murine hippocampal slices. To manipulate cAMP levels at medial perforant path to granule neuron (MPP-DG) synapses by light, we generated transgenic (Tg) mice expressing photoactivatable adenylyl cyclase (PAC) in DG granule neurons. Using these Tg(CMV-Camk2a-RFP/bPAC)3Koka mice, we recorded field excitatory postsynaptic potentials (fEPSPs) from MPP-DG synapses and found that photoactivation of PAC during tetanic stimulation enabled synaptic potentiation that persisted for at least 30 min. This form of LTP was induced without the need for GABA receptor blockade that is typically required for inducing DG plasticity. The paired-pulse ratio (PPR) remained unchanged, indicating the cAMP-dependent LTP was likely postsynaptic. By employing fast fluorescent voltage-sensitive dye (VSD: di-4-ANEPPS) and fluorescence imaging, we found that photoactivation of the PAC actuator enhanced the intensity and extent of dentate gyrus depolarization triggered following tetanic stimulation. These results demonstrate that the elevation of cAMP in granule neurons is capable of rapidly enhancing synaptic strength and neuronal depolarization. The powerful actions of cAMP are consistent with this second messenger having a critical role in the regulation of synaptic function.

Abstract: The zebrafish (Danio rerio) is a popular vertebrate model organism to investigate molecular mechanisms driving development and disease. Due to its transparency at embryonic and larval stages, investigations in the living organism are possible with subcellular resolution using intravital microscopy. The beneficial optical characteristics of zebrafish not only allow for passive observation, but also active manipulation of proteins and cells by light using optogenetic tools. Initially, photosensitive ion channels have been applied for neurobiological studies in zebrafish to dissect complex behaviors on a cellular level. More recently, exciting non-neural optogenetic tools have been established to control gene expression or protein localization and activity, allowing for unprecedented non-invasive and precise manipulation of various aspects of cellular physiology. Zebrafish will likely be a vertebrate model organism at the forefront of in vivo application of non-neural optogenetic tools and pioneering work has already been performed. In this review, we provide an overview of non-neuromodulatory optogenetic tools successfully applied in zebrafish to control gene expression, protein localization, cell signaling, migration and cell ablation.

Abstract: Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light. To overcome these problems, here, we have developed FRET biosensors with red-shifted excitation and emission wavelengths. We chose mKOκ and mKate2 as the favorable donor and acceptor pair by calculating the Förster distance. By optimizing the order of fluorescent proteins and modulatory domains of the FRET biosensors, we developed a FRET biosensor backbone named "Booster". The performance of the protein kinase A (PKA) biosensor based on the Booster backbone (Booster-PKA) was comparable to that of AKAR3EV, a previously developed FRET biosensor comprising CFP and YFP. For the proof of concept, we first showed simultaneous monitoring of activities of two protein kinases with Booster-PKA and ERK FRET biosensors based on CFP and YFP. Second, we showed monitoring of PKA activation by Beggiatoa photoactivated adenylyl cyclase, an optogenetic generator of cyclic AMP. Finally, we presented PKA activity in living tissues of transgenic mice expressing Booster-PKA. Collectively, the results demonstrate the effectiveness and versatility of Booster biosensors as an imaging tool in vitro and in vivo.

Abstract: Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light-oxygen-voltage domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254 and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp.. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic AMP production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.

Abstract: Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.

Abstract: A plethora of studies indicate the important role of cAMP and cGMP cascades in neuronal plasticity and memory function. As a result, altered cyclic nucleotide signaling has been implicated in the pathophysiology of mnemonic dysfunction encountered in several diseases. In the present review we provide a wide overview of studies regarding the involvement of cyclic nucleotides, as well as their upstream and downstream molecules, in physiological and pathological mnemonic processes. Next, we discuss the regulation of the intracellular concentration of cyclic nucleotides via phosphodiesterases, the enzymes that degrade cAMP and/or cGMP, and via A-kinase-anchoring proteins that refine signal compartmentalization of cAMP signaling. We also provide an overview of the available data pointing to the existence of specific time windows in cyclic nucleotide signaling during neuroplasticity and memory formation and the significance to target these specific time phases for improving memory formation. Finally, we highlight the importance of emerging imaging tools like Förster resonance energy transfer imaging and optogenetics in detecting, measuring and manipulating the action of cyclic nucleotide signaling cascades.

Abstract: Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.

Abstract: Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

Abstract: Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.

Abstract: The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.

Abstract: In optogenetics, light-sensitive proteins are specifically expressed in target cells and light is used to precisely control the activity of these proteins at high spatiotemporal resolution. Optogenetics initially used naturally occurring photoreceptors to control neural circuits, but has expanded to include carefully designed and engineered photoreceptors. Several optogenetic constructs are based on plant photoreceptors, but their application to plant systems has been limited. Here, we present perspectives on the development of plant optogenetics, considering different levels of design complexity. We discuss how general principles of light-driven signal transduction can be coupled with approaches for engineering protein folding to develop novel optogenetic tools. Finally, we explore how the use of computation, networks, circular permutation, and directed evolution could enrich optogenetics.

Abstract: Pharmacological augmentation of glucose-stimulated insulin secretion (GSIS), for example, to overcome insulin resistance in type 2 diabetes is linked to suboptimal regulation of blood sugar. Cultured β-cells and islets expressing a photoactivatable adenylyl cyclase (PAC) are amenable to GSIS potentiation with light. However, whether PAC-mediated enhancement of GSIS can improve the diabetic state remains unknown. To this end, β-cells were engineered with stable PAC expression that led to over 2-fold greater GSIS upon exposure to blue light while there were no changes in the absence of glucose. Moreover, the rate of oxygen consumption was unaltered despite the photoinduced elevation of GSIS. Transplantation of these cells into streptozotocin-treated mice resulted in improved glucose tolerance, lower hyperglycemia, and higher plasma insulin when subjected to illumination. Embedding optogenetic networks in β-cells for physiologically relevant control of GSIS will enable novel solutions potentially overcoming the shortcomings of current treatments for diabetes.

Abstract: Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.

Abstract: cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.

Abstract: Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.