Curated Optogenetic Publication Database

Abstract: The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology‐inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light‐controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non‐neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light‐sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light‐controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.

Abstract: On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.

Abstract: The birth and subsequent evolution of optogenetics has resulted in an unprecedented advancement in our understanding of the brain. Its outstanding success does usher wider applications; however, the tool remains still largely relegated to neuroscience. Here, we introduce selected aspects of optogenetics with potential applications in infection biology that will not only answer long-standing questions about intracellular pathogens (parasites, bacteria, viruses) but also broaden the dimension of current research in entwined models. In this essay, we illustrate how a judicious integration of optogenetics with routine methods can illuminate the host-pathogen interactions in a way that has not been feasible otherwise.

Abstract: Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Abstract: Protein/RNA clusters arise frequently in spatially regulated biological processes, from the asymmetric distribution of P granules and PAR proteins in developing embryos to localized receptor oligomers in migratory cells. This co-occurrence suggests that protein clusters might possess intrinsic properties that make them a useful substrate for spatial regulation. Here, we demonstrate that protein droplets show a robust form of spatial memory, maintaining the spatial pattern of an inhibitor of droplet formation long after it has been removed. Despite this persistence, droplets can be highly dynamic, continuously exchanging monomers with the diffuse phase. We investigate the principles of biophysical spatial memory in three contexts: a computational model of phase separation; a novel optogenetic system where light can drive rapid, localized dissociation of liquid-like protein droplets; and membrane-localized signal transduction from clusters of receptor tyrosine kinases. Our results suggest that the persistent polarization underlying many cellular and developmental processes could arise through a simple biophysical process, without any additional biochemical feedback loops.

Abstract: The field of optogenetics uses genetically encoded, light-responsive proteins to control physiological processes. This technology has been hailed as the one of the ten big ideas in brain science in the past decade,[1] the breakthrough of the decade,[2] and the method of the year in 2010[3] and again in 2014[4]. The excitement evidenced by these proclamations is confirmed by a couple of impressive numbers. The term "optogenetics" was coined in 2006.[5] As of December 2017, "optogenetics" is found in the title or abstract of almost 1600 currently funded National Institutes of Health grants. In addition, nearly 600 reviews on optogenetics have appeared since 2006, which averages out to approximately one review per week! However, in spite of these impressive numbers, the potential applications and implications of optogenetics are not even close to being fully realized. This is due, in large part, to the challenges associated with the design of optogenetic analogs of endogenous proteins. This review is written from a chemist's perspective, with a focus on the molecular strategies that have been developed for the construction of optogenetic proteins.

Abstract: In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living bacteria to autonomically form patterned biofilms following prescribed illumination. The results showed that through optogenetic manipulation, patterned bacterial communities with high spatial resolution (approximately 10 μm) could be constructed in 6 h. Thus, optogenetic manipulation greatly increases the range of available bioprinting techniques.

Abstract: Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision.

Abstract: The discovery and domestication of biomolecules that respond to light has taken a light of its own, providing new molecular tools with incredible spatio-temporal resolution to manipulate cellular behavior. Areas covered: The authors herein analyze the current optogenetic tools in light of their current, and potential, uses in cancer drug discovery, biosafety and cancer biology. Expert opinion: The pipeline from drug discovery to the clinic is plagued with drawbacks, where most drugs fail in either efficacy or safety. These issues require the redesign of the pipeline and the development of more controllable/personalized therapies. Light is, aside from inexpensive, almost harmless if used appropriately, can be directed to single cells or organs with controllable penetration, and comes in a variety of wavelengths. Light-responsive systems can activate, inhibit or compensate cell signaling pathways or specific cellular events, allowing the specific control of the genome and epigenome, and modulate cell fate and transformation. These synthetic molecular tools have the potential to revolutionize drug discovery and cancer research.

Abstract: Photoreceptor proteins have been used to study how protein conformational changes are induced by alterations in their environments and how their signals are transmitted to downstream factors to dictate physiological responses. These proteins are attractive models because their signal transduction aspects and structural changes can be precisely regulated in vivo and in vitro based on light intensity. Among the known photoreceptors, members of the blue light-using flavin (BLUF) protein family have been well characterized with regard to how they control various light-dependent physiological responses in several microorganisms. Herein, we summarize our current understanding of their photoactivation and signal-transduction mechanisms. For signal transduction, we review recent studies concerning how the BLUF protein, PixD, transmits a light-induced signal to its downstream factor, PixE, to modulate phototaxis of the cyanobacterium Synechocystis sp. PCC6803.

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: Cyclic nucleotide signaling pathway plays a significant role in various biological processes such as cell growth, transcription, inflammation, in microbial pathogenesis, etc. Modulation of cyclic nucleotide levels by optogenetic tools has overcome certain limitations of studying transduction cascade by pharmacological agents and has allowed several ways to modulate biological processes in a spatiotemporal manner. Here, we have shown the optogenetic modulation of the cyclooxygenase 2 (Cox-2) gene expression and their downstream effector molecule (PGE2) in HEK-293T cells and the development process of Dictyostelium discoideum via modulating the cyclic nucleotide (cAMP) signaling pathway utilizing photoactivated adenylyl cyclases (PACs) as an optogenetic tool. Light-induced activation of PACs in HEK-293T cells increases the cAMP level that leads to activation of cAMP response element-binding protein (CREB) transcription factor and further upregulates downstream Cox-2 gene expression and their downstream effector molecule prostaglandin E2. In D. discoideum, the light-regulated increase in cAMP level affects the starvation-induced developmental process. These PACs could modulate the cAMP levels in a light-dependent manner and have a potential to control gene expression and their downstream effector molecules with varying magnitude. It would enable one to utilize PAC as a tool to decipher cyclic nucleotide mediated signaling pathway regulations and their mechanism.

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: Pancreatic β-cell insulin production is orchestrated by a complex circuitry involving intracellular elements including cyclic AMP (cAMP). Tackling aberrations in glucose-stimulated insulin release such as in diabetes with pharmacological agents, which boost the secretory capacity of β-cells, is linked to adverse side effects. We hypothesized that a photoactivatable adenylyl cyclase (PAC) can be employed to modulate cAMP in β-cells with light thereby enhancing insulin secretion. To that end, the PAC gene from Beggiatoa (bPAC) was delivered to β-cells. A cAMP increase was noted within 5 minutes of photostimulation and a significant drop at 12 minutes post-illumination. The concomitant augmented insulin secretion was comparable to that from β-cells treated with secretagogues. Greater insulin release was also observed over repeated cycles of photoinduction without adverse effects on viability and proliferation. Furthermore, the expression and activation of bPAC increased cAMP and insulin secretion in murine islets and in β-cell pseudoislets, which displayed a more pronounced light-triggered hormone secretion compared to that of β-cell monolayers. Calcium channel blocking curtailed the enhanced insulin response due to bPAC activity. This optogenetic system with modulation of cAMP and insulin release can be employed for the study of β-cell function and for enabling new therapeutic modalities for diabetes.

Abstract: cAMP is a common second messenger that is involved in various physiological processes. To expand the colour palette of available cAMP indicators, we developed a red cAMP indicator named "Pink Flamindo" (Pink Fluorescent cAMP indicator). The fluorescence intensity of Pink Flamindo increases 4.2-fold in the presence of a saturating dose of cAMP, with excitation and emission peaks at 567 nm and 590 nm, respectively. Live-cell imaging revealed that Pink Flamindo is effective for monitoring the spatio-temporal dynamics of intracellular cAMP generated by photoactivated adenylyl cyclase in response to blue light, and in dual-colour imaging studies using a green Ca2+ indicator (G-GECO). Furthermore, we successfully monitored the elevation of cAMP levels in vivo in cerebral cortical astrocytes by two-photon imaging. We propose that Pink Flamindo will facilitate future in vivo, optogenetic studies of cell signalling and cAMP dynamics.

Abstract: The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

Abstract: Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, in particular tools for all-optical electrophysiology, now provide a means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline - from target identification and validation, to target-based and phenotypic screens, to clinical trials.

Abstract: First described about 15 years ago, BLUF (Blue Light Using Flavin) domains are light-triggered switches that control enzyme activity or gene expression in response to blue light, remaining activated for seconds or even minutes after stimulation. The conserved, ferredoxin-like fold holds a flavin chromophore that captures the light and somehow triggers downstream events. BLUF proteins are found in both prokaryotes and eukaryotes and have a variety of architectures and oligomeric forms, but the BLUF domain itself seems to have a well-preserved structure and mechanism that have been the focus of intense study for a number of years. Crystallographic and NMR structures of BLUF domains have been solved, but the conflicting models have led to considerable debate about the atomic details of photo-activation. Advanced spectroscopic and computational methods have been used to analyse the early events after photon absorption, but these too have led to widely differing conclusions. New structural models are improving our understanding of the details of the mechanism and may lead to novel tailor-made tools for optogenetics.

Abstract: Light-regulated enzymes enable organisms to quickly respond to changing light conditions. We characterize a photoactivatable adenylyl cyclase (AC) from Beggiatoa sp. (bPAC) that translates a blue light signal into the production of the second messenger cyclic AMP. bPAC contains a BLUF photoreceptor domain that senses blue light using a flavin chromophore, linked to an AC domain. We present a dark state crystal structure of bPAC that closely resembles the recently published structure of the homologous OaPAC from Oscillatoria acuminata. To elucidate the structural mechanism of light-dependent AC activation by the BLUF domain, we determined the crystal structures of illuminated bPAC and of a pseudo-lit state variant. We use hydrogen-deuterium exchange measurements of secondary structure dynamics and hypothesis-driven point mutations to trace the activation pathway from the chromophore in the BLUF domain to the active site of the cyclase. The structural changes are relayed from the residues interacting with the excited chromophore through a conserved kink of the BLUF β-sheet to a tongue-like extrusion of the AC domain that regulates active site opening and repositions catalytic residues. Our findings not only show the specific molecular pathway of photoactivation in BLUF-regulated ACs but also have implications for the general understanding of signaling in BLUF domains and of the activation of ACs.

Abstract: Many aspects of bacterial physiology and behavior including motility, surface attachment, and cell cycle, are controlled by the c-di-GMP-dependent signaling pathways on the scale of seconds-to-minutes. Interrogation of such processes in real time requires tools for introducing rapid and reversible changes in intracellular c-di-GMP levels. Inducing expression of genes encoding c-di-GMP synthetic (diguanylate cyclases) and degrading (c-di-GMP phosphodiesterase) enzymes by chemicals may not provide adequate temporal control. In contrast, light-controlled diguanylate cyclases and phosphodiesterases can be quickly activated and inactivated. A red/near-infrared light-regulated diguanylate cyclase, BphS, has been engineered earlier, yet a complementary light-activated c-di-GMP phosphodiesterase has been lacking. In search of such a phosphodiesterase, we investigated two homologous proteins from Allochromatium vinosum and Magnetococcus marinus, designated BldP, which contain C-terminal EAL-BLUF modules, where EAL is a c-di-GMP phosphodiesterase domain and BLUF is a blue light sensory domain. Characterization of the BldP proteins in Escherichia coli and in vitro showed that they possess light-activated c-di-GMP phosphodiesterase activities. Interestingly, light activation in both enzymes was dependent on oxygen levels. The truncated EAL-BLUF fragment from A. vinosum BldP lacked phosphodiesterase activity, whereas a similar fragment from M. marinus BldP, designated EB1, possessed such activity that was highly (>30-fold) upregulated by light. Following light withdrawal, EB1 reverted to the inactive ground state with a half-life of ∼6 min. Therefore, the blue light-activated phosphodiesterase, EB1, can be used in combination with the red/near-infrared light-regulated diguanylate cyclase, BphS, for bidirectional regulation of c-di-GMP-dependent processes in E. coli as well as other bacterial and nonbacterial cells.IMPORTANCE Regulation of motility, attachment to surfaces, cell cycle, and other bacterial processes controlled by the c-di-GMP signaling pathways occurs at a fast (seconds-to-minutes) pace. Interrogating these processes at high temporal and spatial resolution using chemicals is difficult-to-impossible, while optogenetic approaches may prove useful. We identified and characterized a robust, blue light-activated c-di-GMP phosphodiesterase (hydrolase) that complements a previously engineered red/near-infrared light-regulated diguanylate cyclase (c-di-GMP synthase). These two enzymes form a dichromatic module for manipulating intracellular c-di-GMP levels in bacterial and nonbacterial cells.

Abstract: Optogenetics allows to non-invasively manipulate cellular functions with spatio-temporal precision by combining genetic engineering with the control of protein function by light. Since the discovery of channelrhodopsin has pioneered the field, the optogenetic toolkit has been ever expanding and allows now not only to control neuronal activity by light, but rather a multitude of other cellular functions. One important application that has been established in recent years is the light-dependent control of second messenger signaling. The optogenetic toolkit now allows to control cyclic nucleotide-dependent signaling by light in vitro and in vivo.

Abstract: Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger, c-di-GMP, as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared light-regulated diguanylate cyclase and a blue-light regulated c-di-GMP phosphodiesterase. It allows generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the timescale of chemotaxis signaling. We provide experimental evidence that c-di-GMP binding to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense changes with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates their contribution to the overall chemotaxis response. Here, we used an optogenetic system to perturb intracellular concentrations of the bacterial second messenger, c-di-GMP, to show that in some chemotaxis receptors, c-di-GMP functions in a similar feedback loop to connect metabolic status of the cells to sensory activity of chemotaxis receptors.

Abstract: The optogenetic revolution enabled spatially-precise and temporally-precise control over protein function, signaling pathway activation, and animal behavior with tremendous success in the dissection of signaling networks and neural circuits. Very recently, optogenetic methods have been paired with optical reporters in novel drug screening platforms. In these all-optical platforms, light remotely activated ion channels and kinases thereby obviating the use of electrophysiology or reagents. Consequences were remarkable operational simplicity, throughput, and cost-effectiveness that culminated in the identification of new drug candidates. These blueprints for all-optical assays also revealed potential pitfalls and inspire all-optical variants of other screens, such as those that aim at better understanding dynamic drug action or orphan protein function.

Abstract: Cyclic AMP (cAMP) signaling augments synaptic transmission, but because many targets of cAMP and protein kinase A (PKA) may be involved, mechanisms underlying this pathway remain unclear. To probe this mechanism, we used optogenetic stimulation of cAMP signaling by Beggiatoa-photoactivated adenylyl cyclase (bPAC) in Caenorhabditis elegans motor neurons. Behavioral, electron microscopy (EM), and electrophysiology analyses revealed cAMP effects on both the rate and on quantal size of transmitter release and led to the identification of a neuropeptidergic pathway affecting quantal size. cAMP enhanced synaptic vesicle (SV) fusion by increasing mobilization and docking/priming. cAMP further evoked dense core vesicle (DCV) release of neuropeptides, in contrast to channelrhodopsin (ChR2) stimulation. cAMP-evoked DCV release required UNC-31/Ca(2+)-dependent activator protein for secretion (CAPS). Thus, DCVs accumulated in unc-31 mutant synapses. bPAC-induced neuropeptide signaling acts presynaptically to enhance vAChT-dependent SV loading with acetylcholine, thus causing increased miniature postsynaptic current amplitudes (mPSCs) and significantly enlarged SVs.