Curated Optogenetic Publication Database

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: 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.

Abstract: In eukaryotes, protein kinase A (PKA) is a master regulator of cell proliferation and survival. The activity of PKA is subject to elaborate control and exhibits complex time dynamics. To probe the quantitative attributes of PKA dynamics in the yeast Saccharomyces cerevisiae, we developed an optogenetic strategy that uses a photoactivatable adenylate cyclase to achieve real-time regulation of cAMP and the PKA pathway. We capitalize on the precise and rapid control afforded by this optogenetic tool, together with quantitative computational modeling, to study the properties of feedback in the PKA signaling network and dissect the nonintuitive dynamic effects that ensue from perturbing its components. Our analyses reveal that negative feedback channeled through the Ras1/2 GTPase is delayed, pinpointing its time scale and its contribution to the dynamic features of the cAMP/PKA signaling network.

Abstract: Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca(2+) and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.

Abstract: Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Abstract: The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca(2+) oscillations and Ca(2+)-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca(2+)-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca(2+)-modulated activities with tailored function.

Abstract: Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.

Abstract: Ca(2+) signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca(2+) channel switch (BACCS), as an optogenetic tool for generating Ca(2+) signals. BACCS opens Ca(2+)-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca(2+) concentration more rapidly, such that Ca(2+) elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca(2+). Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca(2+) signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.

Abstract: Blastocladiomycota fungi form motile zoospores that are guided by sensory photoreceptors to areas of optimal light conditions. We showed that the microbial rhodopsin of Blastocladiella emersonii is a rhodopsin-guanylyl cyclase (RhGC), a member of a previously uncharacterized rhodopsin class of light-activated enzymes that generate the second messenger cyclic guanosine monophosphate (cGMP). Upon application of a short light flash, recombinant RhGC converted within 8 ms into a signaling state with blue-shifted absorption from which the dark state recovered within 100 ms. When expressed in Xenopus oocytes, Chinese hamster ovary cells, or mammalian neurons, RhGC generated cGMP in response to green light in a light dose-dependent manner on a subsecond time scale. Thus, we propose RhGC as a versatile tool for the optogenetic analysis of cGMP-dependent signaling processes in cell biology and the neurosciences.

Abstract: Zebrafish offer an opportunity to study conserved mechanisms underlying the ontogeny and physiology of the hypothalamic-pituitary-adrenal/interrenal axis. As the final effector of the hypothalamic-pituitary-adrenal/interrenal axis, glucocorticoids exert both rapid and long-term regulatory functions. To elucidate their specific effects in zebrafish, transgenic approaches are necessary to complement pharmacological studies. Here, we report a robust approach to specifically manipulate endogenous concentrations of cortisol by targeting heterologous proteins to interrenal cells using a promoter element of the steroidogenic acute regulatory protein. To test this approach, we first used this regulatory region to generate a transgenic line expressing the bacterial nitroreductase protein, which allows conditional targeted ablation of interrenal cells. We demonstrate that this line can be used to specifically ablate interrenal cells, drastically reducing both basal and stress-induced cortisol concentrations. Next, we coupled this regulatory region to an optogenetic actuator, Beggiatoa photoactivated adenylyl cyclase, to increase endogenous cortisol concentrations in a blue light-dependent manner. Thus, our approach allows specific manipulations of steroidogenic interrenal cell activity for studying the effects of both hypo- and hypercortisolemia in zebrafish.

Abstract: Precise spatiotemporal control of physiological processes by optogenetic devices inspired by synthetic biology may provide novel treatment opportunities for gene- and cell-based therapies. An erectile optogenetic stimulator (EROS), a synthetic designer guanylate cyclase producing a blue-light-inducible surge of the second messenger cyclic guanosine monophosphate (cGMP) in mammalian cells, enabled blue-light-dependent penile erection associated with occasional ejaculation after illumination of EROS-transfected corpus cavernosum in male rats. Photostimulated short-circuiting of complex psychological, neural, vascular, and endocrine factors to stimulate penile erection in the absence of sexual arousal may foster novel advances in the treatment of erectile dysfunction.

Abstract: Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.

Abstract: Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.

Abstract: A light-regulated adenylyl cyclase, mPAC, was previously identified from the cyanobacterium Microcoleus chthonoplastes PCC7420. MPAC consists of a flavin-based blue light-sensing LOV domain and a catalytic domain. In this work, we expressed mPAC in an adenylate cyclase A null mutant (aca-) of the eukaryote Dictyostelium discoideum and tested to what extent light activation of mPAC could restore the cAMP-dependent developmental programme of this organism. Amoebas of Dictyostelium, a well-established model organism, generate and respond to cAMP pulses, which cause them to aggregate and construct fruiting bodies. mPAC was expressed under control of a constitutive actin-15 promoter in D. discoideum and displayed low basal adenylyl cyclase activity in darkness that was about five-fold stimulated by blue light. mPAC expression in aca- cells marginally restored aggregation and fruiting body formation in darkness. However, more and larger fruiting bodies were formed when mPAC expressing cells were incubated in light. Extending former applications of light-regulated AC, these results demonstrate that mPAC can be used to manipulate multicellular development in eukaryotes in a light dependent manner.

Abstract: Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.

Abstract: Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.

Abstract: Calcium ion (Ca2+) is an important second messenger implicated in the control of many different cellular processes in living organisms. Ca2+ is typically studied by direct visualization using chemically or genetically encoded indicators. A complementary, and perhaps more useful, approach involves direct manipulation of Ca2+ concentration; tools for this exist but are rather poorly developed compared to the indicators at least. Here, we report a photoactivatable Ca2+-releasing protein, photoactivatable Ca2+ releaser (PACR), made by the insertion of a photosensitive protein domain (LOV2) into a Ca2+ binding protein (calmodulin fused with the M13 peptide). As the PACR is genetically encoded, and unlike conventional optical control tools (e.g., channel rhodopsin) not membrane bound, we are able to restrict expression within the cell, to allow subcellular perturbation of Ca2+ levels. In whole animals, we are able to control the behavior of Caenorhabditis elegans with light by expressing the PACR only in the touch neuron.

Abstract: Enormous potential of cell-based therapeutics is hindered by the lack of effective means to control genetically engineered cells in mammalian tissues. Here, we describe a synthetic module for remote photocontrol of engineered cells that can be adapted for such applications. The module involves photoactivated synthesis of cyclic dimeric GMP (c-di-GMP), a stable small molecule that is not produced by higher eukaryotes and therefore is suitable for orthogonal regulation. The key component of the photocontrol module is an engineered bacteriophytochrome diguanylate cyclase, which synthesizes c-di-GMP from GTP in a light-dependent manner. Bacteriophytochromes are particularly attractive photoreceptors because they respond to light in the near-infrared window of the spectrum, where absorption by mammalian tissues is minimal, and also because their chromophore, biliverdin IXα, is naturally available in mammalian cells. The second component of the photocontrol module, a c-di-GMP phosphodiesterase, maintains near-zero background levels of c-di-GMP in the absence of light, which enhances the photodynamic range of c-di-GMP concentrations. In the E. coli model used in this study, the intracellular c-di-GMP levels could be upregulated by light by >50-fold. Various c-di-GMP-responsive proteins and riboswitches identified in bacteria can be linked downstream of the c-di-GMP-mediated photocontrol module for orthogonal regulation of biological activities in mammals as well as in other organisms lacking c-di-GMP signaling. Here, we linked the photocontrol module to a gene expression output via a c-di-GMP-responsive transcription factor and achieved a 40-fold photoactivation of gene expression.

Abstract: Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2 kDa in size) characteristic of a BL (blue light)-regulated adenylate (adenylyl) cyclase function. The photoreceptive part showed signatures of a LOV (light, oxygen, voltage) domain. The gene product, mPAC (Microcoleus photoactivated adenylate cyclase), exhibited the LOV-specific three-peaked absorption band (λmax=450 nm) and underwent conversion into the photoadduct form (λmax=390 nm) upon BL-irradiation. The lifetime for thermal recovery into the parent state was determined as 16 s at 20°C (25 s at 11°C). The adenylate cyclase function showed a constitutive activity (in the dark) that was in-vitro-amplified by a factor of 30 under BL-irradiation. Turnover of the purified protein at saturating light and pH 8 is estimated to 1 cAMP/mPAC per s at 25°C (2 cAMP/mPAC per s at 35°C). The lifetime of light-activated cAMP production after a BL flash was ~14 s at 20°C. The temperature optimum was determined to 35°C and the pH optimum to 8.0. The value for half-maximal activating light intensity is 6 W/m2 (at 35°C). A comparison of mPAC and the BLUF (BL using FAD) protein bPAC (Beggiatoa PAC), as purified proteins and expressed in Xenopus laevis oocytes, yielded higher constitutive activity for mPAC in the dark, but also when illuminated with BL.

Abstract: Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. Here, we characterize a neuromodulatory circuit that generates long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states.

Abstract: The stress response is a suite of physiological and behavioral processes that help to maintain or reestablish homeostasis. Central to the stress response is the hypothalamic-pituitary-adrenal (HPA) axis, as it releases crucial hormones in response to stress. Glucocorticoids (GCs) are the final effector hormones of the HPA axis, and exert a variety of actions under both basal and stress conditions. Despite their far-reaching importance for health, specific GC effects have been difficult to pin-down due to a lack of methods for selectively manipulating endogenous GC levels. Hence, in order to study stress-induced GC effects, we developed a novel optogenetic approach to selectively manipulate the rise of GCs triggered by stress. Using this approach, we could induce both transient hypercortisolic states and persistent forms of hypercortisolaemia in freely behaving larval zebrafish. Our results also established that transient hypercortisolism leads to enhanced locomotion shortly after stressor exposure. Altogether, we present a highly specific method for manipulating the gain of the stress axis with high temporal accuracy, altering endocrine and behavioral responses to stress as well as basal GC levels. Our study offers a powerful tool for the analysis of rapid (non-genomic) and delayed (genomic) GC effects on brain function and behavior, feedbacks within the stress axis and developmental programming by GCs.

Abstract: Ca(2+) influx via L-type Ca(v)1.2 channels is essential for multiple physiological processes, including gene expression, excitability, and contraction. Amplification of the Ca(2+) signals produced by the opening of these channels is a hallmark of many intracellular signaling cascades, including excitation-contraction coupling in heart. Using optogenetic approaches, we discovered that Ca(v)1.2 channels form clusters of varied sizes in ventricular myocytes. Physical interaction between these channels via their C-tails renders them capable of coordinating their gating, thereby amplifying Ca(2+) influx and excitation-contraction coupling. Light-induced fusion of WT Ca(v)1.2 channels with Ca(v)1.2 channels carrying a gain-of-function mutation that causes arrhythmias and autism in humans with Timothy syndrome (Ca(v)1.2-TS) increased Ca(2+) currents, diastolic and systolic Ca(2+) levels, contractility and the frequency of arrhythmogenic Ca(2+) fluctuations in ventricular myocytes. Our data indicate that these changes in Ca(2+) signaling resulted from Ca(v)1.2-TS increasing the activity of adjoining WT Ca(v)1.2 channels. Collectively, these data support the concept that oligomerization of Ca(v)1.2 channels via their C termini can result in the amplification of Ca(2+) influx into excitable cells.

Abstract: Ca(2+) signals regulate diverse physiological processes through tightly regulated fluxes varying in location, time, frequency, and amplitude. Here, we developed LOVS1K, a genetically encoded and photoactivated synthetic protein to generate local or global Ca(2+) signals. With 300 ms blue light exposure, LOVS1K translocated to Orai1, a plasma membrane Ca(2+) channel, within seconds, generating a local Ca(2+) signal on the plasma membrane, and returning to the cytoplasm after tens of seconds. With repeated photoactivation, global Ca(2+) signals in the cytoplasm were generated to modulate engineered Ca(2+)-inducible proteins. Although Orai1 is typically associated with global store-operated Ca(2+) entry, we demonstrate that Orai1 can also generate local Ca(2+) influx on the plasma membrane. Our photoactivation system can be used to generate spatially and temporally precise Ca(2+) signals and to engineer synthetic proteins that respond to specific Ca(2+) signals.

Abstract: Photoactivated adenylyl cyclase α (PACα) was originally isolated from the flagellate Euglena gracilis. Following stimulation by blue light it causes a rapid increase in cAMP levels. In the present study, we expressed PACα in cholinergic neurons of Caenorhabditis elegans. Photoactivation led to a rise in swimming frequency, speed of locomotion, and a decrease in the number of backward locomotion episodes. The extent of the light-induced behavioral effects was dependent on the amount of PACα that was expressed. Furthermore, electrophysiological recordings from body wall muscle cells revealed an increase in miniature post-synaptic currents during light stimulation. We conclude that the observed effects were caused by cAMP synthesis because of photoactivation of pre-synaptic PACα which subsequently triggered acetylcholine release at the neuromuscular junction. Our results demonstrate that PACα can be used as an optogenetic tool in C. elegans for straightforward in vivo manipulation of intracellular cAMP levels by light, with good temporal control and high cell specificity. Thus, using PACα allows manipulation of neurotransmitter release and behavior by directly affecting intracellular signaling.

Abstract: Cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers that regulate metabolic and behavioral responses in diverse organisms. We describe purification, engineering, and characterization of photoactivated nucleotidyl cyclases that can be used to manipulate cAMP and cGMP levels in vivo. We identified the blaC gene encoding a putative photoactivated adenylyl cyclase in the Beggiatoa sp. PS genome. BlaC contains a BLUF domain involved in blue-light sensing using FAD and a nucleotidyl cyclase domain. The blaC gene was overexpressed in Escherichia coli, and its product was purified. Irradiation of BlaC in vitro resulted in a small red shift in flavin absorbance, typical of BLUF photoreceptors. BlaC had adenylyl cyclase activity that was negligible in the dark and up-regulated by light by 2 orders of magnitude. To convert BlaC into a guanylyl cyclase, we constructed a model of the nucleotidyl cyclase domain and mutagenized several residues predicted to be involved in substrate binding. One triple mutant, designated BlgC, was found to have photoactivated guanylyl cyclase in vitro. Irradiation with blue light of the E. coli cya mutant expressing BlaC or BlgC resulted in the significant increases in cAMP or cGMP synthesis, respectively. BlaC, but not BlgC, restored cAMP-dependent growth of the mutant in the presence of light. Small protein sizes, negligible activities in the dark, high light-to-dark activation ratios, functionality at broad temperature range and physiological pH, as well as utilization of the naturally occurring flavins as chromophores make BlaC and BlgC attractive for optogenetic applications in various animal and microbial models.