Curated Optogenetic Publication Database

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.

Abstract: The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.

Abstract: Olfactory stimulation induces an odor-guided crawling behavior of Drosophila melanogaster larvae characterized by either an attractive or a repellent reaction. In order to understand the underlying processes leading to these orientations we stimulated single olfactory receptor neurons (ORNs) through photo-activation within an intact neuronal network. Using the Gal4-UAS system two light inducible proteins, the light-sensitive cation channel channelrhodopsin-2 (ChR-2) or the light-sensitive adenylyl cyclase (Pacalpha) were expressed in all or in individual ORNs of the larval olfactory system. Blue light stimulation caused an activation of these neurons, ultimately producing the illusion of an odor stimulus. Larvae were tested in a phototaxis assay for their orientation toward or away from the light source. Here we show that activation of Pacalpha expressing ORNs bearing the receptors Or33b or Or45a in blind norpA mutant larvae induces a repellent behavior away from the light. Conversely, photo-activation of the majority of ORNs induces attraction towards the light. Interestingly, in wild type larvae two ligands of Or33b and Or45a, octyl acetate and propionic ethylester, respectively, have been found to cause an escape reaction. Therefore, we combined light and odor stimulation to analyze the function of Or33b and Or45a expressing ORNs. We show that the larval olfactory system contains a designated neuronal pathway for repellent odorants and that activation of a specific class of ORNs already determines olfactory avoidance behavior.

Abstract: In neural mechanisms of animal learning, intracellular cAMP has been known to play an important role. In the present experiments we attempted functional transplant of a photoactivated adenylyl cyclase (PAC) isolated from Euglena into Aplysia neurons, and explored whether PAC can produce cAMP in the neurons by light stimulation. Serotonergic modulation of mechanoafferent sensory neurons in Aplysia pleural ganglia has been reported to increase intracellular cAMP level and promotes synaptic transmission to motor neurons by increasing spike width of sensory neurons. When cAMP was directly injected into the sensory neurons, spike amplitude temporarily decreased while spike width temporarily increased. This effect was not substituted by injection of 5'AMP, and maintained longer in a bath solution containing IBMX, the phosphodiesterase inhibitor. We, therefore, explored these changes as indicators of appearance of the PAC function. PAC or the PAC expression vector (pNEX-PAC) was injected into cell bodies of sensory neurons. Spike amplitude decreased in both cases and spike width increased in the PAC injection when the neurons were stimulated with light, suggesting that the transplanted PAC works well in Aplysia neurons. These results indicate that we can control cAMP production in specific neurons with light by the functional transplant of PAC.

Abstract: The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACalpha and PACbeta. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.