Curated Optogenetic Publication Database

Abstract: The possibility offered by photocontrolling the activity of biomolecules in vivo while recording physiological parameters is opening up new opportunities for the study of physiological processes at the single-cell level in a living organism. For the last decade, such tools have been mainly used in neuroscience, and their application in freely moving animals has revolutionized this field. New photochemical approaches enable the control of various cellular processes by manipulating a wide range of protein functions in a noninvasive way and with unprecedented spatiotemporal resolution. We are at a pivotal moment where biologists can adapt these cutting-edge technologies to their system of study. This user-oriented review presents the state of the art and highlights technical issues to be resolved in the near future for wide and easy use of these powerful approaches.

Abstract: The absorption and emission spectroscopic behavior of the photo-activated adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied. The flavin cofactor was found to be partly fully oxidized and partly fully reduced. The typical BLUF domain (BLUF = Blue Light sensor Using Flavin) oxidized flavin absorption photo-cycle dynamics with about 14 nm flavin absorption red-shift in the signaling state was observed. The quantum efficiency of signaling state formation was determined to be s = 0.66 ± 0.03. A bi-exponential signaling state recovery to the dark-adapted receptor state was observed with the time constants rec,f = 275 s and rec,sl = 45 min. The thermal irreversible protein unfolding was studied and an apparent protein melting temperature of ϑm ≈ 50 ◦C was found. The photodynamic behavior of NgPAC3 is compared with the behavior of the previously investigated photo-activated cyclases NgPAC1 (nPAC) and NgPAC2 from the same N. gruberi NEG-M strain. Purified recombinant NgPAC3 showed light-gated adenylate cyclase activity upon illumination with blue light. Its cyclase activity is compared with those of NgPAC1 and NgPAC2.

Abstract: The brain is a large network of interconnected neurons where each cell functions as a nonlinear processing element. Unraveling the mysteries of information processing in the complex networks of the brain requires versatile neurostimulation and imaging techniques. Optogenetics is a new stimulation method which allows the activity of neurons to be modulated by light. For this purpose, the cell-types of interest are genetically targeted to produce light-sensitive proteins. Once these proteins are expressed, neural activity can be controlled by exposing the cells to light of appropriate wavelengths. Optogenetics provides a unique combination of features, including multimodal control over neural function and genetic targeting of specific cell-types. Together, these versatile features combine to a powerful experimental approach, suitable for the study of the circuitry of psychiatric and neurological disorders. The advent of optogenetics was followed by extensive research aimed to produce new lines of light-sensitive proteins and to develop new technologies: for example, to control the distribution of light inside the brain tissue or to combine optogenetics with other modalities including electrophysiology, electrocorticography, nonlinear microscopy, and functional magnetic resonance imaging. In this paper, the authors review some of the recent advances in the field of optogenetics and related technologies and provide their vision for the future of the field.

Abstract: We developed a novel technique for manipulating the activity of transcription factors with blue light (termed "PICCORO") using the bacterial BLUF-type photoreceptor protein PixD. The chimeric dominant-negative T-box transcription factor No Tail formed heterologous complexes with a PixD decamer in a light-dependent manner, and these complexes affected transcription repressor activity. When applied to zebrafish embryos, PICCORO permitted regulation of the activity of the mutant No Tail in response to 472-nm light provided by a light-emitting diode.

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: Light is fundamental to life on earth. Therefore, nature has evolved a multitude of photoreceptors that sense light across all kingdoms. This natural resource provides synthetic biology with a vast pool of light-sensing components with distinct spectral properties that can be harnessed to engineer novel optogenetic tools. These devices enable control over gene expression, cell morphology and signaling pathways with superior spatiotemporal resolution and are maturing towards elaborate applications in basic research, in the production of biopharmaceuticals and in biomedicine. This article provides a summary of the recent advances in optogenetics that use light for the precise control of biological functions in mammalian cells.

Abstract: Optogenetics arises from the innovative application of microbial opsins in mammalian neurons and has since been a powerful technology that fuels the advance of our knowledge in neuroscience. In recent years, there has been growing interest in designing optogenetic tools extendable to broader cell types and biochemical signals. To date, a variety of photoactivatable proteins (refers to induction of protein activity in contrast to fluorescence) have been developed based on the understanding of plant and microbial photoreceptors including phototropins, blue light sensors using flavin adenine dinucleotide proteins, cryptochromes, and phytochromes. Such tools offered researchers reversible, quantitative, and precise spatiotemporal control of enzymatic activity, protein-protein interaction, protein translocation, as well as gene transcription in cells and in whole animals. In this review, we will briefly introduce these photosensory proteins, describe recent developments in optogenetics, and compare and contrast different methods based on their advantages and limitations.

Abstract: PixD is a blue light-using flavin (BLUF) photoreceptor that controls phototaxis in the cyanobacterium Synechocystis sp. PCC6803. PixD interacts with the response regulator-like protein PixE in a light-dependent manner, and this interaction is critical for light signal transduction in vivo. However, the structure of the PixD-PixE complex has not been determined. To improve our understanding of how PixD transmits its captured light signal to PixE, we used blue-native polyacrylamide gel electrophoresis to characterize the molecular mass of a recombinant PixD-PixE complex purified from Escherichia coli and found it to be 342 kDa, suggesting that the complex contains 10 PixD and 4 PixE monomers. The stoichiometry of the complex was confirmed by Western blotting. Specifically, three intermediate states, PixD(10)-PixE(1), PixD(10)-PixE(2), and PixD(10)-PixE(3), were detected. The apparent dissociation constant for PixE and PixD is ~5 μM. A docking simulation was performed using a modeled PixE structure and the PixD(10) crystal structure. The docking simulation showed how the molecules in the PixD(10)-PixE(4) structure interact. To verify the accuracy of the docked model, a site-directed mutagenesis study was performed in which Arg80 of PixE, which appears to be capable of interacting electrostatically with Asp135 of PixD in the predicted structure, was shown to be critical for complex formation as mutation of PixE Arg80 to Asp or Ala prevented PixD-PixE complex formation. This study provides a structural basis for future investigations of the light signal transduction mechanism involving PixD and PixE.

Abstract: The absorption and emission spectroscopic behavior of the photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied in the dark, during blue-light exposure and after blue-light exposure. The typical BLUF domain (BLUF = Blue Light sensor Using Flavin) flavin cofactor absorption and fluorescence photo-cycle dynamics was observed. For fresh samples a reversible concentration dependent protein oligomerization occurred showing up in free flavin binding and protein color center formation with increasing protein concentration. Thermal and temporal irreversible protein unfolding with loss of BLUF domain activity was investigated. Temperature dependent protein melting times and the apparent protein melting temperature were determined. The photodynamic behavior of the NgPAC2 is compared with the behavior of the previously investigated photo-activated cyclase NgPAC1 (nPAC) from the same N. gruberi NEG-M strain.

Abstract: BLUF (sensor of blue light using FAD) domain-containing proteins are one of three types of flavin-binding, blue-light-sensing proteins found in many bacteria and some algae. The other types of blue-light-sensing proteins are the cryptochromes and the light, oxygen, voltage (LOV) domain-containing proteins. BLUF proteins control a wide variety of light-dependent physiological activities including photosystem synthesis, biofilm formation and the photoavoidance response. The BLUF domain photochemical reaction is unique in that only small chromophore structural changes are involved in the light activation process, because the rigid flavin moiety is involved, rather than an isomerizable chromophore (e.g. phytochromobilin in phytochromes and retinal in rhodopsins). Recent spectroscopic, biochemical and structural studies have begun to elucidate how BLUF domains transmit the light-induced signal and identify related, subsequent changes in the domain structures. Herein, I review progress made to date concerning the physiological functions and the phototransduction mechanism of BLUF proteins.

Abstract: PixD (Slr1694) is a blue light receptor that contains a BLUF (blue light sensors using a flavin chromophore) domain. A protein-protein interaction between PixD and a response regulator PixE (Slr1693) is essential to achieve light signal transduction for phototaxis of the species. Although the initial photochemical reaction of PixD, the red shift of the flavin absorption spectrum, has been investigated, the subsequent reaction dynamics remain largely unresolved. Only the disassembly of the PixD(10)-PixE(5) dark complex has been characterized by static size exclusion chromatography. In this report, interprotein reaction dynamics were examined using time-resolved transient grating spectroscopy. The dissociation process was clearly observed as the light-induced diffusion coefficient change in the time domain, and the kinetics was determined. More strikingly, disassembly was found to take place only after photoactivation of two PixD subunits in the complex. This result suggests that the biological response of PixD does not follow a linear correlation with the light intensity but appears to be light-intensity-dependent.

Abstract: Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.

Abstract: Molecular switches are the fundamental building blocks in the field of synthetic biology. The majority of these switches is based on protein-protein, protein-DNA or protein-RNA interactions that are responsive towards endogenous metabolites or external stimuli like small molecules or light. By the rational and predictive reassembling of multiple compatible molecular switches, complex synthetic signaling networks can be engineered. Here we review how these switches were used for the regulation of important cellular processes at every level of the signaling cascade. In the second part we review how these switches can be assembled to open- and closed-loop control signaling networks and how these networks can be applied to facilitate cattle reproduction, to treat diabetes or to autonomously detect and cure disease states like gouty arthritis or cancer.

Abstract: Tools for optical control of proteins offer an unprecedented level of spatiotemporal control over biological processes, adding a new layer of experimental opportunity. While use of light-activated cation channels and anion pumps has already revolutionized neurobiology, an emerging class of more general optogenetic tools may have similar transformative effects. These tools consist of light-dependent protein interaction modules that allow control of target protein interactions and localization with light. Such tools are modular and can be applied to regulate a wide variety of biological activities. This chapter reviews the different properties of light-induced dimerization systems, based on plant phytochromes, cryptochromes, and light-oxygen-voltage domain proteins, exploring advantages and limitations of the different systems and practical considerations related to their use. Potential applications of these tools within the neurobiology field, including light control of various signaling pathways, neuronal activity, and DNA recombination and transcription, are discussed.

Abstract: Photoreceptor flavoproteins of the LOV, BLUF, and cryptochrome families are ubiquitous among the three domains of life and are configured as UVA/blue-light systems not only in plants-their original arena-but also in prokaryotes and microscopic algae. Here, we review these proteins' structure and function, their biological roles, and their evolution and impact in the living world, and underline their growing application in biotechnologies. We present novel developments such as the interplay of light and redox stimuli, emerging enzymatic and biological functions, lessons on evolution from picoalgae, metagenomics analysis, and optogenetics applications.

Abstract: The knowledge on the mechanisms by which blue light (BL) is sensed by diverse and numerous organisms, and of the physiological responses elicited by the BL photoreceptors, has grown remarkably during the last two decades. The basis for this "blue revival" was set by the identification and molecular characterization of long sought plant BL sensors, employing flavins as chromophores, chiefly cryptochromes and phototropins. The latter photosensors are the foundation members of the so-called light, oxygen, voltage (LOV)-protein family, largely spread among archaea, bacteria, fungi and plants. The accumulation of sequenced microbial genomes during the last years has added the BLUF (Blue Light sensing Using FAD) family to the BL photoreceptors and yielded the opportunity for intense "genome mining," which has presented to us the intriguing wealth of BL sensing in prokaryotes. In this contribution we provide an update of flavin-based BL sensors of the LOV and BLUF type, from prokaryotic microorganisms, with special emphasis to their light-activation pathways and molecular signal-transduction mechanisms. Rather than being a fully comprehensive review, this research collects the most recent discoveries and aims to unveil and compare signaling pathways and mechanisms of BL sensors.

Abstract: Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed.

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: The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.

Abstract: BLUF domains (sensors of blue light using flavin adenine dinucleotide) are a group of flavin-containing blue light photosensory domains from a variety of bacterial and algal proteins. While spectroscopic studies have indicated that these domains reorganize their interactions with an internally bound chromophore upon illumination, it remains unclear how these are converted into structural and functional changes. To address this, we have solved the solution structure of the BLUF domain from Klebsiella pneumoniae BlrP1, a light-activated c-di-guanosine 5'-monophosphate phosphodiesterase which consists of a sensory BLUF and a catalytic EAL (Glu-Ala-Leu) domain [Schmidt et. al. (2008) J. Bacteriol. 187, 4774-4781]. Our dark state structure of the sensory domain shows that it adopts a standard BLUF domain fold followed by two C-terminal alpha helices which adopt a novel orientation with respect to the rest of the domain. Comparison of NMR spectra acquired under dark and light conditions suggests that residues throughout the BlrP1 BLUF domain undergo significant light-induced chemical shift changes, including sites clustered on the beta(4)beta(5) loop, beta(5) strand, and alpha(3)alpha(4) loop. Given that these changes were observed at several sites on the helical cap, over 15 A from chromophore, our data suggest a long-range signal transduction process in BLUF domains.

Abstract: The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FADox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FADox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FADox in the signalling state to the fully reduced hydroquinone form FADredH and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.