Curated Optogenetic Publication Database

Abstract: Reactive Oxygen Species (ROS) are known to cause oxidative damage to DNA, proteins and lipids. In addition, recent evidence suggests that ROS can also initiate signaling cascades that respond to stress and modify specific redox-sensitive moieties as a regulatory mechanism. This suggests that ROS are physiologically-relevant signaling molecules. However, these sensor/effector molecules are not uniformly distributed throughout the cell. Moreover, localized ROS damage may elicit site-specific compensatory measures. Thus, the impact of ROS can be likened to that of calcium, a ubiquitous second messenger, leading to the prediction that their effects are exquisitely dependent upon their location, quantity and even the timing of generation. Despite this prediction, ROS signaling is most commonly intuited through the global administration of chemicals that produce ROS or by ROS quenching through global application of antioxidants. Optogenetics, which uses light to control the activity of genetically-encoded effector proteins, provides a means of circumventing this limitation. Photo-inducible genetically-encoded ROS-generating proteins (RGPs) were originally employed for their phototoxic effects and cell ablation. However, reducing irradiance and/or fluence can achieve sub-lethal levels of ROS that may mediate subtle signaling effects. Hence, transgenic expression of RGPs as fusions to native proteins gives researchers a new tool to exert spatial and temporal control over ROS production. This review will focus on the new frontier defined by the experimental use of RGPs to study ROS signaling.

Abstract: Algae, plants, bacteria and fungi contain Light-Oxygen-Voltage (LOV) domains that function as blue light sensors to control cellular responses to light. All LOV domains contain a bound flavin chromophore that is reduced upon photon absorption and forms a reversible, metastable covalent bond with a nearby cysteine residue. In Avena sativa LOV2 (AsLOV2), the photocycle is accompanied by an allosteric conformational change that activates the attached phototropin kinase in the full-length protein. Both the conformational change and formation of the cysteinyl-flavin adduct are stabilized by the reduction of the N5 atom in the flavin's isoalloxazine ring. In this study, we perform a mutational analysis to investigate the requirements for LOV2 to photocycle. We mutated all the residues that interact with the chromophore isoalloxazine ring to inert functional groups but none could fully inhibit the photocycle except those to the active-site cysteine. However, electronegative side chains in the vicinity of the chromophore accelerate the N5 deprotonation and the return to the dark state. Mutations to the N414 and Q513 residues identify a potential water gate and H₂O coordination sites. These residues affect the electronic nature of the chromophore and photocycle time by helping catalyze the N5 reduction leading to the completion of the photocycle. In addition, we demonstrate that dehydration leads to drastically slower photocycle times. Finally, to investigate the requirements of an active-site cysteine for photocycling, we moved the nearby cysteine to alternative locations and found that some variants can still photocycle. We propose a new model of the LOV domain photocycle that involves all of these components.

Abstract: Vivid (VVD) is a photoreceptor derived from Neurospora Crassa that rapidly forms a homodimer in response to blue light. Although VVD has several advantages over other photoreceptors as photoinducible homodimerization system, VVD has a critical limitation in its low dimer-forming efficiency. To overcome this limitation of wild-type VVD, here we conduct site-directed saturation mutagenesis in the homodimer interface of VVD. We have found that the Ile52Cys mutation of VVD (VVD-52C) substantially improves its homodimer-forming efficiency up to 180%. We have demonstrated the utility of VVD-52C for making a light-inducible gene expression system more robust. In addition, using VVD-52C, we have developed photoactivatable caspase-9, which enables optical control of apoptosis of mammalian cells. The present genetically engineered photoinducible homodimerization system can provide a powerful tool to optically control a broad range of molecular processes in the cell.

Abstract: Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

Abstract: Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K(+) channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.

Abstract: Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3 × 10(3) s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.

Abstract: LOV domains act as versatile photochromic switches servicing multiple effector domains in a variety of blue light sensing photoreceptors abundant in a multitude of organisms from all kingdoms of life. The perception of light is realized by a flavin chromophore that upon illumination reversibly switches from the non-covalently bound dark-state to a covalently linked flavin-LOV adduct. It is usually assumed that most LOV domains preferably bind FMN, but heterologous expression frequently results in the incorporation of all natural occurring flavins, i.e. riboflavin, FMN and FAD. Over recent years, the structures, photochemical properties, activation mechanisms and physiological functions of a multitude of LOV proteins have been studied intensively, but little is known about its affinities to physiologically relevant flavins or the thermodynamics of the flavin-LOV interaction. We have investigated the interaction of the LOV domain of the well characterized bacterial photoreceptor YtvA with riboflavin, FMN and FAD by ITC experiments providing binding constants and thermodynamic profiles of these interactions. For this purpose, we have developed a protocol for the production of the apo forms of YtvA and its isolated LOV domain and we demonstrate that the latter can be used as a molecular probe for free flavins in cell lysates. Furthermore, we show here using NMR spectroscopic techniques and Analytical Ultracentrifugation that the flavin moiety stabilizes the conformation of the LOV domain and that dimerization of YtvA is caused not only by intermolecular LOV-LOV but also by STAS-STAS contacts.

Abstract: Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.

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: Spatiotemporal control of transgene expression in living cells provides new opportunities for the characterization of gene function in complex biological processes. We previously reported a synthetic, light-switchable transgene expression system called LightOn that can be used to control gene expression using blue light. In the present study, we modified the different promoter segments of the light switchable transcription factor GAVPO and the target gene, and assayed their effects on protein expression under dark or light conditions. The results showed that the LightOn system maintained its high on/off ratio under most modifications, but its induction efficiency and background gene expression level can be fine-tuned by modifying the core promoter, the UASG sequence number, the length of the spacer between UASG and the core promoter of the target protein, and the expression level of the GAVPO transcription factor. Thus, the LightOn gene expression system can be adapted to a large range of applications according to the requirements of the background and the induced gene expression.

Abstract: Enabling optical control over biological processes is a defining goal of the new field of optogenetics. Control of membrane voltage by natural rhodopsin family ion channels has found widespread acceptance in neuroscience, due to the fact that these natural proteins control membrane voltage without further engineering. In contrast, optical control of intracellular biological processes has been a fragmented effort, with various laboratories engineering light-responsive properties into proteins in different manners. In the present article, we review the various systems that have been developed for controlling protein functions with light based on vertebrate rhodopsins, plant photoregulatory proteins and, most recently, the photoswitchable fluorescent protein Dronpa. By allowing biology to be controlled with spatiotemporal specificity and tunable dynamics, light-controllable proteins will find applications in the understanding of cellular and organismal biology and in synthetic biology.

Abstract: With their utilization of light-driven allostery to control biochemical activities, photosensory proteins are of great interest as model systems and novel reagents for use by the basic science and engineering communities. One such protein, the light-activated EL222 transcription factor, from the marine bacterium Erythrobacter litoralis HTCC2594, is appealing for such studies, as it harnesses blue light to drive the reorientation of light-oxygen-voltage (LOV) sensory and helix-turn-helix (HTH) effector domains to allow photoactivation of gene transcription in natural and artificial systems. The protein conformational changes required for this process are not well understood, in part because of the relatively short lifetime of the EL222 photoexcited state (τ ∼ 29 s), which complicates its characterization via certain biophysical methods. Here we report how we have circumvented this limitation by creating an EL222 variant harboring V41I, L52I, A79Q, and V121I point mutations (AQTrip) that stabilizes the photoactivated state. Using the wild-type and AQTrip EL222 proteins, we have probed EL222 activation using a combination of solution scattering, nuclear magnetic resonance (NMR), and electromobility shift assays. Size-exclusion chromatography and light scattering indicate that AQTrip oligomerizes in the absence of DNA and selects for an EL222 dimer-DNA complex in the presence of DNA substrates. These results are confirmed in wild-type EL222 with a high-affinity DNA-binding site that stabilizes the complex. NMR analyses of the EL222-DNA complex confirm a 2:1 stoichiometry in the presence of a previously characterized DNA substrate. Combined, these novel approaches have validated a key mechanistic step, whereby blue light induces EL222 dimerization through LOV and HTH interfaces.

Abstract: Dendritic spines are the primary sites of excitatory synaptic transmission in the vertebrate brain, and the morphology of these actin-rich structures correlates with synaptic function. Here we demonstrate a unique method for inducing spine enlargement and synaptic potentiation in dispersed hippocampal neurons, and use this technique to identify a coordinator of these processes; Ras-specific guanine nucleotide releasing factor 2 (RasGRF2). RasGRF2 is a dual Ras/Rac guanine nucleotide exchange factor (GEF) that is known to be necessary for long-term potentiation in situ. Contrary to the prevailing assumption, we find RasGRF2's Rac-GEF activity to be essential for synaptic potentiation by using a molecular replacement strategy designed to dissociate Rac- from Ras-GEF activities. Furthermore, we demonstrate that Rac1 activity itself is sufficient to rapidly modulate postsynaptic strength by using a photoactivatable derivative of this small GTPase. Because Rac1 is a major actin regulator, our results support a model where the initial phase of long-term potentiation is driven by the cytoskeleton.

Abstract: Optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. In the current study, we engineered an optogenetic technique that directly inhibits neurotransmitter release. We used a genetically encoded singlet oxygen generator, miniSOG, to conduct chromophore assisted light inactivation (CALI) of synaptic proteins. Fusions of miniSOG to VAMP2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. In cultured neurons and hippocampal organotypic slices, synaptic release was reduced up to 100%. Such inhibition lasted >1 hr and had minimal effects on membrane electrical properties. When miniSOG-VAMP2 was expressed panneuronally in Caenorhabditis elegans, movement of the worms was reduced after illumination, and paralysis was often observed. The movement of the worms recovered overnight. We name this technique Inhibition of Synapses with CALI (InSynC). InSynC is a powerful way to silence genetically specified synapses with light in a spatially and temporally precise manner.

Abstract: We have developed an optogenetic technique for the activation of diaphanous-related formins. Our approach is based on fusion of the light-oxygen-voltage 2 domain of Avena sativa Phototrophin1 to an isolated Diaphanous Autoregulatory Domain from mDia1. This "caged" diaphanous auto-regulatory domain was inactive in the dark but in the presence of blue light rapidly activated endogenous diaphanous-related formins. Using an F-actin reporter, we observed filopodia and lamellipodia formation as well as a steady increase in F-actin along existing stress fibers, starting within minutes of photo-activation. Interestingly, we did not observe the formation of new stress fibers. Remarkably, a 1.9-fold increase in F-actin was not paralleled by an increase in myosin II along stress fibers and the amount of tension generated by the fibers, as judged by focal adhesion size, appeared unchanged. Our results suggest a decoupling between F-actin accumulation and contractility in stress fibers and demonstrate the utility of photoactivatable diaphanous autoregulatory domain for the study of diaphanous-related formin function in cells.

Abstract: The development and progress in synthetic biology has been remarkable. Although still in its infancy, synthetic biology has achieved much during the past decade. Improvements in genetic circuit design have increased the potential for clinical applicability of synthetic biology research. What began as simple transcriptional gene switches has rapidly developed into a variety of complex regulatory circuits based on the transcriptional, translational and post-translational regulation. Instead of compounds with potential pharmacologic side effects, the inducer molecules now used are metabolites of the human body and even members of native cell signaling pathways. In this review, we address recent progress in mammalian synthetic biology circuit design and focus on how novel designs push synthetic biology toward clinical implementation. Groundbreaking research on the implementation of optogenetics and intercellular communications is addressed, as particularly optogenetics provides unprecedented opportunities for clinical application. Along with an increase in synthetic network complexity, multicellular systems are now being used to provide a platform for next-generation circuit design.

Abstract: The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

Abstract: Light perception is indispensable for plants to respond adequately to external cues and is linked to proteolysis of key transcriptional regulators. To provide synthetic light control of protein stability, we developed a generic photosensitive degron (psd) module combining the light-reactive LOV2 domain of Arabidopsis thaliana phot1 with the murine ornithine decarboxylase-like degradation sequence cODC1. Functionality of the psd module was demonstrated in the model organism Saccharomyces cerevisiae. Generation of conditional mutants, light regulation of cyclin-dependent kinase activity, light-based patterning of cell growth, and yeast photography exemplified its versatility. In silico modeling of psd module behavior increased understanding of its characteristics. This engineered degron module transfers the principle of light-regulated degradation to nonplant organisms. It will be highly beneficial to control protein levels in biotechnological or biomedical applications and offers the potential to render a plethora of biological processes light-switchable.

Abstract: In this issue of Chemistry & Biology, Renicke et al. report a photosensitive degron (psd) consisting of the LOV2 domain fused to a protein degradation sequence. This design enabled light-dependent protein degradation in yeast. When psd was fused to cell-cycle-dependent proteins, it controlled cell cycle by light with spatiotemporal precision.

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: Formins are potent activators of actin filament assembly in the cytoplasm. In turn, cytoplasmic actin polymerization can promote release of actin from megakaryocytic acute leukemia (MAL) protein for serum response factor (SRF) transcriptional activity. We found that formins polymerized actin inside the mammalian nucleus to drive serum-dependent MAL-SRF activity. Serum stimulated rapid assembly of actin filaments within the nucleus in a formin-dependent manner. The endogenous formin mDia was regulated with an optogenetic tool, which allowed for photoreactive release of nuclear formin autoinhibition. Activated mDia promoted rapid and reversible nuclear actin network assembly, subsequent MAL nuclear accumulation, and SRF activity. Thus, a dynamic polymeric actin structure within the nucleus is part of the serum response.

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: Near-infrared light is favourable for imaging in mammalian tissues due to low absorbance of hemoglobin, melanin, and water. Therefore, fluorescent proteins, biosensors and optogenetic constructs for optimal imaging, optical readout and light manipulation in mammals should have fluorescence and action spectra within the near-infrared window. Interestingly, natural Bacterial Phytochrome Photoreceptors (BphPs) utilize the low molecular weight biliverdin, found in most mammalian tissues, as a photoreactive chromophore. Due to their near-infrared absorbance BphPs are preferred templates for designing optical molecular tools for applications in mammals. Moreover, BphPs spectrally complement existing genetically-encoded probes. Several BphPs were already developed into the near-infrared fluorescent variants. Based on the analysis of the photochemistry and structure of BphPs we suggest a variety of possible BphP-based fluorescent proteins, biosensors, and optogenetic tools. Putative design strategies and experimental considerations for such probes are discussed.

Abstract: Light-oxygen-voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of anti-sigma factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV domain protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N- and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light- and dark-state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin-cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single-residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb the oligomeric state. Size exclusion chromatography, multiangle light scattering, small-angle X-ray scattering, and cross-linking studies indicate that RsLOV dimerizes in the dark but, upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove to be useful for engineering molecular associations in controlled cellular settings.

Abstract: Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometre precision within cells. While light-actuated chemicals such as photolabile 'caged' compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.