Curated Optogenetic Publication Database

Abstract: In this work we evaluate the interaction of two optogenetic protein variants (CIB1, CIBN) with their complementary protein CRY2 by single-molecule tools in cell-free extracts. After validating the blue light induced co-localization of CRY2 and CIB1/N by Förster resonance energy transfer (FRET) in live cells, a fluorescence correlation spectroscopy (FCS) based method was developed to quantitatively determine the in vitro association of the extracted proteins. Our experiments suggest that CIB1, in comparison with CIBN, possesses a better coupling efficiency with CRY2 due to its intact protein structure and lower diffusion rate within 300s detection window.

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 regulation of gene expression is crucial in diverse areas of biological science, engineering, and medicine. A genetically encoded system based on the RNA binding domain of the Pumilio and FBF (PUF) proteins was developed for the bidirectional regulation (i.e., either upregulation or downregulation) of the translation of a target mRNA. PUF domains serve as designable scaffolds for the recognition of specific RNA elements and the specificity can be easily altered to target any 8-nucleotide RNA sequence. The expression of a reporter could be varied by over 17-fold when using PUF-based activators and repressors. The specificity of the method was established by using wild-type and mutant PUF domains. Furthermore, this method could be used to activate the translation of target mRNA downstream of PUF binding sites in a light-dependent manner. Such specific bidirectional control of mRNA translation could be particularly useful in the fields of synthetic biology, developmental biology, and metabolic engineering.

Abstract: It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.

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: The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.

Abstract: As light-regulated actuators, sensory photoreceptors underpin optogenetics and numerous applications in synthetic biology. Protein engineering has been applied to fine-tune the properties of photoreceptors and to generate novel actuators. For the blue-light-sensitive light-oxygen-voltage (LOV) photoreceptors, mutations near the flavin chromophore modulate response kinetics and the effective light responsiveness. To probe for potential, inadvertent effects on receptor activity, we introduced these mutations into the engineered LOV photoreceptor YF1 and determined their impact on light regulation. While several mutations severely impaired the dynamic range of the receptor (e.g., I39V, R63K, and N94A), residue substitutions in a second group were benign with little effect on regulation (e.g., V28T, N37C, and L82I). Electron paramagnetic resonance and absorption spectroscopy identified correlated effects for certain of the latter mutations on chromophore environment and response kinetics in YF1 and the LOV2 domain from Avena sativa phototropin 1. Carefully chosen mutations provide a powerful means to adjust the light-response function of photoreceptors as demanded for diverse applications.

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: Perturbations in the concentration of a specific protein are often used to study and control biological networks. The ability to "dial-in" and programmatically control the concentration of a desired protein in cultures of cells would be transformative for applications in research and biotechnology. We developed a culturing apparatus and feedback control scheme which, in combination with an optogenetic system, allows us to generate defined perturbations in the intracellular concentration of a specific protein in microbial cell culture. As light can be easily added and removed, we can control protein concentration in culture more dynamically than would be possible with long-lived chemical inducers. Control of protein concentration is achieved by sampling individual cells from the culture apparatus, imaging and quantifying protein concentration, and adjusting the inducing light appropriately. The culturing apparatus can be operated as a chemostat, allowing us to precisely control microbial growth and providing cell material for downstream assays. We illustrate the potential for this technology by generating fixed and time-varying concentrations of a specific protein in continuous steady-state cultures of the model organism Saccharomyces cerevisiae. We anticipate that this technology will allow for quantitative studies of biological networks as well as external tuning of synthetic gene circuits and bioprocesses.

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: 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: 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: Living systems are fundamentally dependent on the ability of proteins to respond to external stimuli. The mechanism, the underlying structural dynamics, and the time scales for regulation of this response are central questions in biochemistry. Here we probe the structural dynamics of the BLUF domain found in several photoactive flavoproteins, which is responsible for light activated functions as diverse as phototaxis and gene regulation. Measurements have been made over 10 decades of time (from 100 fs to 1 ms) using transient vibrational spectroscopy. Chromophore (flavin ring) localized dynamics occur on the pico- to nanosecond time scale, while subsequent protein structural reorganization is observed over microseconds. Multiple time scales are observed for the dynamics associated with different vibrations of the protein, suggesting an underlying hierarchical relaxation pathway. Structural evolution in residues directly H-bonded to the chromophore takes place more slowly than changes in more remote residues. However, a point mutation which suppresses biological function is shown to 'short circuit' this structural relaxation pathway, suppressing the changes which occur further away from the chromophore while accelerating dynamics close to it.

Abstract: The extracellular signal-regulated kinase (ERK) plays a central role in the signaling cascades of cell growth. Here, we show that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner. A fluorescence resonance energy transfer (FRET) biosensor revealed that stochastic ERK activity pulses fired spontaneously or propagated from adjacent cells. Frequency, but not amplitude, of ERK activity pulses exhibited a bell-shaped response to the cell density and correlated with cell proliferation rates. Consistently, synthetic ERK activity pulses generated by a light-switchable CRaf protein accelerated cell proliferation. A mathematical model clarified that 80% and 20% of ERK activity pulses are generated by the noise and cell-to-cell propagation, respectively. Finally, RNA sequencing analysis of cells subjected to the synthetic ERK activity pulses suggested the involvement of serum responsive factor (SRF) transcription factors in the gene expression driven by the ERK activity pulses.

Abstract: Arabidopsis thaliana cryptochrome 2 (CRY2) mediates light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with CRY2 in response to blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the canonical E-box (CACGTG, also referred to as G-box) with much higher affinity than its interaction with non-canonical E-box (CANNTG) DNA sequences. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the non-canonical E-box sequences. Here, we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. Our genetic analysis demonstrates that CIB1, CIB2, CIB4, and CIB5 act redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated flowering. More importantly, CIB1 and other CIBs proteins form heterodimers, and some of the heterodimers have a higher binding affinity than the CIB homodimers to the non-canonical E-box in the in vitro DNA-binding assays. This result explains why in vitro CIB1 and other CIBs bind to the canonical E-box (G-box) with a higher affinity, whereas they are all associated with the non-canonical E-boxes at the FT promoter in vivo. Consistent with the hypothesis that different CIB proteins play similar roles in the CRY2-midiated blue light signaling, the expression of CIB proteins is regulated specifically by blue light. Our study demonstrates that CIBs function redundantly in regulating CRY2-dependent flowering, and that different CIBs form heterodimers to interact with the non-canonical E-box DNA in vivo.

Abstract: Plants possess multiple photoreceptors to mediate light regulation of growth and development, but it is not well understood how different photoreceptors coordinate their actions to jointly regulate developmental responses, such as flowering time. In Arabidopsis, the photoexcited cryptochrome 2 interacts with the transcription factor CRYPTOCHROME-INTERACTING basic helix-loop-helix 1 (CIB1) to activate transcription and floral initiation. We show that the CIB1 protein expression is regulated by blue light; CIB1 is highly expressed in plants exposed to blue light, but levels of the CIB1 protein decreases in the absence of blue light. We demonstrate that CIB1 is degraded by the 26S proteasome and that blue light suppresses CIB1 degradation. Surprisingly, although cryptochrome 2 physically interacts with CIB1 in response to blue light, it is not the photoreceptor mediating blue-light suppression of CIB1 degradation. Instead, two of the three light-oxygen-voltage (LOV)-domain photoreceptors, ZEITLUPE and LOV KELCH PROTEIN 2, but not FLAVIN-BINDING KELCH REPEAT 1, are required for the function and blue-light suppression of degradation of CIB1. These results support the hypothesis that the evolutionarily unrelated blue-light receptors, cryptochrome and LOV-domain F-box proteins, mediate blue-light regulation of the same transcription factor by distinct mechanisms.

Abstract: Light-dependent dimerization is the basis for recently developed noninvasive optogenetic tools. Here we present a novel tool combining optogenetics with the control of protein kinase activity to investigate signal transduction pathways. Mediated by Arabidopsis thaliana photoreceptor cryptochrome 2, we activated the protein kinase C-RAF by blue light-dependent dimerization, allowing for decoupling from upstream signaling events induced by surface receptors. The activation by light is fast, reversible, and not only time but also dose dependent as monitored by phosphorylation of ERK1/2. Additionally, light-activated C-RAF controls serum response factor-mediated gene expression. Light-induced heterodimerization of C-RAF with a kinase-dead mutant of B-RAF demonstrates the enhancing role of B-RAF as a scaffold for C-RAF activity, which leads to the paradoxical activation of C-RAF found in human cancers. This optogenetic tool enables reversible control of protein kinase activity in signal duration and strength. These properties can help to shed light onto downstream signaling processes of protein kinases in living cells.

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: A genetically encoded optogenetic system was constructed that activates mRNA translation in mammalian cells in response to light. Blue light induces the reconstitution of an RNA binding domain and a translation initiation domain, thereby activating target mRNA translation downstream of the binding sites.