Curated Optogenetic Publication Database

Abstract: To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.

Abstract: Optogenetics allows to non-invasively manipulate cellular functions with spatio-temporal precision by combining genetic engineering with the control of protein function by light. Since the discovery of channelrhodopsin has pioneered the field, the optogenetic toolkit has been ever expanding and allows now not only to control neuronal activity by light, but rather a multitude of other cellular functions. One important application that has been established in recent years is the light-dependent control of second messenger signaling. The optogenetic toolkit now allows to control cyclic nucleotide-dependent signaling by light in vitro and in vivo.

Abstract: The Reelin-Dab1 signaling pathway regulates development of the mammalian brain, including neuron migrations in various brain regions, as well as learning and memory in adults. Extracellular Reelin binds to cell surface receptors and activates phosphorylation of the intracellular Dab1 protein. Dab1 is required for most effects of Reelin, but Dab1-independent pathways may contribute. Here we developed a single-component, photoactivatable Dab1 (opto-Dab1) by using the blue light-sensitive dimerization/oligomerization property of A. thaliana Cryptochrome 2 (Cry2). Opto-Dab1 can activate downstream signals rapidly, locally, and reversibly upon blue light illumination. The high spatiotemporal resolution of the opto-Dab1 probe also allows us to control membrane protrusion, retraction and ruffling by local illumination in both COS7 cells and in primary neurons. This shows that Dab1 activation is sufficient to orient cell movement in the absence of other signals. Opto-Dab1 may be useful to study the biological functions of the Reelin-Dab1 signaling pathway both in vitro and in vivo.

Abstract: Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger, c-di-GMP, as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared light-regulated diguanylate cyclase and a blue-light regulated c-di-GMP phosphodiesterase. It allows generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the timescale of chemotaxis signaling. We provide experimental evidence that c-di-GMP binding to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense changes with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates their contribution to the overall chemotaxis response. Here, we used an optogenetic system to perturb intracellular concentrations of the bacterial second messenger, c-di-GMP, to show that in some chemotaxis receptors, c-di-GMP functions in a similar feedback loop to connect metabolic status of the cells to sensory activity of chemotaxis receptors.

Abstract: The optogenetic revolution enabled spatially-precise and temporally-precise control over protein function, signaling pathway activation, and animal behavior with tremendous success in the dissection of signaling networks and neural circuits. Very recently, optogenetic methods have been paired with optical reporters in novel drug screening platforms. In these all-optical platforms, light remotely activated ion channels and kinases thereby obviating the use of electrophysiology or reagents. Consequences were remarkable operational simplicity, throughput, and cost-effectiveness that culminated in the identification of new drug candidates. These blueprints for all-optical assays also revealed potential pitfalls and inspire all-optical variants of other screens, such as those that aim at better understanding dynamic drug action or orphan protein function.

Abstract: We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein-protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca(2+)/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation.

Abstract: Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.

Abstract: Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

Abstract: Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca(2+), lipid, and protein cargo signals in the cell to deploy in a site-specific manner.

Abstract: Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the 'plug-and-play' nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

Abstract: Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space.

Abstract: Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.

Abstract: Cyclic AMP (cAMP) signaling augments synaptic transmission, but because many targets of cAMP and protein kinase A (PKA) may be involved, mechanisms underlying this pathway remain unclear. To probe this mechanism, we used optogenetic stimulation of cAMP signaling by Beggiatoa-photoactivated adenylyl cyclase (bPAC) in Caenorhabditis elegans motor neurons. Behavioral, electron microscopy (EM), and electrophysiology analyses revealed cAMP effects on both the rate and on quantal size of transmitter release and led to the identification of a neuropeptidergic pathway affecting quantal size. cAMP enhanced synaptic vesicle (SV) fusion by increasing mobilization and docking/priming. cAMP further evoked dense core vesicle (DCV) release of neuropeptides, in contrast to channelrhodopsin (ChR2) stimulation. cAMP-evoked DCV release required UNC-31/Ca(2+)-dependent activator protein for secretion (CAPS). Thus, DCVs accumulated in unc-31 mutant synapses. bPAC-induced neuropeptide signaling acts presynaptically to enhance vAChT-dependent SV loading with acetylcholine, thus causing increased miniature postsynaptic current amplitudes (mPSCs) and significantly enlarged SVs.

Abstract: Light-oxygen-voltage (LOV) domains sense blue light through the photochemical formation of a cysteinyl-flavin covalent adduct. Concurrent protonation at the flavin N5 position alters the hydrogen bonding interactions of an invariant Gln residue that has been proposed to flip its amide side chain as a critical step in the propagation of conformational change. Traditional molecular dynamics (MD) and replica-exchange MD (REMD) simulations of the well-characterized LOV protein Vivid (VVD) demonstrate that the Gln182 amide indeed reorients by ∼180° in response to either adduct formation or reduction of the isoalloxazine ring to the neutral semiquinone, both of which involve N5 protonation. Free energy simulations reveal that the relative free energies of the flipped Gln conformation and the flipping barrier are significantly lower in the light-adapted state. The Gln182 flip stabilizes an important hinge-bβ region between the PAS β-sheet and the N-terminal cap helix that in turn destabilizes an N-terminal latch region against the PAS core. Release of the latch, observed both experimentally and in the simulations, is known to mediate light-induced VVD dimerization. This computational study of a LOV protein, unprecedented in its agreement with experiment, provides an atomistic view of long-range allosteric coupling in a photoreceptor.

Abstract: The molecular composition of the postsynaptic membrane is sculpted by synaptic activity. During synaptic plasticity at excitatory synapses, numerous structural, signaling, and receptor molecules concentrate at the postsynaptic density (PSD) to regulate synaptic strength. We developed an approach that uses light to tune the abundance of specific molecules in the PSD. We used this approach to investigate the relationship between the number of AMPA-type glutamate receptors in the PSD and synaptic strength. Surprisingly, adding more AMPA receptors to excitatory contacts had little effect on synaptic strength. Instead, we observed increased excitatory input through the apparent addition of new functional sites. Our data support a model where adding AMPA receptors is sufficient to activate synapses that had few receptors to begin with, but that additional remodeling events are required to strengthen established synapses. More broadly, this approach introduces the precise spatiotemporal control of optogenetics to the molecular control of synaptic function.

Abstract: The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the β-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

Abstract: Animal development is characterized by signaling events that occur at precise locations and times within the embryo, but determining when and where such precision is needed for proper embryogenesis has been a long-standing challenge. Here we address this question for extracellular signal regulated kinase (Erk) signaling, a key developmental patterning cue. We describe an optogenetic system for activating Erk with high spatiotemporal precision in vivo. Implementing this system in Drosophila, we find that embryogenesis is remarkably robust to ectopic Erk signaling, except from 1 to 4 hr post-fertilization, when perturbing the spatial extent of Erk pathway activation leads to dramatic disruptions of patterning and morphogenesis. Later in development, the effects of ectopic signaling are buffered, at least in part, by combinatorial mechanisms. Our approach can be used to systematically probe the differential contributions of the Ras/Erk pathway and concurrent signals, leading to a more quantitative understanding of developmental signaling.

Abstract: Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.

Abstract: Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.

Abstract: Calcium acts as a second messenger to regulate a myriad of cell functions, ranging from short-term muscle contraction and cell motility to long-term changes in gene expression and metabolism. To study the impact of Ca2+-modulated 'ON' and 'OFF' reactions in mammalian cells, pharmacological tools and 'caged' compounds are commonly used under various experimental conditions. The use of these reagents for precise control of Ca2+ signals, nonetheless, is impeded by lack of reversibility and specificity. The recently developed optogenetic tools, particularly those built upon engineered Ca2+ release-activated Ca2+ (CRAC) channels, provide exciting opportunities to remotely and non-invasively modulate Ca2+ signaling due to their superior spatiotemporal resolution and rapid reversibility. In this review, we briefly summarize the latest advances in the development of optogenetic tools (collectively termed as 'genetically encoded Ca2+ actuators', or GECAs) that are tailored for the interrogation of Ca2+ signaling, as well as their applications in remote neuromodulation and optogenetic immunomodulation. Our goal is to provide a general guide to choosing appropriate GECAs for optical control of Ca2+ signaling in cellulo, and in parallel, to stimulate further thoughts on evolving non-opsin-based optogenetics into a fully fledged technology for the study of Ca2+-dependent activities in vivo.

Abstract: Cell ablation is a strategy to study cell lineage and function during development. Optogenetic methods are an important cell-ablation approach, and we have previously developed a mini singlet oxygen generator (miniSOG) tool that works in the living Caenorhabditis elegans. Here, we use directed evolution to generate miniSOG2, an improved tool for cell ablation via photogenerated reactive oxygen species. We apply miniSOG2 to a far more complex model animal system, Drosophila melanogaster, and demonstrate that it can be used to kill a single neuron in a Drosophila larva. In addition, miniSOG2 is able to photoablate a small group of cells in one of the larval wing imaginal discs, resulting in an adult with one incomplete and one normal wing. We expect miniSOG2 to be a useful optogenetic tool for precision cell ablation at a desired developmental time point in live animals, thus opening a new window into cell origin, fate and function, tissue regeneration, and developmental biology.

Abstract: Protein trans-splicing is a posttranslational modification that joins two protein fragments together via a peptide a bond in a process that does not require exogenous cofactors. Towards achieving cellular control, synthetically engineered systems have used a variety of stimuli such as small molecules and light. Recently, split inteins have been engineered to be photoactive by the LOV2 domain (named LOVInC). Herein, we discuss (1) designing of LOV2-activated target proteins (e.g., inteins), (2) selecting feasible splice sites for the extein, and (3) imaging cells that express LOVInC-based target exteins.

Abstract: Ca2+ signals regulate a plethora of cellular functions that include muscle contraction, heart beating, hormone secretion, lymphocyte activation, gene expression, and metabolism. To study the impact of Ca2+ signals on biological processes, pharmacological tools and caged compounds have been commonly applied to induce fluctuations of intracellular Ca2+ concentrations. These conventional approaches, nonetheless, lack rapid reversibility and high spatiotemporal resolution. To overcome these disadvantages, we and others have devised a series of photoactivatable genetically encoded Ca2+ actuators (GECAs) by installing light sensitivities into a bona fide highly selective Ca2+ channel, the Ca2+ release-activated Ca2+ (CRAC) channel. Store-operated CRAC channel serves as a major route for Ca2+ entry in many cell types. These GECAs enable remote and precise manipulation of Ca2+ signaling in both excitable and non-excitable cells. When combined with nanotechnology, it becomes feasible to wirelessly photo-modulate Ca2+-dependent activities in vivo. In this chapter, we briefly review most recent advances in engineering CRAC channels to achieve optical control over Ca2+ signaling, outline their design principles and kinetic features, and present exemplary applications of GECAs engineered from CRAC channels.

Abstract: Although spatial and temporal elements of immune activation mediate the intensity of the immune response, few tools exist to directly examine these effects. To elucidate the spatiotemporal aspects of innate immune responses, we designed an optogenetic pattern recognition receptor that activates in response to blue light. We demonstrate direct receptor activation, leading to spatial and temporal control of downstream signaling pathways in a variety of relevant cell types. We combined our platform with Bi-molecular Fluorescence Complementation (BiFC), resulting in selective fluorescent labeling of cells in which receptor activation has occurred.

Abstract: Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions but also their link to pathological aggregates.