Curated Optogenetic Publication Database

Abstract: Biological phenomena, such as cellular differentiation and phagocytosis, are fundamental processes that enable cells to fulfill important physiological roles in multicellular organisms. In the field of synthetic biology, the study of these behaviors relies on the use of a broad range of molecular tools that enable the real-time manipulation and measurement of key components in the underlying signaling pathways. This Review will focus on a subset of synthetic biology tools known as bottom-up techniques, which use technologies such as optogenetics and chemically induced dimerization to reconstitute cellular behavior in cells. These techniques have been crucial not only in revealing causal relationships within signaling networks but also in identifying the minimal signaling components that are necessary for a given cellular function. We discuss studies that used these systems in a broad range of cellular and molecular phenomena, including the time-dependent modulation of protein activity in cellular proliferation and differentiation, the reconstitution of phagocytosis, the reconstitution of chemotaxis, and the regulation of actin reorganization. Finally, we discuss the potential contribution of synthetic biology to medicine.

Abstract: Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology.

Abstract: The design of synthetic optogenetic tools that allow precise spatiotemporal control of biological processes previously inaccessible to optogenetic control has developed rapidly over the last years. Rational design of such tools requires detailed knowledge of allosteric light signaling in natural photoreceptors. To understand allosteric communication between sensor and effector domains, characterization of all relevant signaling states is required. Here, we describe the mechanism of light-dependent DNA binding of the light-oxygen-voltage (LOV) transcription factor Aureochrome 1a from Phaeodactylum tricornutum (PtAu1a) and present crystal structures of a dark state LOV monomer and a fully light-adapted LOV dimer. In combination with hydrogen/deuterium-exchange, solution scattering data and DNA-binding experiments, our studies reveal a light-sensitive interaction between the LOV and basic region leucine zipper DNA-binding domain that together with LOV dimerization results in modulation of the DNA affinity of PtAu1a. We discuss the implications of these results for the design of synthetic LOV-based photosensors with application in optogenetics.

Abstract: Rhythmicity is prevalent in the cortical dynamics of diverse single and multicellular systems. Current models of cortical oscillations focus primarily on cytoskeleton-based feedbacks, but information on signals upstream of the actin cytoskeleton is limited. In addition, inhibitory mechanisms--especially local inhibitory mechanisms, which ensure proper spatial and kinetic controls of activation--are not well understood. Here, we identified two phosphoinositide phosphatases, synaptojanin 2 and SHIP1, that function in periodic traveling waves of rat basophilic leukemia (RBL) mast cells. The local, phase-shifted activation of lipid phosphatases generates sequential waves of phosphoinositides. By acutely perturbing phosphoinositide composition using optogenetic methods, we showed that pulses of PtdIns(4,5)P2 regulate the amplitude of cyclic membrane waves while PtdIns(3,4)P2 sets the frequency. Collectively, these data suggest that the spatiotemporal dynamics of lipid metabolism have a key role in governing cortical oscillations and reveal how phosphatidylinositol 3-kinases (PI3K) activity could be frequency-encoded by a phosphatase-dependent inhibitory reaction.

Abstract: Photoreceptors are found in all kingdoms of life and mediate crucial responses to environmental challenges. Nature has evolved various types of photoresponsive protein structures with different chromophores and signaling concepts for their given purpose. The abundance of these signaling proteins as found nowadays by (meta-)genomic screens enriched the palette of optogenetic tools significantly. In addition, molecular insights into signal transduction mechanisms and design principles from biophysical studies and from structural and mechanistic comparison of homologous proteins opened seemingly unlimited possibilities for customizing the naturally occurring proteins for a given optogenetic task. Here, a brief overview on the photoreceptor concepts already established as optogenetic tools in natural or engineered form, their photochemistry and their signaling/design principles is given. Finally, so far not regarded photosensitive modules and protein architectures with potential for optogenetic application are described.

Abstract: Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.

Abstract: Programmable transcription factors can enable precise control of gene expression triggered by a chemical inducer or light. To obtain versatile transgene system with combined benefits of a chemical inducer and light inducer, we created various chimeric promoters through the assembly of different copies of the tet operator and Gal4 operator module, which simultaneously responded to a tetracycline-responsive transcription factor and a light-switchable transactivator. The activities of these chimeric promoters can be regulated by tetracycline and blue light synergistically or antagonistically. Further studies of the antagonistic genetic circuit exhibited high spatiotemporal resolution and extremely low leaky expression, which therefore could be used to spatially and stringently control the expression of highly toxic protein Diphtheria toxin A for light regulated gene therapy. When transferring plasmids engineered for the gene switch-driven expression of a firefly luciferase (Fluc) into mice, the Fluc expression levels of the treated animals directly correlated with the tetracycline and light input program. We suggest that dual-input genetic circuits using TET and light that serve as triggers to achieve expression profiles may enable the design of robust therapeutic gene circuits for gene- and cell-based therapies.

Abstract: The optogenetics techniques that have long been used in neuroscience are now giving biologists the power to probe cellular structures with unprecedented precision.

Abstract: Light-oxygen-voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.

Abstract: The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca(2+) oscillations and Ca(2+)-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca(2+)-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca(2+)-modulated activities with tailored function.

Abstract: Morphogenesis of multicellular organisms is driven by localized cell shape changes. How, and to what extent, changes in behavior in single cells or groups of cells influence neighboring cells and large-scale tissue remodeling remains an open question. Indeed, our understanding of multicellular dynamics is limited by the lack of methods allowing the modulation of cell behavior with high spatiotemporal precision. Here, we developed an optogenetic approach to achieve local modulation of cell contractility and used it to control morphogenetic movements during Drosophila embryogenesis. We show that local inhibition of apical constriction is sufficient to cause a global arrest of mesoderm invagination. By varying the spatial pattern of inhibition during invagination, we further demonstrate that coordinated contractile behavior responds to local tissue geometrical constraints. Together, these results show the efficacy of this optogenetic approach to dissect the interplay between cell-cell interaction, force transmission, and tissue geometry during complex morphogenetic processes.

Abstract: Reactive oxygen species (ROS) can modify and damage DNA. Here we report an optogenetic mutagenesis approach that is free of toxic chemicals and easy to perform by taking advantage of a genetically encoded ROS generator. This method relies on the potency of ROS generation by His-mSOG, the mini singlet oxygen generator, miniSOG, fused to a histone. Caenorhabditis elegans expressing His-mSOG in the germline behave and reproduce normally, without photoinduction. Following exposure to blue light, the His-mSOG animals produce progeny with a wide range of heritable phenotypes. We show that optogenetic mutagenesis by His-mSOG induces a broad spectrum of mutations including single-nucleotide variants (SNVs), chromosomal deletions, as well as integration of extrachromosomal transgenes, which complements those derived from traditional chemical or radiation mutagenesis. The optogenetic mutagenesis expands the toolbox for forward genetic screening and also provides direct evidence that nuclear ROS can induce heritable and specific genetic mutations.

Abstract: LOV domains act as biomolecular sensors for light, oxygen or the environment's redox potential. Conformational changes upon the formation of a covalent cysteinyl flavin adduct are propagated through hydrogen-bonding networks in the core of designed hybrid phototropin LOV2 domains that incorporate the Bcl homology region 3 (BH3) of the key pro-apoptotic protein BH3-interacting-domain death agonist (BID). The resulting change in conformation of a flanking amphiphilic α-helix creates a light-dependent optogenetic tool for the modulation of interactions with the anti-apoptotic B-cell leukaemia-2 (Bcl-2) family member Bcl-xL .

Abstract: Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events.

Abstract: The resting and signaling structures of the blue-light sensing using flavin (BLUF) photoreceptor domains are still controversially debated due to differences in the molecular models obtained by crystal and NMR structures. Photocycles for the given preferred structural framework have been established, but a unifying picture combining experiment and theory remains elusive. We summarize present work on the AppA BLUF domain from both experiment and theory. We focus on IR and UV/vis spectra, and to what extent theory was able to reproduce experimental data and predict the structural changes upon formation of the signaling state. We find that the experimental observables can be theoretically reproduced employing any structural model, as long as the orientation of the signaling essential Gln63 and its tautomer state are a choice of the modeler. We also observe that few approaches are comparative, e.g., by considering all structures in the same context. Based on recent experimental findings and a few basic calculations, we suggest the possibility for a BLUF activation mechanism that only relies on electron transfer and its effect on the local electrostatics, not requiring an associated proton transfer. In this regard, we investigate the impact of dispersion correction on the interaction energies arising from weakly bound amino acids.

Abstract: Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.

Abstract: The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times.

Abstract: Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.

Abstract: High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.

Abstract: YtvA, a photosensory LOV (light-oxygen-voltage) protein from Bacillus subtilis, exists as a dimer that previously appeared to undergo surprisingly small structural changes after light illumination compared with other light-sensing proteins. However, we now report that light induces significant structural perturbations in a series of YtvA-LOV domain derivatives in which the Jα helix has been truncated or replaced. Results from native gel analysis showed significant mobility changes in these derivatives after light illumination; YtvA-LOV without the Jα helix dimerized in the dark state but existed as a monomer in the light state. The absence of the Jα helix also affected the dark regeneration kinetics and the stability of the flavin mononucleotide (FMN) binding to its binding site. Our results demonstrate an alternative way of photo-induced signal propagation that leads to a bigger functional response through dimer/monomer conversions of the YtvA-LOV than the local disruption of Jα helix in the As-LOV domain.

Abstract: The dynamic activity of the serine/threonine kinase Akt is crucial for the regulation of diverse cellular functions, but the precise spatiotemporal control of its activity remains a critical issue. Herein, we present a photo-activatable Akt (PA-Akt) system based on a light-inducible protein interaction module of Arabidopsis thaliana cryptochrome2 (CRY2) and CIB1. Akt fused to CRY2phr, which is a minimal light sensitive domain of CRY2 (CRY2-Akt), is reversibly activated by light illumination in several minutes within a physiological dynamic range and specifically regulates downstream molecules and inducible biological functions. We have generated a computational model of CRY2-Akt activation that allows us to use PA-Akt to control the activity quantitatively. The system provides evidence that the temporal patterns of Akt activity are crucial for generating one of the downstream functions of the Akt-FoxO pathway; the expression of a key gene involved in muscle atrophy (Atrogin-1). The use of an optical module with computational modeling represents a general framework for interrogating the temporal dynamics of biomolecules by predictive manipulation of optogenetic modules.

Abstract: To study the molecular mechanism of complex biological systems, it is important to be able to artificially manipulate gene expression in desired target sites with high precision. Based on the light dependent binding of cryptochrome 2 and a cryptochrome interacting bHLH protein, we developed a split lexA transcriptional activation system for use in Drosophila that allows regulation of gene expression in vivo using blue light or two-photon excitation. We show that this system offers high spatiotemporal resolution by inducing gene expression in tissues at various developmental stages. In combination with two-photon excitation, gene expression can be manipulated at precise sites in embryos, potentially offering an important tool with which to examine developmental processes.

Abstract: Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.

Abstract: Techniques allowing precise spatial and temporal control of gene expression in the brain are needed. Herein we describe optogenetic approaches using a photo-activatable Cre recombinase (PA-Cre) to stably modify gene expression in the mouse brain. Blue light illumination for 12 hours via optical fibers activated PA-Cre in the hippocampus, a deep brain structure. Two-photon illumination through a thinned skull window for 100 minutes activated PA-Cre within a sub-millimeter region of cortex. Light activation of PA-Cre may allow permanent gene modification with improved spatiotemporal precision compared to standard methods.

Abstract: Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), that can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning task induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble.