Curated Optogenetic Publication Database

Abstract: The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light-oxygen-voltage photoreceptor with cognate RNA aptamers. Here light-responsive ribozymes, denoted optozymes, which undergo light-dependent self-cleavage and thereby control gene expression are described. This approach transcends existing aptamer-ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non-invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.

Abstract: The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.

Abstract: Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.

Abstract: T cells regulate adaptive immune responses through complex signaling pathways mediated by T cell receptor (TCR). The functional domains of the TCR are combined with specific antibodies for the development of chimeric antigen receptor (CAR) T cell therapy. In this review, we first overview current understanding on the T cell signaling pathways as well as traditional methods that have been widely used for the T cell study. These methods, however, are still limited to investigating dynamic molecular events with spatiotemporal resolutions. Therefore, genetically encoded biosensors and optogenetic tools have been developed to study dynamic T cell signaling pathways in live cells. We review these cutting-edge technologies that revealed dynamic and complex molecular mechanisms at each stage of T cell signaling pathways. They have been primarily applied to the study of dynamic molecular events in TCR signaling, and they will further aid in understanding the mechanisms of CAR activation and function. Therefore, genetically encoded biosensors and optogenetic tools offer powerful tools for enhancing our understanding of signaling mechanisms in T cells and CAR-T cells.

Abstract: Early life stress (ELS) is one of the strongest risk factors for developing psychiatric disorders in humans. As conserved key stress hormones of vertebrates, glucocorticoids (GCs) are thought to play an important role in mediating the effects of ELS exposure in shaping adult phenotypes. In this process, early exposure to high level of GCs may induce molecular changes that alter developmental trajectory of an animal and primes differential adult responses. However, comprehensive characterization of identities of molecules that are targeted by developmental GC exposure is currently lacking. In our study, we describe lifelong molecular consequences of high level of developmental GC exposure using an optogenetic zebrafish model. First, we developed a new double-hit stress model using zebrafish by combining exposure to a high endogenous GC level during development and acute adulthood stress exposure. Our results establish that similar to ELS-exposed humans and rodents, developmental GC exposed zebrafish model shows altered behavior and stress hypersensitivity in adulthood. Second, we generated time-series gene expression profiles of the brains in larvae, in adult, and upon stress exposure to identify molecular alterations induced by high developmental GC exposure at different developmental stages. Third, we identify a set of GC-primed genes that show altered expression upon acute stress exposure only in animals exposed to a high developmental GC. Interestingly, our datasets of GC primed genes are enriched in risk factors identified for human psychiatric disorders. Lastly, we identify potential epigenetic regulatory elements and associated post-transcriptional modifications following high developmental GC exposure. Thus, we present a translationally relevant zebrafish model for studying stress hypersensitivity and alteration of behavior induced by exposure to elevated GC levels during development. Our study provides comprehensive datasets delineating potential molecular targets underlying the impact of developmental high GC exposure on adult responses.

Abstract: Biomolecular condensates are membraneless compartments that impart spatial and temporal organization to cells. Condensates can undergo maturation, transitioning from dynamic liquid-like states into solid-like states associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington's disease. Despite their important roles, many aspects of condensate biology remain incompletely understood, requiring tools for acutely manipulating condensate-relevant processes within cells. Here we used the BCL6 BTB domain and its ligands BI-3802 and BI-3812 to create a chemical genetic platform, BTBolig, allowing inducible condensate formation and dissolution. We also developed optogenetic and chemical methods for controlled induction of condensate maturation, where we surprisingly observed recruitment of chaperones into the condensate core and formation of dynamic biphasic condensates. Our work provides insights into the interaction of condensates with proteostasis pathways and introduces a suite of chemical-genetic approaches to probe the role of biomolecular condensates in health and disease.

Abstract: The structural plasticity of synapses is crucial for regulating brain functions. However, currently available methods for studying synapse organization based on split fluorescent proteins (FPs) have been limited in assessing synaptic dynamics in vivo due to the irreversible binding of split FPs. Here, we develop 'SynapShot', a method for visualizing the structural dynamics of intact synapses by combining dimerization-dependent FPs (ddFPs) with engineered synaptic adhesion molecules. SynapShot allows real-time monitoring of reversible and bidirectional changes of synaptic contacts under physiological stimulation. The application of green and red ddFPs in SynapShot enables simultaneous visualization of two distinct populations of synapses. Notably, the red-shifted SynapShot is highly compatible with blue light-based optogenetic techniques, allowing for visualization of synaptic dynamics while precisely controlling specific signaling pathways. Furthermore, we demonstrate that SynapShot enables real-time monitoring of structural changes in synaptic contacts in the mouse brain during both primitive and higher-order behaviors.

Abstract: Cellular processes can be modulated by physical means, such as light, which offers advantages over chemically inducible systems with respect to spatiotemporal control. Here we introduce an optogenetic gene expression system for Lactococcus lactis that utilizes a split T7 RNA polymerase linked to two variants of the Vivid regulators. Depending on the chosen photoreceptor variant, either ‘Magnets’ or ‘enhanced Magnets’, this system can achieve either high protein expression levels or low basal activity in the absence of light, exhibiting a fold induction close to 30, rapid expression kinetics, and heightened light sensitivity. This system functions effectively in liquid cultures and within cells embedded in hydrogel matrices, highlighting its potential in the development of novel engineered living materials capable of responding to physical stimuli such as light. The optogenetic component of this system is highly customizable, allowing for the adjustment of expression patterns through modifications to the promoters and/or engineered T7 RNA polymerase variants. We anticipate that this system can be broadly adapted to other Gram-positive hosts with minimal modifications required.

Abstract: Prostaglandin E2 (PGE2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin–Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE2 discharge in response to the increasing cytoplasmic Ca2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE2 discharge is regulated temporally by calcium transients and ERK activity.

Abstract: When animals are infected by a pathogen, peripheral sensors of infection signal to the brain to coordinate a set of adaptive behavioral changes known as sickness behaviors. While the pathways that signal from the periphery to the brain have been intensively studied in recent years, how central circuits are reconfigured to elicit sickness behaviors is not well understood. Here we find that neuromodulatory systems linked to stress and satiety are recruited upon infection to drive sickness behaviors in C. elegans. Upon chronic infection by the bacterium Pseudomonas aeruginosa PA14, C. elegans decrease their feeding behavior, then display reversible bouts of quiescence, and eventually die. The ALA neuron and its neuropeptides FLP-7, FLP-24, and NLP-8, which control stress-induced sleep in uninfected animals, promote the PA14-induced feeding reduction. However, the ALA neuropeptide FLP-13 instead acts to delay quiescence and death in infected animals. Cell-specific genetic perturbations show that the neurons that release FLP-13 to delay quiescence in infected animals are distinct from ALA. A brain-wide imaging screen reveals that infection-induced quiescence involves ASI and DAF-7/TGF-beta, which control satiety-induced quiescence in uninfected animals. Our results suggest that a common set of neuromodulators are recruited across different physiological states, acting from distinct neural sources and in distinct combinations to drive state-dependent behaviors.

Abstract: The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

Abstract: Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.

Abstract: The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.

Abstract: Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system, and introduce a powerful new tool for bacterial optogenetics.

Abstract: Engineering bacterial properties requires precision and fine-tuning for optimal control of the desired application. In consequence, it is essential to accurately turn the function of interest from OFF to ON state and vice versa, avoiding any type of residual activation. For this type of purpose, light switches have revealed a clean and powerful tool in which control does not depend on the addition of chemical compounds that may remain in the media. To reach this degree of directed regulation through light, the switch based on the cyanobacterial two-component system CcaSR system was previously adapted to manipulate Pseudomonas putida for transcription of a gene of interest. In this chapter, we describe how to induce biofilm formation by placing the expression of the c-di-GMP-producing diguanylate cyclase PleD from Caulobacter sp. under the control of the CcaSR system. The regulation through optogenetics accomplished with this protocol promotes higher exploitation of biofilm beneficial features in a cheaper and cleaner way compared to chemical induction.

Abstract: Chimeric antigen receptor (CAR)-T cell immunotherapy emerges as an effective cancer treatment. However, significant safety concerns remain, such as cytokine release syndrome (CRS) and "on-target, off-tumor" cytotoxicity, due to a lack of precise control over conventional CAR-T cell activity. To address this issue, a nano-optogenetic approach has been developed to enable spatiotemporal control of CAR-T cell activity. This system is comprised of synthetic light-sensitive CAR-T cells and upconversion nanoparticles acting as an in situ nanotransducer, allowing near-infrared light to wirelessly control CAR-T cell immunotherapy.

Abstract: By applying sensory photoreceptors, optogenetics realizes the light-dependent control of cellular events and state. Given reversibility, noninvasiveness, and exquisite spatiotemporal precision, optogenetic approaches enable innovative use cases in cell biology, synthetic biology, and biotechnology. In this chapter, we detail the implementation of the pREDusk, pREDawn, pCrepusculo, and pAurora optogenetic circuits for controlling bacterial gene expression by red and blue light, respectively. The protocols provided here guide the practical use and multiplexing of these circuits, thereby enabling graded protein production in bacteria at analytical and semi-preparative scales.

Abstract: CRISPR-Cas effectors are powerful tools for genome and transcriptome targeting and editing. Naturally, these protein-RNA complexes are part of the microbial innate immune system, which emerged from the evolutionary arms race between microbes and phages. This coevolution has also given rise to so-called anti-CRISPR (Acr) proteins that counteract the CRISPR-Cas adaptive immunity. Acrs constitutively block cognate CRISPR-Cas effectors, e.g., by interfering with guide RNA binding, target DNA/RNA recognition, or target cleavage. In addition to their important role in microbiology and evolution, Acrs have recently gained particular attention for being useful tools and switches to regulate or fine-tune the activity of CRISPR-Cas effectors. Due to their commonly small size, high inhibition potency, and structural and mechanistic versatility, Acrs offer a wide range of potential applications for controlling CRISPR effectors in heterologous systems, including mammalian cells.Here, we review the diverse applications of Acrs in mammalian cells and organisms and discuss the underlying engineering strategies. These applications include (i) persistent blockage of CRISPR-Cas function to create write-protected cells, (ii) reduction of CRISPR-Cas off-target editing, (iii) focusing CRISPR-Cas activity to specific cell types and tissues, (iv) spatiotemporal control of CRISPR effectors based on engineered, opto-, or chemogenetic Acrs, and (v) the use of Acrs for selective binding and detection of CRISPR-Cas effectors in complex samples. We will also highlight potential future applications of Acrs in a biomedical context and point out present challenges that need to be overcome on the way.

Abstract: The ability of biological systems to convert inputs from their environment into information to guide future decisions is central to life and a matter of great importance. While we know the components of many of the signaling networks that make these decisions, our understanding of the dynamic flow of information between these parts remains far more limited. T cells are an essential white blood cell type of an adaptive immune response and can discriminate between healthy and infected cells with remarkable sensitivity. This chapter describes the use of a synthetic T-cell receptor (OptoCAR) that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T-cell signaling dynamics. Optical control can also provide control over signaling with high spatial precision, and the OptoCAR is likely to find application more generally when modulating T-cell function with imaging approaches.

Abstract: Mononegaviruses are promising tools as oncolytic and transgene vectors for gene therapy and regenerative medicine. However, when mononegaviruses are used for therapeutic applications, the viral activity must be strictly controlled due to concerns about toxicity and severe side effects. With this technology, mononegavirus vectors can be grown where they are intended and can be easily removed when they are no longer needed. In particular, a photoswitch protein called Magnet (consisting of two magnet domains) is incorporated into the hinge region between the connector and methyltransferase domains of the mononegavirus polymerase protein (L protein) to disrupt the L protein functions. Blue light (470 ± 20 nm) irradiation causes the dimerization of the two magnet domains, and the L protein is restored to activity, allowing viral gene expression and virus replication. Since the magnet domains' dimerization is reversible, viral gene expression and replication cease when blue light irradiation is stopped.

Abstract: A given protein can perform numerous roles in a cell with its participation in protein complexes and distinct localization within the cell playing a critical role in its diverse functions. Thus, the ability to artificially dimerize proteins and recruit proteins to specific locations in a cell has become a powerful tool for the investigation of protein function and the understanding of cell biology. Here, we discuss two systems that have been used to activate signal transduction pathways, a chemically inducible dimerization (CID) and a light-inducible (LI) system to control signaling and cytoskeletal regulation in a spatial and temporal manner.

Abstract: Optogenetic tools provide a means for controlling cellular processes that is rapid, noninvasive, and spatially and temporally precise. With the increase in available optogenetic systems, quantitative comparisons of their performances become important to guide experiments. In this chapter, we first discuss how photoreceptors can be repurposed for light-mediated control of transcription. Then, we provide a detailed protocol for characterizing light-regulated transcriptional systems in budding yeast using fluorescence time-lapse microscopy and mathematical modeling, expanding on our recent publication (Gligorovski et al., Nat Commun 14:3810, 2023).

Abstract: Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.

Abstract: Avena Sativa phototropin 1 Light-oxygen-voltage 2 domain (AsLOV2) is the model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change is initiated by the unfolding of the N-terminal helix in the dark state followed by the unfolding of the C-terminal helix. The light state is defined by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, β-sheets have been identified as crucial components for mediating allosteric signal transmission between the two termini. In this study, we combined microsecond all-atm molecular dynamics simulations and Markov state modeling of conformational states to quantify molecular basis of mutation-induced allostery in the AsLOV2 protein. Through a combination of computational investigations, we determine that the Hβ and Iβ strands are the most critical structural elements involved in the allosteric mechanism. To elucidate the role of these β-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive simulation analysis. The results highlighted the role of two hydrogen bond Asn482-Leu453 and Gln479-Val520 in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. The comprehensive atomistic-level analysis of the conformational landscapes revealed the critical functional role of β-sheet segments in the transmission of the allosteric signal upon the photoactivation of the light state.

Abstract: Mural cells are critical components of the cerebral vasculature. They are categorized into three primary subsets: arteriole smooth muscle cells (aSMCs), pericytes (PCs) and venule smooth muscle cells (vSMCs). It is well known that aSMCs can directly regulate cerebral blood flow (CBF) with their own contraction and dilation mechanisms. On the other hand, the direct involvement of PCs or vSMCs in CBF regulation is controversial. This ambiguity is largely due to the lack of specifically manipulable tools to isolate their function. To address this issue, we employed a set-subtraction approach by using a combination of tTA-mediated gene induction and Cre-mediated gene excision. We developed transgenic mice expressing optical actuators, channelrhodopsin-2 (ChR2) and photoactivated adenylyl cyclase (PAC) in smooth muscle actin (SMA)-negative mural cells that lack the machinery for SMA-mediated vasoregulation. Using these mouse models, we assessed CBF alterations in response to optical stimulation using laser Doppler techniques. Our results showed that optical stimulation induced notable CBF changes in both models. This study provides evidence for the potential regulatory role of PCs and vSMCs in cerebral hemodynamics and introduces powerful tools to specifically manipulate these cell types in vascular neurobiology.