Curated Optogenetic Publication Database

Abstract: Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.

Abstract: Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

Abstract: CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.

Abstract: Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform – Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially-designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Abstract: Cells communicate with each other to jointly regulate cellular processes during cellular differentiation and tissue morphogenesis. This multiscale coordination arises through spatiotemporal activity of morphogens to pattern cell signaling and transcriptional factor activity. This coded information controls cell mechanics, proliferation, and differentiation to shape the growth and morphogenesis of organs. While many of the molecular components and physical interactions have been identified in key model developmental systems, there are still many unresolved questions related to the dynamics involved due to challenges in precisely perturbing and quantitatively measuring signaling dynamics. Recently, a broad range of synthetic optogenetic tools have been developed and employed to quantitatively define relationships between signal transduction and downstream cellular responses. These optogenetic tools can control intracellular activities at the single cell or whole tissue scale to direct subsequent biological processes. In this brief review, we highlight a selected set of studies that develop and implement optogenetic tools to unravel quantitative biophysical mechanisms for tissue growth and morphogenesis across a broad range of biological systems through the manipulation of morphogens, signal transduction cascades, and cell mechanics. More generally, we discuss how optogenetic tools have emerged as a powerful platform for probing and controlling multicellular development.

Abstract: The cGAS-STING signaling pathway has emerged as a promising target for immunotherapy development. Here, we introduce a light-sensitive optogenetic device for control of the cGAS/STING signaling to conditionally modulate innate immunity, called 'light-inducible SMOC-like repeats' (LiSmore). We demonstrate that photo-activated LiSmore boosts dendritic cell (DC) maturation and antigen presentation with high spatiotemporal precision. This non-invasive approach photo-sensitizes cytotoxic T lymphocytes to engage tumor antigens, leading to a sustained antitumor immune response. When combined with an immune checkpoint blocker (ICB), LiSmore improves antitumor efficacy in an immunosuppressive lung cancer model that is otherwise unresponsive to conventional ICB treatment. Additionally, LiSmore exhibits an abscopal effect by effectively suppressing tumor growth in a distal site in a bilateral mouse model of melanoma. Collectively, our findings establish the potential of targeted optogenetic activation of the STING signaling pathway for remote immunomodulation in mice.

Abstract: The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.

Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Abstract: Intra-thymic T cell development is coordinated by the regulatory actions of SATB1 genome organizer. In this report, we show that SATB1 is involved in the regulation of transcription and splicing, both of which displayed deregulation in Satb1 knockout murine thymocytes. More importantly, we characterized a novel SATB1 protein isoform and described its distinct biophysical behavior, implicating potential functional differences compared to the commonly studied isoform. SATB1 utilized its prion-like domains to transition through liquid-like states to aggregated structures. This behavior was dependent on protein concentration as well as phosphorylation and interaction with nuclear RNA. Notably, the long SATB1 isoform was more prone to aggregate following phase separation. Thus, the tight regulation of SATB1 isoforms expression levels alongside with protein post-translational modifications, are imperative for SATB1's mode of action in T cell development. Our data indicate that deregulation of these processes may also be linked to disorders such as cancer.

Abstract: Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ('cell doublet'). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

Abstract: Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro's components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Abstract: High-performance biosensors are crucial for elucidating the spatiotemporal regulatory roles and dynamics of membrane lipids, but there is a lack of improvement strategies for biosensors with low sensitivity and low-content substrates detection. Here we developed universal optogenetic strategies to improve a set of membrane biosensors by trapping them into specific region and further reducing the background signal, or by optically-controlled phase separation for membrane lipids detection and tracking. These improved biosensors were superior to typical tools and light simulation would enhance their detection performance and resolution, which might contribute to the design and optimization of other biosensors.

Abstract: An increasing experimental evidence points to physiological importance of space-time correlations in signalling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of bio-molecules between cells allows the collectives to perform more complex tasks and better tackle environmental challenges. To understand this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signalling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We make a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells induce ERK activity waves with different size, duration, and frequency. The open-source implementations of ARCOS are available as R and Python packages, and as a plugin for napari image viewer to interactively quantify collective phenomena without prior programming experience.

Abstract: Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.

Abstract: Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, the mechanical properties of embryonic epithelial tissues and the factors that control these properties are not well understood. Actomyosin generates contractile tensions and contributes to the mechanical properties of cells and cytoskeletal networks in vitro, but it remains unclear how the levels and patterns of actomyosin activity contribute to embryonic epithelial tissue mechanics in vivo. To dissect the roles of cell-generated tensions in the mechanics of flowing epithelial tissues, we use optogenetic tools to manipulate actomyosin contractility with spatiotemporal precision in the Drosophila germband epithelium, which rapidly flows during body axis elongation. We find that manipulating actomyosin-dependent tensions by either optogenetic activation or deactivation of actomyosin alters the solid-fluid mechanical properties of the germband epithelium, leading to changes in cell rearrangements and tissue-level flows. Optogenetically activating actomyosin leads to increases in the overall level but decreases in the anisotropy of tension in the tissue, whereas optogenetically deactivating actomyosin leads to decreases in both the level and anisotropy of tension compared to in wild-type embryos. We find that optogenetically activating actomyosin results in more solidlike (less fluidlike) tissue properties, which is associated with reduced cell rearrangements and tissue flow compared to in wild-type embryos. Optogenetically deactivating actomyosin also results in more solidlike properties than in wild-type embryos but less solidlike properties compared to optogenetically activating actomyosin. Together, these findings indicate that increasing the overall tension level is associated with more solidlike properties in tissues that are relatively isotropic, whereas high-tension anisotropy fluidizes the tissue. Our results reveal that epithelial tissue flows in developing embryos involve the coordinated actomyosin-dependent regulation of the mechanical properties of tissues and the tensions driving them to flow in order to achieve rapid tissue remodeling.

Abstract: Regulated cell death plays a key role in immunity, development, and homeostasis, but is also associated with a number of pathologies such as autoinflammatory and neurodegenerative diseases and cancer. However, despite the extensive mechanistic research of different cell death modalities, the direct comparison of different forms of cell death and their consequences on the cellular and tissue level remain poorly characterized. Comparative studies are hindered by the mechanistic and kinetic differences between cell death modalities, as well as the inability to selectively induce different cell death programs in an individual cell within cell populations or tissues. In this method, we present a protocol for rapid and specific optogenetic activation of three major types of programmed cell death: apoptosis, necroptosis, and pyroptosis, using light-induced forced oligomerization of their major effector proteins (caspases or kinases).

Abstract: The integrated stress response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered a photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics, we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR, which demonstrated the dependence of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. A record of this paper's transparent peer review process is included in the supplemental information.

Abstract: Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are primarily neurodegenerative diseases with progressive decline in motor function. Aggregates composed of alpha-synuclein, which are known as Lewy bodies, are a neuropathological hallmark of synucleinopathies; their pathogenesis has been attributed to neuronal loss owing to intracellular alpha-synuclein accumulation. However, the mechanism of alpha-synuclein aggregation remains unclear. Here we show that the RNA G-quadruplexes assembly forms scaffolds for alpha-synuclein aggregation, thereby contributing to neurodegeneration. RNA G-quadruplexes undergo phase separation and form scaffolds for co-aggregation with & alpha-synuclein. Upon pathogenic alpha-synuclein seeds-induced cellular stress and an optogenetic assembly of RNA G-quadruplexes, phase-separated RNA G-quadruplexes served as scaffolds for & alpha-synuclein phase transition, and the co-aggregates initiated synaptic dysfunction and Parkinsonism in mice. Treatment with 5-aminolevulinic acid and protoporphyrin IX, which prevents RNA G-quadruplexes phase separation, attenuates alpha-synuclein phase transition, neurodegeneration, and motor deficits in synucleinopathy model mice. Together, the RNA G-quadruplexes assembly accelerates alpha-synuclein phase transition and aggregation owing to intracellular Ca2+ homeostasis, thereby contributing to the pathogenesis of synucleinopathies.

Abstract: Optogenetic systems use genetically encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light; however, these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to the high-throughput characterization of optogenetic systems across a range of biological systems and applications.

Abstract: Biomolecular condensates, including membraneless organelles, are ubiquitously observed in subcellular compartments. However, the accumulation and dynamic properties of arbitrarily in-duced condensates remain elusive. Here, we show the size, amount, and dynamic properties of subcellular condensates using various fluorescence spectroscopic imaging analyses. Spatial image correlation spectroscopy showed that the size of blue-light-induced condensates of cryptochrome 2-derived oligomerization tag (CRY2olig) tagged with a red fluorescent protein in the nucleus was not different from that in the cytoplasm. Fluorescence intensity measurements showed that the condensates in the nucleus were more prone to accumulation than those in the cytoplasm. Sin-gle-particle tracking analysis showed that the condensates in the nucleus are predisposed to be stationary dynamics compared to those in the cytoplasm. Therefore, the subcellular compartment may, in part, affect the characteristics of self-recruitment of biomolecules in the condensates and their movement property.

Abstract: The inflammasome is a conserved structure for the intracellular detection of danger or pathogen signals. As a large intracellular multiprotein signaling platform, it activates downstream effectors that initiate a rapid necrotic programmed cell death (PCD) termed pyroptosis and activation and secretion of pro-inflammatory cytokines to warn and activate surrounding cells. However, inflammasome activation is difficult to control experimentally on a single-cell level using canonical triggers. We constructed Opto-ASC, a light-responsive form of the inflammasome adaptor protein ASC (Apoptosis-Associated Speck-Like Protein Containing a CARD) which allows tight control of inflammasome formation in vivo. We introduced a cassette of this construct under the control of a heat shock element into zebrafish in which we can now induce ASC inflammasome (speck) formation in individual cells of the skin. We find that cell death resulting from ASC speck formation is morphologically distinct from apoptosis in periderm cells but not in basal cells. ASC-induced PCD can lead to apical or basal extrusion from the periderm. The apical extrusion in periderm cells depends on Caspb and triggers a strong Ca2+ signaling response in nearby cells.

Abstract: Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid β-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.

Abstract: In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging, and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition, and TGF-β signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish an hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.

Abstract: RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

Abstract: Collective cell migration during embryonic development, wound healing, and cancer metastasis entails the emergence of leader cells at the migration front. These cells with conspicuous lamellipodial structures provide directional guidance to the collective. Despite their physiological relevance, the mechanisms underlying the emergence of leader cells remain elusive. Here we report that in diverse model systems for wound healing, including cultured epithelial monolayer, Drosophila embryo, and mouse embryonic skin, leader cells display a peripheral accumulation of lysosomes. This accumulation appears essential for leader cell emergence, involves lysosomal movement along microtubules, and depends on the actomyosin contractility-generated cellular forces. Peripheral lysosomes associate with inactive Rac1 molecules to remove them from the leading periphery, which increases local Rac1-activity, triggering actin polymerization and promoting lamellipodium formation. Taken together, we demonstrate that beyond their catabolic role, lysosomes act as the intracellular platform that links mechanical and biochemical signals to control the emergence of leader cells.