Curated Optogenetic Publication Database

Abstract: Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

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: Cyclic adenosine monophosphate (cAMP) is a second messenger that mediates diverse intracellular signals. Studies of cAMP transport in cells have produced wildly different results, from reports of nearly free diffusion to reports that cAMP remains localized in nanometer-scale domains. We developed an all-optical toolkit, termed cAMP-SITES, to locally perturb and map cAMP transport. In MDCK cells and in cultured neurons, cAMP had a diffusion coefficient of ~120 μm2/s, similar to the diffusion coefficients of other small molecules in cytoplasm. In neuronal dendrites, a balance between diffusion and degradation led to cAMP domains with a length scale of ~30 μm. Geometrical confinement by membranes led to subcellular variations in cAMP concentration, but we found no evidence of nanoscale domains or of distinct membrane-local and cytoplasmic pools. We introduce theoretical relations between cell geometry and small-molecule reaction-diffusion dynamics and transport to explain our observations.

Abstract: We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.

Abstract: Wnt/β-catenin signaling and its dysregulation play critical roles in the fate determination of stem cells and the pathology of various diseases. However, the application of translated Wnt ligand in regenerative medicine is hampered by its hydrophobicity and cross-reactivity with Frizzled (FZD) receptors. Here, we generate an engineered water-soluble, FZD subtype-specific agonist, RRP-pbFn, for high-efficiency Wnt/β-catenin signaling activation. In the absence of direct binding to LRP5/6, RRP-pbFn stimulates Wnt/β-catenin signaling more potently than surrogate Wnt. RRP-pbFn supports the growth of a variety of mouse and human organoids, and induces the expansion of liver and intestine progenitors in vivo. Meanwhile, we develop a synthetic LRP antagonist, RRP-Dkk1c, which exhibits heightened effectiveness in attenuating Wnt/β-catenin signaling activity compared to Dkk1, thereby abolishing the formation of CT26-derived colon cancer xenograft in vivo. Together, these two paired Wnt/β-catenin signaling manipulators hold great promise for biomedical research and potential therapeutics.

Abstract: The RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (A1) regulates RNA metabolism, which is crucial to maintaining cellular homeostasis. A1 dysfunction mechanistically contributes to reduced cell viability and loss, but molecular mechanisms of how A1 dysfunction affects cell viability and loss, and methodologies to attenuate its dysfunction, are lacking. Utilizing in silico molecular modeling and an in vitro optogenetic system, this study examined the consequences of RNA oligonucleotide (RNAO) treatment on attenuating A1 dysfunction and its downstream cellular effects. In silico and thermal shift experiments revealed that binding of RNAOs to the RNA Recognition Motif 1 of A1 is stabilized by sequence- and structure-specific RNAO-A1 interactions. Using optogenetics to model A1 cellular dysfunction, we show that sequence- and structure-specific RNAOs significantly attenuated abnormal cytoplasmic A1 self-association kinetics and A1 cytoplasmic clustering. Downstream of A1 dysfunction, we demonstrate that A1 clustering affects the formation of stress granules, activates cell stress, and inhibits protein translation. With RNAO treatment, we show that stress granule formation is attenuated, cell stress is inhibited, and protein translation is restored. This study provides evidence that sequence- and structure-specific RNAO treatment attenuates A1 dysfunction and its downstream effects, thus allowing for the development of A1-specific therapies that attenuate A1 dysfunction and restore cellular homeostasis.

Abstract: Optogenetic tools can provide fine spatial and temporal control over many biological processes. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. We demonstrate the approach's utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

Abstract: Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Abstract: Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of 'superPLDs' with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

Abstract: The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

Abstract: Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury.

Abstract: Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, Photo-induced Modulation of Gα protein – Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. We alter the behavior of C. elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gαi signaling.

Abstract: In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

Abstract: The light-regulated Gal4-UAS system has offered new ways to control cellular activities with precise spatial and temporal resolution in zebrafish and Drosophila. However, the existing optogenetic Gal4-UAS systems suffer from having multiple protein components and a dependence on extraneous light-sensitive cofactors, which increase the technical complexity and limit the portability of these systems. To overcome these limitations, we herein describe the development of a novel optogenetic Gal4-UAS system (ltLightOn) for both zebrafish and Drosophila based on a single light-switchable transactivator, termed GAVPOLT, which dimerizes and binds to gene promoters to activate transgene expression upon blue light illumination. The ltLightOn system is independent of exogenous cofactors and exhibits a more than 2400-fold ON/OFF gene expression ratio, allowing quantitative, spatial, and temporal control of gene expression. We further demonstrate the usefulness of the ltLightOn system in regulating zebrafish embryonic development by controlling the expression of lefty1 by light. We believe that this single-component optogenetic system will be immensely useful in understanding the gene function and behavioral circuits in zebrafish and Drosophila.

Abstract: Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Abstract: The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.

Abstract: Exogenous insulin therapy is the mainstay treatment for Type-1 diabetes (T1D) caused by insulin deficiency. A fine-tuned insulin supply system is important to maintain the glucose homeostasis. In this study, we present a designed cell system that produces insulin under an AND gate control, which is triggered only in the presence of both high glucose and blue light illumination. The glucose-sensitive GIP promoter induces the expression of GI-Gal4 protein, which forms a complex with LOV-VP16 in the presence of blue light. The GI-Gal4:LOV-VP16 complex then promotes the expression of UAS-promoter-driven insulin. We transfected these components into HEK293T cells, and demonstrated the insulin was secreted under the AND gate control. Furthermore, we showed the capacity of the engineered cells to improve the blood glucose homeostasis through implantation subcutaneously into Type-1 diabetes mice.

Abstract: Secreted reporters have been demonstrated to be simple and useful tools for analyzing transcriptional regulation in mammalian cells. The distinctive feature of these assays is the ability to detect reporter gene expression in the culture supernatant without affecting the cell physiology or leading to cell lysis, which allows repeated experimentation and sampling of the culture medium using the same cell cultures. Secreted embryonic alkaline phosphatase (SEAP) is one of the most widely used reporter, which can be easily detected using colorimetry following incubation with a substrate, such as p-nitrophenol phosphate. In this report, we present detailed procedures for detection and quantification of the SEAP reporter. We believe that this step-by-step protocol can be easily used by researchers to monitor and measure molecular genetic events in a variety of mammalian cells due to its simplicity and ease of handling. Graphical abstract Schematic overview of the workflow described in this protocol.

Abstract: Bovine parainfluenza virus type 3 (BPIV3) is a promising vaccine vector against various respiratory virus infections, including the human PIV3, respiratory syncytial virus, and severe acute respiratory syndrome-coronavirus 2 infections. In this study, we combined the Magnet system and reverse genetic approach to generate photocontrollable BPIV3. An optically controllable Magnet gene was inserted into the H2 region of the BPIV3 large protein gene, which encodes an RNA-dependent RNA polymerase. The generated photocontrollable BPIV3 grew in specific regions of the cell sheet only when illuminated with blue light, suggesting that spatiotemporal control can aid in safe clinical applications of BPIV3.

Abstract: Pyroptosis has been identified as a pro-inflammatory form of programmed cell death. It can be triggered by different stimuli including pathogen invasion or cell stress/danger signals releasing hundreds of proteins upon lysis that cause complex responses in neighboring cells. Pyroptosis is executed by the gasdermin (GSDM) family of proteins which, upon cleavage by caspases, form transmembrane pores that release cytokines to induce inflammation. However, despite the importance of gasdermins in the development of inflammatory diseases and cancer, a lot is still to be understood in the downstream consequences of this cell death pathway. Currently, conventional methods, such as drug treatments or chemically forced oligomerization, are limited in the spatiotemporal analysis of pyroptosis signaling in the cellular population, since all cells are primed for undergoing pyroptosis. Here, we provide a protocol for the application of a novel optogenetics tool called NLS_PhoCl_N-GSDMD_mCherry that enables precise temporal and spatial pyroptosis induction in a confocal microscopy setup, followed by imaging of the cell death process and subsequent quantitative analysis of the experiment. This tool opens new opportunities for the study of pyroptosis activation and of its effects on the bystander cell responses.

Abstract: The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.

Abstract: Photosensory protein domains are the basis of optogenetic protein engineering. These domains originate from natural sources where they fulfill specific functions ranging from the protection against photooxidative damage to circadian rhythms. When used in synthetic biology, the features of these photosensory domains can be specifically tailored towards the application of interest, enabling their full exploitation for optogenetic regulation in basic research and applied bioengineering. In this work, we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity. We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants. For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted. We functionally characterize the variants in vivo in bacteria on the single-cell and the population levels. We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells. We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments. Our findings expand the applicability of the widely used Magnets photosensors by enabling a tuning towards the needs of specific optogenetic regulation strategies. More generally, our approach will aid optogenetic approaches by making the adaptation of photosensor properties possible to better suit specific experimental or bioprocess needs.

Abstract: When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Abstract: The experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research.