Curated Optogenetic Publication Database

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.

Abstract: Hyperactivation of phosphatidylinositol 3-kinase (PI3K) signaling is a prominent feature in cancer cells. However, the mechanism underlying malignant behaviors in the state remains unknown. Here, we describe a mechanism of cancer drug resistance through the protein synthesis pathway, downstream of PI3K signaling. An optogenetic tool (named PPAP2) controlling PI3K signaling was developed. Melanoma cells stably expressing PPAP2 (A375-PPAP2) acquired resistance to a cancer drug in the hyperactivation state. Proteome analyses revealed that expression of the antiapoptotic factor tumor necrosis factor alpha-induced protein 8 (TNFAIP8) was upregulated. TNFAIP8 upregulation was mediated by protein translation from preexisting mRNA. These results suggest that cancer cells escape death via upregulation of TNFAIP8 expression from preexisting mRNA even though alkylating cancer drugs damage DNA.

Abstract: A wide array of optogenetic tools is available that allow for precise spatiotemporal control over many cellular processes. These tools have been especially popular among zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and apoptosis.

Abstract: The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Abstract: The GGGGCC (G4C2) hexanucleotide repeat expansion in the C9ORF72 gene is a major cause of both hereditary amyotrophic lateral sclerosis and familial frontotemporal dementia. Recent studies have shown that G4C2 hexanucleotide repeat-containing RNA transcripts ((G4C2)n RNA) could go through liquid-liquid phase separation to form RNA foci, which may elicit neurodegeneration. However, the direct causality between these abnormal RNA foci and neuronal toxicity remains to be demonstrated. Here we introduce an optogenetic control system that can induce the assembly and phase separation of (G4C2)n RNA foci with blue light illumination in human cells, by fusing a specific (G4C2)n RNA binding protein as the linker domain to Cry2, a protein that oligomerizes in response to blue light. Our results demonstrate that a higher number of G4C2 repeats have the potential to be induced into more RNA foci in the cells. Both spontaneous and induced RNA foci display liquid-like properties according to FRAP measurements. Computational simulation shows strong consistency with the experimental results and supports the effect of our system to promote the propensity of (G4C2)n RNA towards phase separation. This system can thus be used to investigate whether (G4C2)n RNA foci would disrupt normal cellular processes and lead to pathological phenotypes relevant to repeat expansion disorders.

Abstract: Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate β-catenin stability. Overexpressed DC scaffolds undergo liquid-liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in β-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of β-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3β partitioning on the centrosome controls β-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle.

Abstract: BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse-BAR) domain containing tools derived from the fusion of the A. thaliana Cryptochrome 2 photoreceptor and I-BAR protein domains ('CRY-BARs') with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamics changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and PIP2 binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

Abstract: Many transcription factors (TFs) translocate to the nucleus with varied dynamic patterns in response to different inputs. A notable example of such behavior is RelA, a subunit of NF-κB, which translocates to the nucleus with either pulsed or sustained dynamics, depending on the stimulus. Our understanding of how these dynamics are interpreted by downstream genes has remained incomplete, partly because ubiquitously used environmental inputs activate other transcriptional regulators in addition to RelA. Here, we use an optogenetic tool, CLASP (controllable light-activated shuttling and plasma membrane sequestration), to control RelA spatiotemporal dynamics in mouse fibroblasts and quantify their effect on downstream genes using RNA-seq. Using RelA-CLASP, we show for the first time that nuclear translocation of RelA, without post-translational modifications or activation of other transcriptional regulators, is sufficient to activate downstream genes. Furthermore, we find that TNFα, a common endogenous input, regulates many genes independently of RelA, and that this gene regulation is different from that induced by RelA-CLASP. Genes responsive to RelA-CLASP show a wide range of dynamics in response to a constant RelA input. We use a simple promoter model to recapitulate these diverse dynamic responses, as well as data collected in response to a pulsed RelA-CLASP input, and extract features of many RelA-responsive promoters. We also pinpoint many genes for which more complex models, involving feedback or multi-step promoters, may be needed to explain their response to constant and pulsed TF inputs. This study introduces a new robust tool for studying mammalian transcriptional regulation and demonstrates the power of optogenetic tools in dissecting the quantitative features of important cellular pathways.

Abstract: Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to control gene expression independently from mammalian transcriptional and/or translational machineries thus working orthogonally to endogenous regulatory mechanisms. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize, and optimize a new optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool is orthogonal to the cellular machinery for transcription and consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7-mOptoT7). In our study we exploited the T7 polymerase's viral origins to tune our system's expression level, reaching up to an almost 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate the gene expression burden when compared to another optogenetic construct. These properties make mOptoT7 a powerful new tool to use when orthogonality and viral RNA species (that lack endogenous RNA modifications) are desired.

Abstract: Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein interactomes to be captured using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to local generation of reactive radical species within cell-permeable probes. When combined with quantitative proteomics, the system generates ‘snapshots’ of protein interactions with high temporal resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein (MVP). The technology should be broadly useful in the biomedical area.

Abstract: YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Abstract: Cells interpret a variety of signals through G protein-coupled receptors (GPCRs), and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different responses, despite generating similar levels of cAMP. We previously showed that GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here, we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to cue different outputs. We show that generating cAMP from the Golgi by an optogenetic approach or activated GPCR leads to regulation of a specific PKA target that increases rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We validated the physiological consequences of these observations in intact zebrafish and mice. Thus, the same GPCR regulates distinct molecular and physiological pathways depending on its subcellular location despite generating cAMP in each case.

Abstract: How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

Abstract: The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

Abstract: Immune cells activate in binary, switch-like fashion via large protein assemblies known as signalosomes, but the molecular mechanism of the switch is not yet understood. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, which governs nuclear transcription factor-κB activation in both innate and adaptive immunity. We found that the switch consists of a sequence-encoded and deeply conserved nucleation barrier to ordered polymerization by the adaptor protein BCL10. The particular structure of the BCL10 polymers did not matter for activity. Using optogenetic tools and single-cell transcriptional reporters, we discovered that endogenous BCL10 is functionally supersaturated even in unstimulated human cells, and this results in a predetermined response to stimulation upon nucleation by activated CARD multimers. Our findings may inform on the progressive nature of age-associated inflammation, and suggest that signalosome structure has evolved via selection for kinetic rather than equilibrium properties of the proteins.

Abstract: Diabetes treatment and rehabilitation are usually a lifetime process. Optogenetic engineered designer cell-therapy holds great promise in regulating blood glucose homeostasis. However, portable, sustainable, and long-term energy supplementation has previously presented a challenge for the use of optogenetic stimulation in vivo. Herein, we purpose a self-powered optogenetic system (SOS) for implantable blood glucose control. The SOS consists of a biocompatible far-red light (FRL) source, FRL-triggered transgene-expressing cells, a power management unit, and a flexible implantable piezoelectric nanogenerator (i-PENG) to supply long-term energy by converting biomechanical energy into electricity. Our results show that this system can harvest energy from body movement and power the FRL source, which then significantly enhanced production of a short variant of human glucagon-like peptide 1 (shGLP-1) in vitro and in vivo. Indeed, diabetic mice equipped with the SOS showed rapid restoration of blood glucose homeostasis, improved glucose, and insulin tolerance. Our results suggest that the SOS is sufficiently effective in self-powering the modulation of therapeutic outputs to control glucose homeostasis and, furthermore, present a new strategy for providing energy in optogenetic-based cell therapy.

Abstract: Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light-activatable photoswitch that consists of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light-activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light-inducible transcriptional regulators based on CRISPR-Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.