Curated Optogenetic Publication Database

Abstract: Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.

Abstract: ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

Abstract: Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.

Abstract: The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca2+ selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. Here, we describe the design of a light-operated Ca2+ channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel, which enables precise optical control over Ca2+ signals and hallmark Ca2+-dependent physiological responses. Furthermore, we demonstrate the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis.

Abstract: Optogenetic systems have been increasingly investigated in the field of biomedicine. Previous studies had found the inhibitory effect of the light-inducible genetic circuits on cancer cell growth. In our study, we applied an AND logic gates to the light-inducible genetic circuits to inhibit the cancer cells more specifically. The circuit would only be activated in the presence of both the human telomerase reverse transcriptase (hTERT) and the human uroplakin II (hUPII) promoter. The activated logic gate led to the expression of the p53 or E-cadherin protein, which could inhibit the biological function of tumor cells. In addition, we split the dCas9 protein to reduce the size of the synthetic circuit compared to the full-length dCas9. This light-inducible system provides a potential therapeutic strategy for future bladder cancer.

Abstract: Membrane receptors play a crucial role in transmitting external signals inside cells. Signal molecule-bound receptors activate multiple downstream pathways, the dynamics of which are modulated by intracellular trafficking. A significant contribution of β-arrestin to intracellular trafficking has been suggested, but the underlying mechanism is poorly understood. Here, we describe a protocol for manipulating β-arrestin-regulated membrane receptor trafficking using photo-induced dimerization of cryptochrome-2 from Arabidopsis thaliana and its binding partner CIBN. Additionally, the protocol guides analytical methods to quantify the changes in localization and modification of membrane receptors during trafficking.

Abstract: The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.

Abstract: With the development of mobile communication technology, smartphones have been used in point-of-care technologies (POCTs) as an important part of telemedicine. Using a multidisciplinary design principle coupling electrical engineering, software development, synthetic biology, and optogenetics, the investigators developed a smartphone-controlled semiautomatic theranostic system that regulates blood glucose homeostasis in diabetic mice in an ultraremote-control manner. The present chapter describes how the investigators tailor-designed the implant architecture "HydrogeLED," which is capable of coharboring a designer-cell-carrying alginate hydrogel and wirelessly powered far-red light LEDs. Using diabetes mellitus as a model disease, the in vivo expression of insulin or human glucagon-like peptide 1 (shGLP-1) from HydrogeLED implants could be controlled not only by pre-set ECNU-TeleMed programs, but also by a custom-engineered Bluetooth-active glucometer in a semiautomatic and glycemia-dependent manner. As a result, blood glucose homeostasis was semiautomatically maintained in diabetic mice through the smartphone-controlled semiautomatic theranostic system. By combining digital signals with optogenetically engineered cells, the present study provides a new method for the integrated diagnosis and treatment of diseases.

Abstract: With the increasing indispensable role of smartphones in our daily lives, the mobile health care system coupled with embedded physical sensors and modern communication technologies make it an attractive technology for enabling the remote monitoring of an individual's health. Using a multidisciplinary design principle coupled with smart electronics, software, and optogenetics, the investigators constructed smartphone-controlled optogenetic switches to enable the ultraremote-control transgene expression. A custom-designed SmartController system was programmed to process wireless signals from smartphones, enabling the regulation of therapeutic outputs production by optically engineered cells via a far-red light (FRL)-responsive optogenetic interface. In the present study, the investigators describe the details of the protocols for constructing smartphone-controlled optogenetic switches, including the rational design of an FRL-triggered transgene expression circuit, the procedure for cell culture and transfection, the implementation of the smartphone-controlled far-red light-emitting diode (LED) module, and the reporter detection assay.

Abstract: The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.

Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Abstract: The spatiotemporal organization of oligomeric protein complexes, such as the supramolecular organizing centers (SMOCs) made of MyDDosome and MAVSome, is essential for transcriptional activation of host inflammatory responses and immunometabolism. Light‐inducible assembly of MyDDosome and MAVSome is presented herein to induce activation of nuclear factor‐kB and type‐I interferons. Engineering of SMOCs and the downstream transcription factor permits programmable and customized innate immune operations in a light‐dependent manner. These synthetic molecular tools will likely enable optical and user‐defined modulation of innate immunity at a high spatiotemporal resolution to facilitate mechanistic studies of distinct modes of innate immune activations and potential intervention of immune disorders and cancer.

Abstract: Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

Abstract: The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatio-temporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light-induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2–5]). Irradiation yielded a 50-230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

Abstract: Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.

Abstract: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGFβ) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic, and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise activation of the BMP signaling pathway. Here, we report the development of an optogenetic BMP signaling system (optoBMP) that enables rapid induction of the canonical BMP signaling pathway driven by illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signaling pathway through variable light stimulation. Optogenetic control of BMP signaling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.

Abstract: The precise control of signaling proteins is a prerequisite to decipher the complexity of the signaling network and to reveal and to study pathways involved in regulating cellular metabolism and gene expression. Optogenetic approaches play an emerging role as they enable the spatiotemporal control of signaling processes. Herein, a multichromatic system is developed by combining the blue light cryptochrome 2 system and the red/far-red light phytochrome B system. The use of three wavelengths allows the orthogonal control of the RAF/ERK and the AKT signaling pathway. Continuous exposure of cells to blue light leads to activation of AKT while simultaneous pulses of red and far-red light enable the modulation of ERK signaling in cells with constantly active AKT signaling. The optimized, orthogonal multichromatic system presented here is a valuable tool to better understand the fine grained and intricate processes involved in cell fate decisions.

Abstract: Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows the feasibility of stimulating neural repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration-competent and -incompetent sensory neurons in the peripheral nervous system but also allowed temporal tuning and proper guidance of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. We conclude that non-neuronal optogenetics target damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.

Abstract: Short regulatory RNA molecules underpin gene expression and govern cellular state and physiology. To establish an alternative layer of control over these processes, we generated chimeric regulatory RNAs that interact reversibly and light-dependently with the light-oxygen-voltage photoreceptor PAL. By harnessing this interaction, the function of micro RNAs (miRs) and short hairpin (sh) RNAs in mammalian cells can be regulated in a spatiotemporally precise manner. The underlying strategy is generic and can be adapted to near-arbitrary target sequences. Owing to full genetic encodability, it establishes optoribogenetic control of cell state and physiology. The method stands to facilitate the non-invasive, reversible and spatiotemporally resolved study of regulatory RNAs and protein function in cellular and organismal environments.

Abstract: Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.

Abstract: Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli as well as of many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, i.e. metazoan opsins, which are light-activated GPCRs. Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.

Abstract: The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A. thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.

Abstract: Protein degradation technology, which is one of the most direct and effective ways to regulate the life activities of cells, is expected to be applied to the treatment of various diseases. However, current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation, making them difficult to transform into clinical applications. In this article, an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation. This system worked via two interconnected parts: 1) the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure; 2) the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation. Experimental results based on model protein (Green Fluorescent Protein, GFP) validated that this system could fulfill protein degradation both in vitro (both Hela and 293T cells) and in vivo (by upconversion optogenetic nanosystem), and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time (0–25 h) and the illumination frequency (the illuminating frequency of 0–30 s every 1 min). We further took another functional protein (The Nonstructural Protein 9, NSP9) into experiment. Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV) was inhibited by degrading the NSP9 in this light-induced system, and PRRSV proliferation was affected by different light conditions (illumination time varies from 0–24 h). We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.

Abstract: A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.

Abstract: Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.