Curated Optogenetic Publication Database

Abstract: The aggregation of α-synuclein (α-SYN) into Lewy bodies (LBs) is a central event in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies1,2. Despite significant advances in understanding α-SYN self-assembly, the precise sequence of early aggregation steps has not been directly visualized in living neurons. Here, we use an optogenetic-induced protein aggregation system with a high temporal resolution to monitor the onset of α-SYN assembly in neurons. We found that the initiation and accumulation of α-SYN aggregates occur predominantly at the lysosomal membrane, an event driven by the α-SYN N-terminus and modulated by the membrane-associated adaptor protein WD repeat-containing protein 44 (WDR44). Remarkably, we demonstrate that WDR44 knockdown markedly reduced de novo α-SYN aggregation in both neuronal cultures and in vivo, whereas WDR44 overexpression enhances α-SYN aggregation in PD patient-derived iPSC neurons. Consistent with its potential pathogenic involvement, WDR44 aberrantly accumulates in vivo and in the brains of PD patients, where it colocalizes with LB inclusions. Finally, we show that lysosome-associated α-SYN aggregates compromised lysosomal structure and function, leading to neuronal impairment, a phenotype worsened by WDR44 overexpression, linking early aggregation events to downstream toxicity. Together, these findings reveal the earliest dynamic stages of α-SYN oligomerization in living neurons and identify the WDR44-α-SYN interaction as a promising therapeutic target for reducing α-SYN pathology and enabling early intervention in PD.

Abstract: Transcription factor nucleocytoplasmic dynamics play a key role in developmental gene regulation. In Dictyostelium , the transcription factor GtaC exhibits nucleocytoplasmic shuttling, but its shuttling trajectory across multicellular aggregation has not been systematically quantified. Using a knock-in strain, we tracked GtaC dynamics from starvation through aggregation and quantified developmental changes in shuttling period, amplitude, and synchrony. Notably, brief-pulse optogenetic activation of cAMP at higher input frequencies reproduced the reported attenuation of GtaC shuttling amplitude with high temporal precision and minimal phototoxicity. Together, long-term quantification and brief-pulse optogenetic cAMP perturbation show that GtaC nucleocytoplasmic shuttling is developmentally tuned in a frequency-dependent manner.

Abstract: Reprogramming tumor-associated macrophages (TAMs) from the pro-tumoral M2-like state to the immunostimulatory M1-like phenotype has emerged as a promising strategy for tumor therapy. However, most M2-like TAMs are preferentially located in hypoxic regions of the tumor, which are poorly accessible to many advanced drug delivery systems, posing a significant challenge to effective TAM reprogramming. Here, leveraging the tropism of facultative anaerobic bacteria to localize and propagate in the hypoxic tumor, an optogenetically engineered Escherichia coli Nissle 1917 strain conjugated with murine STING agonist (EcNflaB@UPD) was developed for cancer-specific immunotherapy. Upon near-infrared light illumination, the blue and UV emissions from upconversion nanoparticles (UCNPs) simultaneously activate the expression of Toll-like receptor (TLR) agonist, flaB, from EcNflaB, and the release of photocaged murine STING agonist, DMXAA, respectively. This spatiotemporally synchronized dual release ensures co-localized STING and TLR5 agonists inside the hypoxic niche, repolarizing TAMs from the M2 to the M1 phenotype via synergistic TLR5-MAPK1-NF-κB and STING-NF-κB signaling. The polarization of TAMs enhances their antigen-presenting capacity and, more importantly, activates the cytotoxic, stem-like and memory CD8+ T cells responses. This subsequently inhibits tumor growth, relapse, and metastasis in the murine 4T1 tumor model. Collectively, our work introduces the bacteria-based system that uses near-infrared light to dual-release immunotherapeutics for systemic anti-tumor immunity, opening new avenues for precise and effective cancer immunotherapy.

Abstract: The mammalian kidney relies on a branched network of collecting ducts for fluid transport and homeostasis. Replicating this network in vitro would parallelize function in synthetic replacement kidneys, yet current organoids have limited branching capacity. Here, we establish a developmentally-informed strategy to control organoid budding through optogenetic control of a receptor tyrosine kinase, RET. We first show pharmacological manipulation of RET signaling controls the extent of branching in mouse embryonic kidneys and human stem cell-derived kidney organoids. Next, we develop an optogenetic RET receptor (optoRET) that signals in a ligand-independent manner via blue light-mediated clustering. Epithelial cells expressing optoRET reproduce stereotyped RET signaling, scattering, and symmetry breaking in response to blue light. Human kidney organoids undergo budding with controllable orientation in response to spatially patterned optoRET stimulation. Our results establish ligand-free optogenetic control of branching and inspire new synthetic biology strategies for epithelial organoid design.

Abstract: The integrated stress response (ISR) is a highly conserved signaling network, allowing cells to adapt and respond to various stressors. With its aggressive spread and high recurrence rates, glioblastoma multiforme (GBM) is one of the toughest cancers to date, yet the role of the ISR is still to be well understood, whether activation may suppress or promote this disease, and drug-treatment of GBM has thus far shown inconclusive results. In this work, we use an optogenetic tool, opto-PKR, to specifically trigger ISR activation via light-induced oligomerizing PKR-kinases, offering high spatiotemporal and reversible control, while avoiding potential upstream damage or side effects from drugs. Using immunofluorescence and RNA-sequencing, we show that targeted ISR activation reaching levels where both adaptive (ATF4) and terminal responses (CHOP) are activated results in subsequent downregulation of genes associated with the extracellular environment and glial cell migration, further supported by ECM-stain and scratch assays. Next, we show inhibition of aggressive spread for ISR-activated GBM spheroids in collagen 3D culture. Photopatterning of ISR activation in spheroids demonstrates a cell-intrinsic effect at the tissue scale, and recovery studies indicate a tunable, non-ablative intervention space. These findings suggest a route to containment and motivate ISR-activating small molecule screening in GBM models.

Abstract: Precise and rapid modeling of aging remains a significant challenge. This review explores the potential of emerging light-based technologies-including direct light exposure, photodynamic therapy (PDT), and optogenetics-as accelerated, mechanistically targeted models for interrogating aging processes. Unlike conventional methods that rely on chronologically aged organisms or non-specific stressors, these light-driven approaches can induce specific hallmark aging phenotypes within days. This offers unprecedented temporal and spatial control, allowing researchers to precisely trigger and define molecular pathways, such as localized oxidative bursts or programmed protein interactions. Such capabilities provide a powerful platform for testing causal hypotheses about aging, especially organ-specific aging. The review also outlines a framework connecting each light modality to aging, including cellular senescence, telomere attrition, proteostasis loss, and epigenetic drift. It discusses a mechanistic matrix linking each light modality to specific molecular targets and age-associated outcomes. Light-based platforms, when combined with genetic and pharmacological tools, are poised to accelerate discovery in aging by enabling high-throughput and organelle-specific perturbation of aging biology. These approaches could ultimately redefine how we experimentally dissect and potentially modulate the aging process either by rapidly recapitulating aging features for study or by revealing leverage points to slow aging.

Abstract: Directed migration and rapid process extension-retraction allow microglia to continuously survey the brain and efficiently identify and phagocytose apoptotic neurons. Defining how cytoskeletal regulators coordinate these behaviors could guide strategies for targeted modulation of microglial activity. To this aim, using in vivo imaging in zebrafish, we identified a mechanistic framework in which myosin II-dependent contractility governs transitions between surveillance, migratory, and phagocytic states. Building on this, we engineered an optogenetic RhoA actuator (opto-ArhGEF25) that enables reversible, spatiotemporally precise manipulation of microglial behavior in vivo. We show that patterned RhoA activation can modulate process dynamics and induce front-rear polarity that drives rapid repulsive migration away from the applied light source, overriding injury-evoked cues and preventing migration toward lesions. Together, these results establish optogenetic control of the cytoskeleton as a powerful approach to probe and ultimately modulate microglial function in the living brain.

Abstract: The cytoskeleton is a dynamic intracellular network that governs cell shape, migration, division, and mechanotransduction. Precise spatiotemporal control of cytoskeletal regulation is essential for understanding how these processes are coordinated in physiology and disease, yet conventional pharmacological and genetic approaches often lack sufficient resolution or reversibility. Optogenetic technologies provide a powerful alternative by enabling light-controlled, noninvasive manipulation of cytoskeletal regulators with high temporal precision and subcellular specificity. This review summarizes recent advances in genetically encoded optogenetic tools for interrogating cytoskeletal dynamics. We discuss core design strategies, including allosteric regulation, light-induced oligomerization, heterodimerization, and dissociation, and highlight representative applications targeting actin filaments, microtubules, and upstream signaling pathways such as Rho family GTPases. We conclude by outlining current limitations and emerging directions, including improved tissue penetration, reduced phototoxicity, and multiplexed optical control, which are expected to further expand the utility of optogenetics in cytoskeleton research.

Abstract: The regulation of the actin cytoskeleton is key to controlling cell shape and structure. While the Rho GTPase RhoA is well known to regulate the actomyosin cytoskeleton, its function in controlling the septin cytoskeleton remains unclear. As RhoA interactions can vary in both time and space, they can be challenging to discern from traditional bulk biochemical assays. Here, we use multiple optogenetic tools to spatially and temporally increase myosin localization, stimulate contractile force, and activate RhoA to investigate how RhoA and its downstream effector myosin impact the septin cytoskeleton. We find that neither local accumulation of myosin nor increased activity of myosin is sufficient to alter septin architecture. Local activation of RhoA, however, results in a local increase in septin accumulation. Importantly, this septin increase is independent of the scaffolding protein anillin, which can directly bind both septin and RhoA. Together, these data expand the potential role of septins in mediating RhoA signaling by stimulating the remodeling of the septin cytoskeleton.

Abstract: Light-inducible heterodimerization systems offer precise, reversible control of protein interactions in living cells. Leveraging the high tissue-penetration of red/far-red light, the MagRed system, composed of a bacteriophytochrome Deinococcus radiodurans BphP (DrBphP) and its engineered affibody binder Aff6, achieves robust photoswitchable dimerization. This makes MagRed well-suited for in vivo and deep-tissue optogenetic application. However, the structural mechanism underlying Aff6's photo-state-specific recognition of DrBphP remains elusive. Here, we combine solution NMR spectroscopy, surface plasmon resonance (SPR), molecular docking and mutational analysis to elucidate the light-dependent interaction between a monomeric photosensory core module of DrBphP (DrBphP-PCMmono) and Aff6. We show that DrBphP-PCMmono alone is sufficient for light-inducible heterodimerization with Aff6, exhibiting a ∼ 23-fold affinity difference between the Pfr and Pr states. NMR titration reveals that Aff6 binds primarily to the PHY domain and the C-terminal region of the helical spine. Furthermore, docking and mutagenesis identify a key aromatic interaction (involving F327/H334 of DrBphP and F18 of Aff6) as the molecular basis for this conformational selectivity. Additionally, Aff6 binding stabilizes the Pfr state and retards the Pfr-to-Pr reversion of DrBphP-PCMmono. These findings not only provide critical structural insight into MagRed function but also establish a foundation for rationally engineering next-generation phytochrome-based optogenetic tools.

Abstract: Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise induction of microtubule acetylation within minutes in live cells. Implementing optoTAT in migration assays, we observed striking and rapid responses at both molecular and cellular levels. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation, enabling a dynamic crosstalk between the actin and microtubule cytoskeletal networks in the coordination of cellular motility.

Abstract: Calcium (Ca²+) release from intracellular stores, Ca²+ entry across the plasma membrane and their coordination via store-operated Ca²+ entry (SOCE) are critical for receptor-activated Ca²+ oscillations. However, the precise mechanism of Ca²+ oscillations and whether their control loop resides at the plasma membrane or intracellularly remains unresolved. By examining the dynamics of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER)-localized Ca²+ sensor that activates the Orai1 channel on the plasma membrane for SOCE, in mast cells, we found that a significant proportion of cells exhibited STIM1 oscillations with the same periodicity as Ca²+ oscillations. These cortical oscillations, shared with ER-plasma membrane (ER-PM) contact site proteins, were only detectable using total internal reflection fluorescence microscopy. Notably, STIM1 oscillations could occur independently of Ca²+ oscillations. Simultaneous imaging of cytoplasmic Ca²+ and ER Ca²+ with CEPIA1er revealed that receptor activation does not deplete ER Ca²+, whereas receptor activation without extracellular Ca²+ influx induces cyclic ER Ca²+ depletion. However, under such non-physiological conditions, cyclic ER Ca²+ oscillations lead to sustained STIM1 recruitment, indicating that oscillatory Ca²+ release is neither necessary nor sufficient for STIM1 oscillations. Using optogenetic tools to manipulate ER-PM contact site dynamics, we found that persistent ER-PM contact sites reduced the amplitude of Ca²+ oscillations without alteration of oscillation frequency. Together, these findings suggest an active cortical mechanism governs the rapid dissociation of ER-PM contact sites, thereby controlling amplitude of oscillatory Ca²+ dynamics during receptor-induced Ca²+ oscillations.

Abstract: Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Tailoring their properties enables their full exploitation for optogenetic regulation in basic research and applied bioengineering applications. Here, we present a simple, yet powerful strategy based on random mutagenesis coupled to high-throughput screening that allowed altering the most fundamental properties of the widely used nMag/pMag photodimerization system: its light sensitivity and activation. Variants were characterized in vivo in bacteria by flow cytometry and during the entire growth curve by spectrofluorometry. We identify mutations that either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the light activation and dark-to-light fold change. Notably, light sensitivity and activation levels could be changed independently. In addition, we demonstrated that the shapes of the dose-response curves can be finely tuned. This broadens the applicability of the Magnets photosensors for optogenetic regulation strategies.

Abstract: Inflammatory bowel disease demands spatiotemporally precise drug delivery, yet the variable gut redox environment limits stimuli-responsive nanocarriers. Here we report a living biohybrid platform in which optogenetically engineered Shewanella oneidensis MR-1 is electrostatically conjugated with azo-bond covalent organic frameworks (TA-COFs) loaded with anti-inflammatory drugs magnolol or 4-iodobenzoic acid. Under intestinal conditions and non-invasive red-light irradiation (660 nm), light-induced restoration of the metal-reducing pathway promotes extracellular electron transfer, thereby cleaving azo bonds in the COF. This triggers rapid structural disassembly and a 2.8-fold increase in drug release. Although wild-type Shewanella is thermally inactivated at 37 °C and cannot utilize abundant colonic acetate, expression of heat-shock genes (groES/thiF) and an acetate-to-TCA pathway (ato1/ato2/gltA) confers 37 °C tolerance and robust metabolism in the gut. In DSS-induced colitis mice, oral administration of the biohybrid significantly alleviates inflammation, restores epithelial barrier integrity, rebalances gut microbiota (enrichment of Akkermansia, Muribaculaceae, and Lachnospiraceae). This work presents a generalizable strategy for constructing electroactive living composites by integrating microbial electron generation with stimuli-responsive nanomaterials, offering a new paradigm for light-programmed smart therapeutics and programmable living materials in biomedical applications.

Abstract: Populations of cells regulate gene expression in response to external signals, but their ability to make reliable collective decisions is limited by both intrinsic noise in molecular signaling and variability between individual cells. In this work, we use optogenetic control of the canonical Wnt pathway as an example to study how reliably information about an external signal is transmitted to a population of cells, and determine an optimal encoding strategy to maximize information transmission from Wnt signals to gene expression. We find that it is possible to reach an information capacity beyond 1 bit only through an appropriate, discrete encoding of signals: using no Wnt, a short Wnt pulse, or a sustained Wnt signal. By averaging over an increasing number of outputs, we systematically vary the effective noise in the pathway. As the effective noise decreases, the optimal encoding comprises more discrete input signals. These signals do not need to be fine-tuned to achieve near-optimal information transmission. The optimal code transitions into a continuous code in the small-noise limit, which can be shown to be consistent with the Jeffreys prior. We visualize the performance of different signal encodings using decoding maps. Our results suggest that optogenetic Wnt signaling allows for regulatory control beyond a simple binary switch and provide a framework to apply ideas from information processing to single-cell in vitro experiments.

Abstract: Mechanoreceptors can be motile and actively amplify their mechanical input.1,2,3,4 We here found that the responses of mechanoreceptor cells can also be shaped actively by contractile supporting cells. Drosophila larvae monitor body movements with pentascolopidial chordotonal (lch5) organs that are stretched out between cuticular attachment sites.5,6,7,8 These proprioceptive organs contain five stretch-receptor neurons each that receive mechanical stimuli from supporting cap cells. The elastic cap cells are surrounded by extracellular matrix and contain actin cables and non-muscle myosin II motors, suggesting that the cells might be motile.9,10 We show that the supporting cap cells are pre-strained at rest to about twice their relaxed length, and that the force they transmit is modulated by myosin II in the cap cells. Cap cells contracted upon optogenetic activation of myosin II. Cap cell-specific knockdown of the regulatory light chain of myosin II relieved tension and converted the spiking responses of the stretch receptors from phasic to more tonic, impairing adaptation to sustained stimuli. Our findings thus illustrate that mechanoreceptor responses can be actively tailored by contractile neighboring cells.

Abstract: Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.

Abstract: Recent advances in experimental model systems have improved our ability to study cardiovascular development, function, and disease with high spatial and temporal resolution. The zebrafish (Danio rerio) has emerged as a powerful vertebrate model for cardiovascular research due to its transparency, genetic tractability, and conserved cardiac physiology, similar to humans. These features allow real-time in vivo imaging, the functional assessment of cardiac performance, and the tracking of signaling pathways that are fundamental in cardiovascular development and disease. Recent advances in nanotechnology and optogenetics have introduced complementary tools for probing and manipulating cardiovascular systems with high spatial and temporal precision. Nanoparticle-based platforms enable the tunable delivery of drugs, nucleic acids, and imaging agents, while optogenetic systems allow the light-mediated control of gene expression, signaling pathways, and cardiac electrophysiology. In this review, we summarize recent progress in the application of nanoparticle-based technologies and the emerging optogenetic tools in zebrafish cardiovascular research, including the optical control of cardiac signaling and electrophysiology. We briefly discuss emerging complementary efforts toward nanoparticle and optogenetic approaches, how to overcome key technical limitations, such as light penetration and gene delivery, and how to facilitate the development of fully optical platforms for cardiovascular disease modeling and drug screening.

Abstract: Evolving dynamic, multi-state, and computational protein functionalities is challenging because it requires selection pressure on all the states of a protein of interest (POI) and the transitions between them. To create a continuous directed evolution paradigm for such properties, we genetically engineered budding yeast for optogenetic input to switch a POI "on" and "off," which, in turn, controls a Cdk1 cyclin that is essential for one cell-cycle stage but detrimental for another. The method, "optovolution," generates dynamic selection pressure on POI cycling at the timescale of tens of minutes. We used it to evolve 19 new variants of the LOV transcription factor El222, including in vivo green-light-responsive variants allowing LOV color-multiplexing. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing phycocyanobilin (PCB) unnecessary. Finally, we demonstrated the generality of the method by evolving a non-light-responsive AND gate (PEST-rtTA). Optovolution makes difficult-to-engineer protein functionalities continuously evolvable.

Abstract: As an interdisciplinary frontier integrating optical technologies and genetic principles, optogenetics enables precise spatiotemporal control of gene expression and neuronal activity via light-sensitive molecular assemblies, thereby driving transformative advancements in biomedical fields. Red/far-red light optogenetic tools, by virtue of the advantages of long wavelengths, have emerged as powerful platforms for deep-tissue manipulations for both basic researches and clinical applications. Although a number of in-depth studies on various red/far-red light optogenetic tools and their biomedical applications have been published, there has not yet been a comprehensive review that systematically summarizes the advancements of diverse researches on this type of optogenetics. This article systematically delineates the technology of red/far-red light optogenetics, focusing on the molecular mechanisms and biomedical applications of two core photoreceptor protein families: phytochromes and channelrhodopsins. Phytochromes distributed in plants, bacteria and fungi undergo reversible red/far-red light-driven conformational conversion, initiating downstream signaling cascades that support various optogenetic technologies. Channelrhodopsins, originally microalgal blue-light-gated cation channels, are engineered into red-shifted variants, enabling rapid and non-invasive red/far-red light-controlled neuronal excitability manipulation at precise spatiotemporal resolution. The representative case studies of applications of phytochromes-based optogenetic tools in gene editing, transcriptional regulation, light-gated drug delivery and deep tissue imaging and diagnosis; as well as applications of red-shifted channelrhodopsins-based optogenetic tools in spatiotemporally precise neuromodulation are discussed in detail. Moreover, the main technical challenges in the utilization of red/far-red light optogenetic tools are analyzed. With continuous advancements of wavelength-optimized actuators and closed-loop control architectures, red/far-red light optogenetic techniques are poised to drive multidisciplinary convergence, offering unprecedented tools for decoding cellular dynamics and accelerating therapeutic discoveries.

Abstract: Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways to deliver light into cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a genetically encoded optogenetic circuit for the control of gene expression. Furthermore, we utilized a magneto-sensitive protein to engineer a split-protein version of this luminescent enzyme, where its reconstitution is driven by a 50 mT magnetic stimulus. Thus, resulting in a gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.

Abstract: The bacterial flagellar motor drives bacterial swimming and chemotaxis by rotating helical flagellar filaments. When Escherichia coli navigates chemical gradients, the motor switches from counterclockwise (CCW) during forward swimming to clockwise (CW) during direction-changing tumbles. The motor responds indirectly to extracellular chemosensory input to membrane-bound chemoreceptors using an intervening intracellular signaling pathway. How the motor responds to its direct input signal-the diffusible messenger phosphorylated CheY (CheY-P)-remains poorly understood. Steady-state motor measurements have been modeled as an allosteric switch between CCW/CW states that depends on mean CheY-P levels. Allosteric models have suggested that as many as 20 CheY-P molecules can be bound to the motor when it switches rotational direction. But steady-state models cannot predict the sensitivity of the motor to dynamic changes in CheY-P that essentially modulate chemotactic behavior. We present an optogenetic reagent that precisely controls the direct dynamical input signal to the motor. We designed a "caged" molecule, Opto-CheY, that is transiently activated by photon absorption. We find that activation and binding of one to three additional CheY-P molecules is sufficient to switch the motor from the CCW to CW state. The sensitivity of the motor to small changes in CheY-P occupancy helps resolve a long-standing paradox about the high sensitivity of the chemotactic response to external sensory input. Optogenetic biochemistry by light-activated uncaging of signal molecules is a new strategy to dissect information-processing in the living cell.

Abstract: Light is a pivotal environmental signal regulating diverse plant developmental and physiological processes, including seed germination, hypocotyl elongation, phototropism, metabolite biosynthesis, stress resistance, temperature response, and circadian rhythms. Multiple signal transduction pathways of ultraviolet, blue light, and red/far-red light as well as related protein interaction networks in plants have been identified. Deciphering the mechanisms of light perception and signal transduction is of great significance to crop breeding and optogenetic manipulation. Structural biology has profoundly advanced the studies of light signal transduction by elucidating high-resolution three-dimensional (3D) structures of photoreceptors and their downstream signaling components. These studies uncover the molecular basis underlying perception and transduction of different light signals by plants. This review summarizes key structural findings of plant light signal transduction, highlighting the architectures and molecular functions of photoreceptors and associated signaling factors. We also outline the mechanisms underlying photoreceptor activation, inhibition, and regulatory interactions within light signaling networks and discuss the challenges in this field.

Abstract: In plants, the regulation of transgene transcription is typically achieved using chemical agents. A safe alternative to chemically induced systems may be optogenetic systems. The BphP1-QPAS1 system has distinct advantages over other optogenetic systems, as it is activated by near-infrared (NIR, 780 nm) light, which is beyond the spectrum of plant photoreceptors. This system is based on the use of a split transcription factor (TF), consisting of the DNA-binding and dimerization domains of the yeast TF Gal4, fused to the QPAS1 component, along with the transactivation domain VP16 fused to BphP1. Under NIR light, BphP1 interacts with QPAS1, leading to the formation of the functional TF Gal4-VP16. A primary obstacle to using optogenetic systems in plants is their undesired activation under white light, which is vital for normal plant growth. A potential solution to this issue is temporarily removing one component of the split TF from the nucleus under white light. We modified the BphP1-QPAS1 system to activate reporter gene expression in Nicotiana benthamiana leaves using NIR light. We combined BphP1-QPAS1 with several variants of LOV domain-containing proteins activated by blue light (460-480 nm). The best results were achieved by combining the BphP1-QPAS1 system with the AsLOV2 domain, which carries the degron sequence RRRG at the C-terminal Jα helix and initiates the degradation of the chimeric protein NES-Gal4-QPAS1-AsLOV2-RRRG under white light. This modification induced the BphP1-QPAS1 system in tobacco leaves only under NIR light, but not in the dark or under white light. We believe that, in the future, the BphP1-QPAS1 system could be applied to enhance plant resistance to adverse environmental conditions, pests, and viral diseases.

Abstract: An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.