Curated Optogenetic Publication Database

Abstract: Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions.

Abstract: Optogenetics combines optics and genetics to enable non-invasive interrogation of cell physiology at an unprecedented high spatiotemporal resolution. Here, we introduce Opto-CRAC as a set of genetically-encoded calcium actuators (GECAs) engineered from the calcium release-activated calcium (CRAC) channel, which has been tailored for optical control of calcium entry and calcium-dependent physiological responses in non-excitable cells and tissues. We describe a detailed protocol for applying Opto-CRAC as an optogenetic tool to achieve photo-tunable control over intracellular calcium signals and calcium-dependent gene expression in mammalian cells.

Abstract: The deuterosome is a non-membranous organelle involved in large-scale centriole amplification during multiciliogenesis. Deuterosomes are specifically assembled during the process of multiciliogenesis. However, the molecular mechanisms underlying deuterosome formation are poorly understood. In this study, we investigated the molecular properties of deuterosome protein 1 (Deup1), an essential protein involved in deuterosome assembly. We found that Deup1 has the ability to self-assemble into macromolecular condensates both in vitro and in cells. The Deup1-containing structures formed in multiciliogenesis and the Deup1 condensates self-assembled in vitro showed low turnover of Deup1, suggesting that Deup1 forms highly stable structures. Our biochemical analyses revealed that an increase of the concentration of Deup1 and a crowded molecular environment both facilitate Deup1 self-assembly. The self-assembly of Deup1 relies on its N-terminal region, which contains multiple coiled coil domains. Using an optogenetic approach, we demonstrated that self-assembly and the C-terminal half of Deup1 were sufficient to spatially compartmentalize centrosomal protein 152 (Cep152) and polo like kinase 4 (Plk4), master components for centriole biogenesis, in the cytoplasm. Collectively, the present data suggest that Deup1 forms the structural core of the deuterosome through self-assembly into stable macromolecular condensates.This article has an associated First Person interview with the first author of the paper.

Abstract: Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein-protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light-activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light-tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Genetic engineering strategy for the generation of modular light-activated protein dimerization units Support Protocol 1: Molecular cloning Basic Protocol 2: Cell culture and transfection Support Protocol 2: Production of dark containers for optogenetic samples Basic Protocol 3: Confocal microscopy and light-dependent activation of the dimerization system Alternate Protocol 1: Protein recruitment to intracellular compartments Alternate Protocol 2: Induction of organelles' membrane tethering Alternate Protocol 3: Optogenetic reconstitution of protein function Basic Protocol 4: Image analysis Support Protocol 3: Analysis of apparent on- and off-kinetics Support Protocol 4: Analysis of changes in organelle overlap over time.

Abstract: Vascular endothelial growth factor (VEGF) is the key regulator in neovascular lesions. The anti-VEGF injection is a major way to relieve retinal neovascularization and treat these diseases. However, current anti-VEGF therapeutics show significant drawbacks. The reason is the inability to effectively control its therapeutic effect. Therefore, how to controllably inhibit the VEGF target is a key point for preventing angiogenesis. Here, a CRISPR-dCas9 optogenetic nanosystem was designed for the precise regulation of pathologic neovascularization. This system is composed of a light-controlled regulatory component and transcription inhibition component. They work together to controllably and effectively inhibit the target gene's VEGF. The opto-CRISPR nanosystem achieved precise regulation according to individual differences, whereby the expression and interaction of gene was activated by light. The following representative model laser-induced choroid neovascularization and oxygen-induced retinopathy were taken as examples to verify the effect of this nanosystem. The results showed that the opto-CRISPR nanosystem was more efficacious in the light control group (NV area effectively reduced by 41.54%) than in the dark control group without light treatment. This strategy for the CRISPR-optogenetic gene nanosystem led to the development of approaches for treating severe eye diseases. Besides, any target gene of interest can be designed by merely replacing the guide RNA sequences in this system, which provided a method for light-controlled gene transcriptional repression.

Abstract: The myocardin‐related transcription factor A (MRTF‐A) controls the transcriptional activity of the serum response factor (SRF) in a tightly controlled actin‐dependent manner. In turn, MRTF‐A is crucial for many actin‐dependent processes including adhesion, migration, and contractility and has emerged as novel targets for anti‐tumor strategies. MRTF‐A rapidly shuttles between cytoplasmic and nuclear compartment via dynamic actin interactions within its N‐terminal RPEL domain. Here, optogenetics is used to spatiotemporally control MRTF‐A nuclear localization by blue light using the light‐oxygen‐voltage‐sensing domain 2‐domain based system LEXY (light‐inducible nuclear export system). It is found that light‐regulated nuclear export of MRTF‐A occurs within 10–20 min. Importantly, MRTF‐A‐LEXY shuttling is independent of perturbations of actin dynamics. Furthermore, light‐regulation of MRTF‐A‐LEXY is reversible and repeatable for several cycles of illumination and its subcellular localization correlates with SRF transcriptional activity. As a consequence, optogenetic control of MRTF‐A subcellular localization determines subsequent cytoskeletal dynamics such as non‐apoptotic plasma membrane blebbing as well as invasive tumor‐cell migration through 3D collagen matrix. This data demonstrate robust optogenetic regulation of MRTF as a powerful tool to control SRF‐dependent transcription as well as cell motile behavior.

Abstract: As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. Herein, a generally applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody, respectively, is introduced. These engineered antibody-like modular domains enable rapid and reversible antibody-antigen recognition by utilizing light. By the paralleled insertion of two light-oxygen-voltage domain 2 modules into a single sunbody and the use of bivalent sunbodies, the range of dynamic changes of photoswitchable sunbodies is substantially enhanced. Furthermore, the use of moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base editing by harnessing the power of light is demonstrated.

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: During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

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: 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: 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: Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28oC to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

Abstract: Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

Abstract: The investigation and manipulation of cellular processes with subcellular resolution requires non-invasive tools with spatiotemporal precision and reversibility. Building on the interaction of the photoreceptor PAL with an RNA aptamer, we describe a variation of the CRISPR/dCAS9 system for light-controlled activation of gene expression. This platform significantly reduces the coding space required for genetic manipulation and provides a strong on-switch with almost no residual activity in the dark. It adds to the current set of modular building blocks for synthetic biological circuit design and is broadly applicable.

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: Optogenetics uses light to manipulate protein localization or activity from subcellular to supra-cellular level with unprecedented spatiotemporal resolution. We used it to control the activity of the Cdc42 Rho GTPase, a major regulator of actin polymerization and cell polarity. In this chapter, we describe how to trigger and guide cell migration using optogenetics as a way to mimic EMT in an artificial yet highly controllable fashion.

Abstract: Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools. Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool.

Abstract: RNA has important and diverse biological roles, but the molecular methods to manipulate it spatiotemporally are limited. Here, an engineered photoactivatable RNA N6 -methyladenosine (m6 A) editing system with CRISPR-Cas13 is designed to direct specific m6 A editing. Light-inducible heterodimerizing proteins CIBN and CRY2PHR are fused to catalytically inactive PguCas13 (dCas13) and m6 A effectors, respectively. This system, referred to as PAMEC, enables the spatiotemporal control of m6 A editing in response to blue light. Further optimization of this system to create a highly efficient version, known as PAMECR , allows the manipulation of multiple genes robustly and simultaneously. When coupled with an upconversion nanoparticle film, the optogenetic operation window is extended from the visible range to tissue-penetrable near-infrared wavelengths, which offers an appealing avenue to remotely control RNA editing. These results show that PAMEC is a promising optogenetic platform for flexible and efficient targeting of RNA, with broad applicability for epitranscriptome engineering, imaging, and future therapeutic development.

Abstract: The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species (ROS), and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as amyloid β (Aβ) fibrils and Tau tangles in Alzheimer's disease (AD) are accessible only via invasive cerebrospinal fluid assays, and ROS can be fleeting and challenging to monitor in vivo. Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early-stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the AD brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the "CofActor," we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied, depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early-stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.

Abstract: During mitotic cell division, the actomyosin cytoskeleton undergoes several dynamic changes that play key roles in progression through mitosis. Although the regulators of cytokinetic ring formation and contraction are well established, proteins that regulate cortical stability during anaphase and telophase have been understudied. Here, we describe a role for CLIC4 in regulating actin and actin regulators at the cortex and cytokinetic cleavage furrow during cytokinesis. We first describe CLIC4 as a new component of the cytokinetic cleavage furrow that is required for successful completion of mitotic cell division. We also demonstrate that CLIC4 regulates the remodeling of the sub-plasma-membrane actomyosin network within the furrow by recruiting MST4 kinase (also known as STK26) and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in regulating cortex stiffness and blebbing during the late stages of cytokinetic furrowing.

Abstract: Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.

Abstract: Activation of Fas (CD95) is observed in various neurological disorders and can lead to both apoptosis and prosurvival outputs, yet how Fas signaling operates dynamically in the hippocampus is poorly understood. The optogenetic dissection of a signaling network can yield molecular-level explanations for cellular responses or fates, including the signaling dysfunctions seen in numerous diseases. Here, we developed an optogenetically activatable Fas that works in a physiologically plausible manner. Fas activation in immature neurons of the dentate gyrus triggered mammalian target of rapamycin (mTOR) activation and subsequent brain-derived neurotrophic factor secretion. Phosphorylation of extracellular signal-regulated kinase (Erk) in neural stem cells was induced under prolonged Fas activation. Repetitive activation of this signaling network yielded proliferation of neural stem cells and a transient increase in spatial working memory in mice. Our results demonstrate a novel Fas signaling network in the dentate gyrus and illuminate its consequences for adult neurogenesis and memory enhancement.