Curated Optogenetic Publication Database

Abstract: Memory persistence, the ability to retain information over time, is a fundamental feature of long-term memory. Although astrocytes contribute to synaptic plasticity, the molecular mechanisms by which they support memory persistence remain unclear. Here we show that astrocytic ankyrin-2 (Ank2) is required for memory persistence in adult mice. Astrocyte-specific deletion of Ank2 impaired remote memory without affecting recent memory and disrupted the maintenance of long-term potentiation. Loss of Ank2 reduced astrocyte contacts with engram neurons and impaired astrocyte morphogenesis driven by brain-derived neurotrophic factor (BDNF) signaling through the truncated tropomyosin receptor kinase B receptor (TrkB.T1) and inositol 1,4,5-trisphosphate receptor type 2 (IP3R2). Consistent with this mechanism, astrocytic Ank2 was required for the enhancement of memory persistence by hippocampal BDNF infusion. Furthermore, selective optogenetic activation of astrocytic TrkB.T1 signaling enhanced remote memory, demonstrating that astrocytic BDNF signaling is sufficient to promote memory persistence. These findings identify astrocytic Ank2 as a key regulator of long-term memory persistence.

Abstract: Cells under high confinement migrate efficiently in low-adhesion environments by forming stable, polarized, hydrostatic pressure-driven leader blebs. Here we investigated the basis of polarized bleb morphology in metastatic melanoma cells migrating under low-adhesive and highly confined microenvironments. Using high-resolution live imaging, molecular perturbations and biosensors, we show that EGF signalling through PI3K stabilizes and maintains polarized leader blebs. EGFR and PI3K activities form a gradient within leader blebs that decreases from rear to front, promoting phosphatidylinositol 3,4,5-trisphosphate and Rac1-GTP accumulation at the bleb rear, whereas phosphatidylinositol 4,5-bisphosphate and RhoA-GTP concentrate at the bleb tip, the inverse of the organization observed in integrin-dependent mesenchymal migration. Optogenetic disruption of this gradient triggers bleb retraction, underscoring its functional importance. Mathematical modelling and experiments identified a mechanism whereby during bleb initiation, CD44 and ERM proteins restrict EGFR mobility within a membrane-apposed cortical actin meshwork at the bleb rear, establishing the EGFR-PI3K-Rac gradient. Together, these findings define the biophysical and molecular mechanisms that underlie polarity in bleb-based migration and highlight how alternative spatial organization of signalling modules supports distinct migration modes in different microenvironments.

Abstract: Activation of the NLRP3 inflammasome by infectious or sterile insults culminates in pyroptosis, a lytic and highly inflammatory form of programmed cell death. A safeguarded two-step process tightly regulates pyroptosis: priming, which drives NF-κB signaling, followed by execution, ultimately leading to plasma membrane rupture. Linear (Met1-linked) ubiquitination, catalyzed by the E3 ligase complex LUBAC, was previously shown to participate in pyroptosis, but the underlying mechanisms are not fully understood. In this study, we show that Met1-linked ubiquitin chains can assemble during both priming and execution phases, independently of the inflammasome sensor NLRP3. Genetic deletion of the LUBAC enzymes or pharmacological inhibition impairs pyroptosis. Conversely, cell death is enhanced without the deubiquitinase OTULIN, which selectively removes linear ubiquitination. Finally, using an optogenetic model to bypass priming, we demonstrate that Met-1-linked ubiquitination is required for the execution phase of pyroptosis. These findings offer insights into the regulation of pyroptotic cell death by linear ubiquitination.

Abstract: In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are positioned at opposite poles remains unclear. We leverage optogenetics, mechanical perturbations, and mathematical modelling to reveal a surprising mechanochemical long-range mutual activation between front and back polarity programmes that complements their well-known local mutual inhibition. Rac-based protrusions elevate membrane tension, stimulating an mTORC2-dependent activation of Rho at the opposite side of the cell. Conversely, Rho-mediated contractility induces cortical-flow-based regulation of phosphoinositide signalling that triggers Rac activation distally. We develop a minimal mechanochemical model to explain how long-range facilitation, together with local inhibition, enables robust Rho and Rac partitioning. Our findings demonstrate how the actin cortex and plasma membrane interact as an integrated mechanochemical system for long-range Rac-Rho patterning. This circuit is required for efficient polarity and migration in primary human T cells and is conserved in epithelial cells, highlighting the generality of this mechanism.

Abstract: Glycosylation plays a pivotal role in regulating diverse biological processes. However, the lack of tools capable of controlling the spatiotemporal dynamics of glycosylation has largely hindered its functional elucidation. Here, we introduce an optogenetic approach that employs red/far-red light to dynamically and reversibly control the plasma membrane localization of O-linked N-acetylglucosamine transferase (OGT) in living systems. Red-light-induced translocation of OGT suppresses insulin signaling in both cells and mice. Glycoproteomic and phosphoproteomic analyses reveal a global impact of OGT-mediated glycosylation on signal transduction. Moreover, using protein semisynthesis, cell-based assays, and molecular dynamics simulations, we demonstrate that red-light-induced O-GlcNAcylation of WNK1 at S1949 inhibits downstream cell volume response signaling pathways by suppressing WNK1 biomolecular condensate formation. Together, our findings provide a valuable tool to modulate subcellular O-GlcNAcylation and control cellular signaling in living systems, with broad applicability to the study of glycosylation in cells.

Abstract: In canonical developmental patterning, the embryo is exposed to gradients of signaling activators that elicit different cellular responses depending on the activator's concentration. Recent optogenetic studies of terminal ERK signaling downstream of Torso receptor tyrosine kinase in the early Drosophila embryo reveal that even a brief, 5-minute ERK stimulus is sufficient to rescue the development of larval "tail" structures. Here, we reveal components of the molecular network that defines this sensitive developmental fate response. We find that low ERK doses produce sustained Abdominal-B ( Abd-B ) expression comparable to that of wild-type embryos. Abd-B expression is adjacent to, but non-overlapping with, two other transcriptional repressors: the ERK effector Tailless (Tll) and the gap gene Giant (Gt). Analysis of gene expression patterns in response to optogenetic perturbations suggests that the Tll-dependent repression of gt constitutes the sensitive ERK-responsive step: even low tll expression leads to potent repression of gt in nearby regions, with Abd-B expression arising in a stripe between the tll and gt domains. Our work suggests that the spectrum of phenotypes produced through optogenetic manipulation can be used to define how robust patterning can arise from low doses of inductive signals.

Abstract: The active regulation of tissue material properties via phase transitions is central in morphogenesis. Transitions occur abruptly at critical points in different control parameters, such as cell density, shape or adhesion. Whether these parameters are interdependent, and perform redundant or distinct functions, is unknown. Here we show that depending on the co-regulation of multiple control parameters, a tissue not only tunes its deformability but also its morphogenetic trajectory. We theoretically define a phase diagram capturing the material states of zebrafish pluripotent tissues undergoing epiboly-a tissue movement occurring during gastrulation-and show that they simultaneously cross critical points in cell density, connectivity and adhesion strength. We then combine optogenetics, biophysical measurements and quantitative morphometrics to independently modulate each parameter in vivo, and identify adhesion as the main determinant of tissue rheology. Further decoupling adhesion from density and inducing adhesion-driven rigidification in unjammed pluripotent tissues is sufficient to switch their morphogenetic program and trigger epithelial organization. This switch in tissue reorganization is achieved via tricellular junction formation, followed by lumenogenesis and the initiation of apical polarity. Our work reveals that the nonlinear dynamics of emergent tissue mechanics are mechanisms of tissue organization and morphogenesis.

Abstract: Mitochondrial stress activates the integrated stress response (ISR) and triggers cell-cell communication through the secretion of the metabokine growth differentiation factor 15 (GDF15). However, the gene network underlying the ISR remains poorly defined across metabolically diverse cellular states and tissues. Using RNAseq data from fibroblasts subjected to eleven metabolic perturbations, including genetic and pharmacological mitochondrial OxPhos defects, we show that the ISR has multiple arms. To quantify the GDF15 arm of ISR activation in human cells, we developed an ISRGDF15 index. We validate the ISRGDF15 index in datasets from optogenetic and small molecule activation of ISR kinases, demonstrating its rapid kinetics preceding to GDF15 gene expression. We then deploy the ISRGDF15 index across 44 postmortem human tissues, confirm its correlation with age, and report that the ISRGDF15 is upregulated in the heart of individuals with acute causes of death in the emergency room, whereas it was upregulated in the brain of individuals who died after protracted hospital inpatient stays. These data highlight distinct arms of the ISR and clarify genes related to the GDF15 ISR arm, yielding an ISRGDF15 index that can be used to investigate tissue-specific and age-related ISR activation in both in vitro cultures and human tissues.

Abstract: Optoswitches are of particular interest to the metabolic engineering community, as light has a superior advantage of tunability and reversibility. However, the light-shading effect at industrial scales remains an unsolved challenge. Here, we report optogenetic quorum-sensing (OptoQS) circuits to induce and maintain a sustained gene expression at the population level by transient light stimulation. In particular, we reprogram the pheromone-responsive G-protein coupled receptor (GPCR) signaling cascade in Saccharomyces cerevisiae to effectively record transient light inputs. Once the transient light input is recorded as a form of α-factor accumulation, the surrogate messenger can diffuse and transmit the signal across the cell population. Eventually, we successfully demonstrated the utility of the OptoQS circuit for metabolic regulation of 3-hydroxypropionate biosynthesis. Based on the promising results from OptoQS circuits, we envision that the flexibility of our design might be explored for the future fabrication of various genetic circuits to record other transient physical stimuli.

Abstract: Serotonin (5-HT) signaling is strictly controlled by the serotonin transporter (SERT). The present study aims to establish optogenetic approaches for the control of SERT localization and function by modulating the interaction between SERT and its regulatory protein, soluble guanylate cyclase (sGC). We generated several cell lines that stably express blue light-inducible optogenetic elements fused to sGC or the fourth internal loop (IL4) motif of SERT. Our results indicated that blue light-induced SERT-sGC interaction by heterodimerizing SsrA embedded in the membrane-associated improved light-induced dimer (iLID) and SspB-sGCβ1 decreased SERT localization in the plasma membrane, thus reducing the maximum transport velocity of SERT without affecting its Km for substrate. The light-induced subcellular redistribution of SERT was shown to be attributable to an interference of the SERT-sGC interaction with SERT trafficking but not PKC-mediated internalization. In addition, the light-induced SERT-sGC interaction was blocked by the IL4 peptide or a mutation in the IL4 motif. Furthermore, light-induced exposure of the IL4 motif in iLID decreased the SERT-sGC interaction by displacing SERT from the SERT-sGC complex, thus increasing SERT localization in the membrane and elevating its ability for substrate uptake. This study achieved light-inducible modulation of the protein-protein interaction that allows for the study of biochemical and cellular processes in live cells.

Abstract: Cell signaling regulates a wide range of biological processes including development, homeostasis, and disease. Accessible technologies to precisely manipulate signaling have important applications in basic and translational research. Here, we present an optogenetic toolkit for signaling manipulation in zebrafish embryos. We introduce a zebrafish-optimized optogenetic FGF signaling activator and a single-transcript Nodal signaling activator, and assess them together with a previously established BMP signaling activator. We thoroughly characterize this suite of tools and demonstrate light-dependent spatiotemporal control of signaling in vivo. In response to ∼455 nm (blue) light, zebrafish receptor kinase domains fused to blue light-dimerizing LOV domains enable robust signaling activation with minimal inadvertent activity in the dark or at wavelengths over 495 nm. Each optogenetic tool initiates pathway-specific signaling and activates known target genes. Signaling is activated with rapid on/off kinetics, and activation strength can be tuned by adjusting light irradiance. Finally, we demonstrate spatially localized signaling activation in vivo. Together, our results establish this optogenetic toolkit as a potent experimental platform and provide guidelines for rapid, direct, and adjustable activation of FGF, BMP, and Nodal signaling in zebrafish embryos.

Abstract: Dynamic metabolic engineering enables temporal redirection of microbial metabolism from biomass production to product synthesis. Here, we show that optogenetic control of protein kinase A (PKA) activity via light-regulated modulation of intracellular cyclic AMP (cAMP) levels can enhance heterologous production of β-carotene and cordycepin in Saccharomyces cerevisiae. To enable exclusive, glucose-independent control of cAMP synthesis, the photoactivatable adenylyl cyclase bPAC from Beggiatoa sp. was introduced into cells lacking the endogenous adenylyl cyclase Cyr1 or with lowered Cyr1 levels using an optogenetically controlled degron. Despite being growth-competent under illumination, the bPAC-containing yeast strain showed alterations in energy metabolism under all conditions. Quantitative proteome analysis using timsTOF mass spectrometry revealed profound changes in central carbon metabolism, sulfur homeostasis, energy charge, and ribosome biogenesis upon uncoupling cAMP from nutrient-dependent regulation, particularly under sustained light activation. These results highlight the critical role of dynamic Cyr1-dependent regulation for central metabolism, and underscore the biotechnological promise of refined PKA-targeted strategies for eukaryotic cell factories.IMPORTANCECarbon-footprint-minimized production of fine chemicals, pharmaceuticals, and biofuels requires optimized microbial cell factories with tailored metabolic performance. We employed optogenetic dynamic metabolic engineering in baker's yeast by uncoupling nutrient sensing from cAMP signaling using a light-controlled adenylate cyclase. Precise light regulation of intracellular cAMP levels and PKA activity enabled acute control of the metabolism, redirecting resources toward product synthesis, and boosting the production of valuable compounds such as β-carotene and cordycepin. Quantitative proteomics revealed that uncoupling of the cAMP-PKA axis from glucose sensing profoundly reprograms the central carbon metabolism and other key cellular processes. This approach provides a blueprint for refined, light-tunable strategies targeting the cAMP-PKA axis directly with light, e.g., for enhanced bioethanol production. Moreover, our data provide evidence for the profound influence of the cAMP-PKA axis on metabolism and balanced energy production that are fundamental for efficient production in microbial cell factories.

Abstract: During development, local mechanochemical cues within the cell microenvironment are translated into signalling pathways that drive cell fate decisions. Yet, as cells differentiate collectively, how global tissue-level properties shape these instructive cues remains unclear. Here we show that a tissue-scale rigidity transition guides patterning by tuning the length scales and timescales of morphogen signalling. By combining rigidity percolation theory, reaction-diffusion modelling, quantitative imaging and optogenetics in zebrafish, we uncover dynamical global tissue rigidity patterns that actively shape the Nodal morphogen gradient by locally changing its concentration and accelerating its signalling activity. In this self-generated mechanism, Nodal, besides instructing meso-endoderm fate specification, increases cell-cell adhesion strength via regulating planar cell polarity genes. Once the adhesion strength reaches a critical point, it triggers a rigidity transition which, in turn, induces the collapse of tissue porosity. The abrupt tissue reorganization negatively feeds back on Nodal signalling, impacting both its length scales, by restricting Nodal diffusivity, and its timescales, by speeding up the expression of its antagonist Lefty, thereby ensuring timely signal termination and robust patterning. Overall, we uncover a multiscale regulatory mechanism by which positional information and tissue material properties dynamically tune one another.

Abstract: The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoG’s effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.

Abstract: Store-operated Ca2+ release-activated Ca2+ (CRAC) channels, composed of STIM and ORAI, are essential for immune and developmental processes, and their dysregulation underlies channelopathies such as Stormorken syndrome. Here, we report the engineering of genetically encoded CRAC channel inhibitory binders (CRABs) derived from the ORAI C-terminal tail. Guided by deep mutational scanning, we optimize a membrane-anchored CRAB variant that potently inhibits Ca2+ influx and NFAT signaling, and rescues thrombocytopenia-like phenotypes in a zebrafish model of Stormorken syndrome. To enable tunable inhibition, we further design oligomeric, optogenetic (Opto-CRAB), and chemogenetic (Chemo-CRAB) variants, providing graded and real-time control of CRAC activity. Chemo-CRAB further suppresses Ca2+ signaling downstream of RTKs, GPCRs, and CAR-T cell activation, establishing broad applicability across physiological and synthetic contexts. Together, these programmable peptide-based inhibitors provide a versatile platform to dissect SOCE dynamics and hold promise as a therapeutic strategy against autoimmune, inflammatory, and neoplastic disorders driven by CRAC channel hyperactivity.

Abstract: Emerging evidence suggests that Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) bind to the cytoskeleton or focal adhesions (FAs), controlling spatio-temporal Rho GTPase activity through feedback mechanisms. We explore such feedback in the Rho-specific GAP Deleted in Liver Cancer 1 (DLC1), which binds to FAs through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to globally increased Rho activity and contractility in fibroblasts. Although Rho activity appears macroscopically steady, individual molecules undergo 'signaling flux'-a dynamic cycle of activation and deactivation. To measure this flux, we built a genetic circuit that enables both optogenetic activation of Rho and simultaneous readout of Rho activity. In cells at mechanical steady state, this reveals that DLC1 globally controls the rate of Rho deactivation, both at FAs and at the plasma membrane. Transient induction of local contractility, however, shows DLC1 associating with and dissociating from FAs during their reinforcement and relaxation, which might provide local positive feedback on Rho activity for robust FA disassembly. Together, our results indicate that DLC1 regulates Rho activity both globally at steady state and locally at FAs under tension, highlighting the complexity of spatio-temporal Rho GTPase signaling.

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: 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: 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: In vitro stem cell models of human gastrulation have been an advance for developmental biology, though elucidating mechanisms of germ layer formation remains challenging. While investigating whether spatially-patterned signaling is required for germ layer formation, we tested a "salt-and-pepper" signaling strategy in which WNT was optogenetically activated in a subset of human pluripotent stem cells (hPSC) uniformly mixed into an aggregate. Following mesendodermal specification, WNT-activated cells spatially segregated into a hemisphere, then underwent further differentiation and organization into mesoderm and endoderm. RNAseq-based lineage analysis revealed that WNT activation non-autonomously induced TGFβ/BMP signaling, leading to robust emergence of an anterior visceral endoderm-like population that patterned adjacent neural and mesendodermal fates. Transcriptional profiles and trajectories closely mirrored those observed during human gastrulation. Moreover, TGFβ or cadherin perturbation disrupted germ layer formation or spatial organization, respectively. This simple model thus enables mechanistic dissection of complex human lineage specifications and organization during gastrulation.

Abstract: Optogenetics holds great potential for diverse biological applications, including fundamental research, tissue engineering, and regenerative medicine, by enabling the precise spatial and temporal control of cellular signaling pathways. Transforming growth factor-beta (TGFβ), a multifunctional cytokine, is a critical regulator of cell proliferation, differentiation, and particularly chondrogenesis. Although TGFβ signaling is necessary for effective chondrogenic differentiation, previous studies have primarily relied on recombinant TGFβ ligand supplementation. In this study, we established an advanced optogenetic platform by knocking-in opto-TGFβ receptors in the AAVS1 locus of human embryonic stem cells (hESCs), enabling precise optogenetic activation of endogenous TGFβ signaling. Blue light illumination specifically activated TGFβ signaling, indicated by enhanced SMAD2 phosphorylation. Employing a three-dimensional pellet culture system, we demonstrated that direct optogenetic activation of TGFβ receptors, without exogenous ligand supplementation, is sufficient for robust chondrogenic differentiation of hESC-derived mesenchymal stem cells. The efficiency of optogenetic differentiation was comparable to conventional recombinant TGFβ protein treatment, evidenced by the expression of chondrogenic markers and deposition of cartilage-specific extracellular matrix components, including aggrecan and type II collagen. Our findings directly confirm the sufficiency and critical role of TGFβ receptor activation itself in chondrogenesis. Furthermore, this optogenetic approach provides a theoretical advantage by enabling noninvasive external modulation of TGFβ signaling post-transplantation, potentially facilitating further maturation and functional integration of transplanted chondrocytes. Thus, our results highlight a promising recombinant-protein-free strategy for use in cartilage tissue engineering and regenerative medicine.

Abstract: Estrogen receptor (ER)-mediated genomic actions are crucial for maintaining various physiological functions, and their dysfunction is associated with numerous human diseases. Traditional estrogen replacement therapy (ERT) is commonly used to manage estrogen deficiency-related conditions, such as vulvovaginal atrophy during menopause, but its systemic effects pose notable risks. This study introduces OptoER, an optogenetic tool engineered to precisely modulate ER-mediated genomic pathways through light-induced transcription regulation, offering spatial-temporal control over ER-dependent gene expression. Our in vitro studies demonstrate that OptoER significantly enhances ER-specific gene transcription and protein synthesis, leading to improved cell proliferation and migration. In a proof-of-principle study using ovariectomized (OVX) mice, OptoER demonstrated considerable therapeutic potential for vaginal atrophy, with observed improvement in epithelial thickness and keratinization. These findings suggest that OptoER provides a targeted therapeutic strategy for estrogen deficiency conditions, with significant implications for treating vaginal atrophy and promoting regenerative healing in estrogen-deprived tissues.