Curated Optogenetic Publication Database

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: Cytokinesis, the final step of cell division, relies on ingression of a precisely positioned actomyosin ring. Chromatin-associated Ran-GTP fine-tunes ring position, although the mechanism remains unclear. We hypothesize that depletion of Ran-GTP between segregating chromosomes leads to equatorial enrichment of importins, promoting recruitment of the scaffold protein anillin. However, the role of importins during anaphase is not known. Here, we tested whether importins form a gradient in response to chromatin-associated Ran-GTP and regulate ring assembly in two cultured human cell lines. We endogenously tagged importin-β1 with mNeonGreen in hypotriploid HeLa cells and euploid HCT 116 cells. Live-cell imaging revealed that importin-β1 becomes transiently enriched between segregating chromosomes in anaphase HeLa cells, but not in HCT 116 cells. Using a newly developed optogenetic tool to rapidly disrupt importin-β1 function, we found that importin-β1 is required for ring ingression in HeLa cells. We speculated that the stronger requirement for importin-β1 in HeLa cells reflects differences in chromatin-to-cytosol ratio compared with HCT 116 cells, which could determine whether the Ran-GTP gradient reaches the cortex. Consistently, FLIM-FRET imaging showed that equatorially enriched importin-β1 is Ran-free in HeLa cells, but not in HCT 116 cells. A predictive model of the Ran-free importin-β1 gradient identified factors that modulate gradient formation, including chromatin-to-cytosol ratio. Experimentally decreasing or increasing the chromatin-to-cytosol ratio in HeLa and HCT 116 cells, respectively, altered importin-β1 and anillin localization to resemble the other cell type. Our findings suggest that highly aneuploid cancer cells may depend on importin-mediated anillin recruitment, representing a targetable weakness. VIDEO ABSTRACT.

Abstract: Inducible CRISPR/Cas systems enable spatiotemporal control of genome editing in response to chemical, optical, biological, or physical stimuli. By restricting genome-editing activity to defined conditions, these systems may reduce off-target exposure and immune burden while improving tumor-selective control, making them attractive tools for precision oncology.

Abstract: Investigating astrocyte-neuron dynamics following ischemic stroke is essential for understanding post-stroke recovery mechanisms. However, current methodologies often fail to capture real-time interactions between neurons and astrocytes in animals executing specific behavioral tasks, limiting our ability to investigate the acute phase of stroke pathology. This protocol presents an integrated method that combines photothrombotic stroke modeling with simultaneous multichannel electrophysiology recording and fiber photometry in awake, behaving mice using a custom-fabricated optrode. The protocol includes focal ischemia induction via photothrombosis followed by simultaneous recording of neuronal spikes and astrocytic calcium transients. The optrode enables concurrent delivery of photothrombosis, calcium signal recording, and optogenetic manipulation without requiring separate surgical procedures. Representative results validate the success in simultaneous recording of astrocytic calcium signal and neuronal spiking. Optogenetic manipulation of astrocytes produces measurable changes in neuronal firing patterns (reduction in firing frequency of pyramidal neurons by 1.55 ± 0.45 Hz and interneuron by 3.64 ± 1.37 Hz compared to pre-optogenetic stimulation, n = 2), confirming that the system is capable of investigating astrocyte-neuron interactions. This integrated approach addresses critical gaps in stroke research methodology by providing real-time, multimodal recordings from the acute to chronic stage of stroke in behaving animals.

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: 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: Optogenetics which involves the use of light to control cell functions on a genetic level has found utility in studying cell physiology, biomaterials and metabolic engineering. S. cerevisiae is an industrially relevant model organism that is used in many applications, but due to the large number of genes required and issues relating to cross-activation between different colours, optogenetics for different wavelengths of light have not been multiplexed in S. cerevisiae. In this paper, we develop a compact red light responsive optogenetic system for S. cerevisiae that requires only a single gene and no exogenous cofactors. Through engineering modular protein domains, we reduce the cross-activation of our system by blue light. We integrate our red light optogenetic system with EL222 blue light optogenetics to establish dual channel optogenetics in S. cerevisiae and demonstrate its utility for engineering biology through the light-based control of flavonoid luteolin synthesis and flocculation for ease of product extraction. We also demonstrate our system's potential for the development of living materials by producing dual-coloured optogenetic patterns using S. cerevisiae. This work expands optogenetic applications in S. cerevisiae from single-light to multi-light systems, introducing the potential to multiplex different colours of light for dynamic, orthogonal control of separate cell processes.

Abstract: Neural stem/progenitor cells (NPCs) have primary cilia, which are critical organelles for Sonic hedgehog signaling. However, little is known about the components of primary cilia in NPCs and whether manipulating signaling in the cilia is sufficient to alter dorsal/ventral regional identity. Using a human telencephalic organoid model, we perform comprehensive proteomic profiling of NPC cilia and find enrichment in GTPase signaling. Deletion of the ciliary GTPase ARL13B reduces ciliary localization of GPR161, an orphan G protein-coupled receptor 161 that negatively regulates Sonic hedgehog, resulting in ventralization of NPCs. GPR161 deletion also induces ventralization. To investigate whether manipulation of ciliary signaling is sufficient to restore dorsal identity in this context, we optogenetically elevate ciliary cAMP, rescuing dorsal fate in GPR161 KO organoids. Furthermore, chemogenetic induction of GPR161 removal from cilia is sufficient to increase ventral NPCs. These data indicate that ciliary signaling functions as a critical switch regulating dorsal/ventral fate decisions.

Abstract: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic cancers but encounters challenges, including severe treatment-related toxicities, a highly suppressive tumor microenvironment (TME), limited long-term persistence, and poor trafficking/infiltration into solid tumors. This review outlines recent genetic engineering strategies to address these issues and enhance the safety, durability, and efficacy of CAR-T cell therapy. To reduce cytokine release syndrome and neurotoxicity, methods such as affinity-tuned and humanized scFvs, hinge/TM optimization, and ITAM calibration have been developed, along with programmable "switch-off" and "switch-on" systems that include suicide genes, antibody-bridging switches, and optogenetic or hypoxia-gated circuits. TME remodeling strategies utilize nanomaterials for targeted cytokine delivery, cell-surface "backpack" systems, and engineered oncolytic viruses that release cytokines or checkpoint-blocking agents. For durability and resistance to exhaustion, precise genome engineering techniques, including CRISPR-based editing and multiplexed shRNA platforms, were employed to target inhibitory receptors and exhaustion-driving transcriptional programs. Additionally, chemokine-receptor engineering and local biomaterial-based delivery systems are discussed as ways to enhance CAR-T trafficking and intratumoral persistence. These innovations collectively point toward integrated, patient-specific CAR-T platforms that incorporate safety controls, metabolic and transcriptional flexibility, and enhanced trafficking through the TME to broaden clinical use.

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: Sepsis is a comprehensive ailment of systemic inflammatory response syndrome arising from infection. Activation of CASPASE-1 plays a central role in initiating the inflammatory cascade during sepsis. Herein, we construct optogenetically engineered extracellular vesicles (EVs) that achieve the specific degradation of CASPASE-1 and inhibit sepsis-associated inflammation. Specifically, blue light (460 nm)-induced CRY2/CIBN heterodimerization was applied during the EVs production stage to selectively load GCE-CTM fusion proteins into EVs by EXPLORs technology, yielding EVsGCE-CTM loading efficiency compared to conventional methods. Upon systemic delivery, EVsGCE-C TM preferentially accumulated in macrophages, where the GCE domain selectively bound activated CASPASE-1. The CTM motif then facilitated its lysosomal degradation by chaperone-mediated autophagy, resulting in potent inhibition of CASPASE-1 activity. In a murine model of sepsis, treatment with EVsGCE-CTM effectively attenuated systemic inflammation, reduced multi-organ damage, and significantly improved survival outcomes. This approach enables highly efficient, ubiquitin-independent degradation of intracellular target proteins through macrophage-directed EVs delivery, offering a potential therapeutic approach to address sepsis and other inflammation-related diseases.

Abstract: Advances in optogenetics now allow specific modifications to the DNA of live cells with light. However, successfully using these technologies requires knowing their properties in terms of sensitivity, efficiency, kinetics and mechanism. We previously developed an optogenetic tool made of a single chimeric protein called LiCre that enables the induction of specific changes in the genome with blue light via DNA recombination between loxP sites ( Duplus-Bottin et al., 2021). Here, we used in vitro and in vivo experiments combined with kinetic modeling to provide a deeper characterization of the photoactivated LiCre-loxP recombination reaction. We find that LiCre binds DNA with high affinity in the absence of a light stimulus and that this binding is cooperative, although not as much as for the Cre recombinase from which LiCre was derived. In yeast, the addition of riboflavin to the culture medium had no effect on LiCre's efficiency, even when cells over-expressed riboflavin kinase, suggesting that the abundance of the flavin mononucleotide cofactor is not limiting for the reaction. However, LiCre's efficiency in yeast gradually increased when raising the temperature from 20°C to 37°C. The recombination kinetics observed in live cells are best explained by a model where the photoactivation of two or more DNA-bound LiCre units (happening in seconds) can produce (in several minutes) a functional recombination synapse. This model was able to capture the effect of a point mutation altering LiCre's light cycle. This deeper understanding of the LiCre-loxP system provides additional knowledge for designing experiments where specific genetic changes are induced in live cells with light.

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: Membrane contact sites (MCSs) are dynamic subcellular compartments formed between organelles that coordinate diverse aspects of cellular communication, including signaling, metabolism, and membrane organization. Tools capable of monitoring and controlling the spatially localized and dynamic properties of MCSs are needed to dissect their regulatory mechanisms and physiological roles. Recent advances in protein engineering have begun to address this need. Proximity-based reporters, chemogenetic approaches, and optogenetic systems have been developed to enable the visualization, interrogation, and manipulation of inter-organelle contacts with improved spatial and temporal precision. This minireview highlights key developments in these molecular toolkits and their representative applications in studying MCS biology. These approaches provide new insights into organelle crosstalk and may inform future therapeutic strategies targeting MCSs.

Abstract: Chromatin-modifying enzymes (CMEs) have traditionally been studied in their nuclear context for regulating gene expression. However, recent evidence points to the significant non-canonical functions that they perform in the cytoplasm, mitochondria, and plasma membrane, which can contribute to disease progression and alter cell phenotypes. This review surveys emerging engineering approaches to control protein localization, which could be applied to CMEs, particularly histone-modifying enzymes. Natural regulatory mechanisms include nuclear import/export signals and mechanical force-mediated translocation. Engineering strategies encompass diverse approaches: synthetic localization signals for directional transport, RNA editing systems like SNAP-ADAR, and small molecule platforms including bifunctional compounds, self-localizing ligands, and nanobody-mediated translocation. Optogenetic tools provide spatiotemporal control through light-inducible trapping, while inducible condensates enable reversible protein sequestration. Additional tools provide extra control via protease-based cleavage mechanisms and endogenous secondary messenger coupling. Despite significant advances in protein relocalization technologies, their application to CMEs remains largely unexplored, which would allow us to decode mechanisms of disease and develop targeted therapeutic interventions for those diseases. Future applications of these tools to CMEs will elucidate our understanding of epigenetic regulation and expand how we conceptualize CMEs.

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: Methylotrophic yeasts such as Pichia pastoris are widely used for heterologous protein production because they contain strong and tightly regulated promoters. However, the use of methanol as an inducer presents several practical challenges, including toxicity, flammability, high oxygen demand during fermentation, and increased production costs. To overcome these limitations, researchers have been working on redesigning the AOX1 regulatory system and developing alternative induction strategies that do not rely on methanol. One promising approach is optogenetics, which uses light to control gene expression in a non-invasive way. These systems rely on light-sensitive proteins such as phytochromes, cryptochromes, LOV-domain proteins, and UVR8, allowing gene activity to be regulated in a precise and reversible manner without adding chemical inducers to the culture medium. This review brings together key advances in yeast optogenetics, with a focus on the EL222 system, highlighting its implementation for light-controlled heterologous protein production in P. pastoris and its broad application in synthetic biology and metabolic engineering in Saccharomyces cerevisiae. The growing versatility and scalability of EL222-based circuits highlight their potential to reshape both fundamental research and industrial bioprocessing through safer, more controllable, and energy-efficient gene regulation strategies.

Abstract: Cre recombination is a widely used technique for mechanistic insights in physiology and disease. However, available constitutive and inducible Cre systems present challenges that can be prohibitive for some study designs. For example, Cre expression can result in cell types targeted across numerous tissues and organs, or when a gene is expressed across multiple cell types in a tissue, Cre-Lox restricted knockout will not enable ablation across an entire tissue or organ. Photoactivatable Cre (PA-Cre) systems enable temporally and spatially restricted gene expression control in delimited anatomical regions, typically requiring a micro-LED or fibre optic implantation. Here, we report as proof-of-concept the effective knockout of BDNF in subcutaneous adipose tissue after PA-Cre activation through external blue light illumination in awake, freely moving mice. We demonstrated that for mice with black fur, shaving can be used to anatomically limit PA-Cre activation. BDNF protein expression was decreased by 87% in the inguinal scWAT after blue light exposure, with no effect observed in the perigonadal (deep) or axillary subcutaneous (non-shaved) adipose tissues. We propose blue light induction of PA-Cre as safe and effective to study adipose tissue physiology and pathology across models. Considerations for applying this tool to future studies are also presented.

Abstract: Cells in many naturally occurring organisms routinely cooperate to control their extracellular pH in a dynamic and reversible manner, but this capability has been underexplored in synthetic biology. Here, we sought to engineer a microbial system that switches between two states -high and low extracellular pH- with minimal human intervention. We accomplished this by combining: (1) a genetic circuit that produces recombinant urease under the control of a light-inducible promoter; (2) a degradation tag on urease to accelerate the high-to-low pH transition; and (3) optimization of several environmental factors, including media composition, replenishment rate, and light exposure patterns. The system raises the pH when urease is produced and hydrolyzes urea in the media to produce ammonia; it lowers the pH as a byproduct of the cell's native metabolism when urease production ceases. We demonstrate that the optimized system cycles continuously for up to 14 days with minimal performance loss. Overall, our system demonstrates synthetic pH control in an engineered living system and highlights challenges and potential solutions for using such systems outside of the context of typical laboratory manipulation.

Abstract: Aberrant aggregation of the prion-like RNA binding protein TDP-43 drives several fatal neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). In this work, we define how short, specific RNAs solubilize TDP-43. These short RNAs engage and stabilize the TDP-43 RNA recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregation-resistant conformers. Sequence-space mining identified short RNA chaperones with enhanced activity against TDP-43 and disease-linked variants. Enhanced short RNA chaperones mitigated aberrant TDP-43 phenotypes in optogenetic models and in ALS patient-derived and control motor neurons. In mice with cytoplasmic TDP-43 aggregation and motor neuron loss, an enhanced short RNA chaperone reduced pathological aggregation, restored TDP-43 function, and conferred neuroprotection. These results define a mechanistic and therapeutic framework for RNA-based strategies to counter TDP-43 proteinopathies.

Abstract: Here, we present extrabody, an activatable, modular antibody platform that enables optogenetic or chemical reassembly of split antibody fragments for inducible extracellular antigen recognition. We demonstrate compatibility across diverse targets, including GFP, mCherry, and the tumor-associated antigens EGFR and HER2, and show that both nanobody- and scFv-derived fragments support light-dependent reconstitution. Extrabody enables input-gated cell-cell interactions and antigen transfer, providing external control over intercellular communication. Integration with synNotch receptors and chimeric antigen receptors (CARs) further allows dual-input regulation of downstream responses, including gene expression, cytokine release, and cytotoxicity. Together, these results establish extrabody as a versatile and generalizable interface for externally controlled cellular communication and synthetic signaling.

Abstract: Phosphoinositides are low-abundance regulatory lipids that control a broad range of cellular processes, from membrane trafficking and cytoskeletal remodeling to transcriptional regulation and RNA processing. These lipids are distributed across distinct subcellular compartments, where they carry out compartment-specific regulatory functions. Dysregulation of phosphoinositide metabolism is associated with cancer, neurodegenerative diseases, and immune dysfunction. However, their roles remain difficult to investigate owing to technical limitations in lipid detection and manipulation. This review outlines current strategies for modulating, visualizing, and quantifying phosphoinositide pools, including genetic manipulation techniques such as RNA interference, clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches, and optogenetics. It also evaluates visualization tools such as fluorescent biosensors and live-cell imaging techniques, including superresolution microscopy. In parallel, quantitative methods such as thin-layer chromatography and mass spectrometry for profiling phosphoinositide species, including isomer- and acyl-specific variants, are discussed. By comparing the strengths and limitations of these approaches and highlighting how they can be combined, this review provides a practical framework for dissecting phosphoinositide function in defined subcellular contexts.