Curated Optogenetic Publication Database

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: Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger downstream of G protein-coupled receptors (GPCRs) and a central regulator of cellular signaling. Genetically encoded exchange proteins directly activated by cAMP (Epac)-based Förster resonance energy transfer (FRET) biosensors enable real-time monitoring of cAMP dynamics in living cells, but commonly used cyan/yellow FRET pairs require short-wavelength excitation, limiting compatibility with multiplex imaging and blue-light optogenetic tools such as bacterial photoactivated adenylyl cyclases (bPACs). Here, we engineered and systematically characterized four red-shifted Epac-based single-chain FRET cAMP sensors combining yellow or orange FRET donors with red fluorescent FRET acceptors. Using ratiometric live-cell imaging, we quantified stimulus-evoked FRET responses and identified Epacred4 as the best-performing variant, showing an approximately 55% decrease in normalized FRET after forskolin stimulation. Epacred4 also reliably detected Gi/o-mediated decreases in cAMP following μ-opioid receptor activation. Brief 405 nm light pulses induced graded and reversible cAMP elevations using the low dark-activity variant bPAC-F198Y. Furthermore, Epacred4 enabled analysis of cAMP recovery kinetics during phosphodiesterase inhibition and multiplex imaging of cAMP and intracellular Ca2+ using Fura-2 with minimal spectral and pH-related interference under physiological imaging conditions. Together, Epacred4 represents a robust red-shifted cAMP sensor for optogenetic and multiplex signaling studies.

Abstract: The cardiac sodium channel NaV1.5 initiates each heartbeat by generating the rapid depolarizing upstroke of the action potential. Dysregulation of NaV1.5 gating can produce cardiac arrhythmias by slowing inactivation, increasing late sodium current (INa,L), and impairing electrical stability. Here, we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical membrane cofactor that stabilizes NaV1.5 gating. Acute PIP2 depletion in human iPSC-derived cardiomyocytes, produced by activation of endogenous AT1 receptors, activation of an engineered M3q-DREADD, or optogenetic recruitment of CRY2-pseudojanin, shifted voltage dependence, slowed fast inactivation, and increased INa,L. These effects were prevented by augmenting intracellular PIP2, required PLC activity when driven by Gq-coupled receptors, and were independent of downstream Ca2 or PKC signaling. Unlike the skeletal-muscle isoform NaV1.4, NaV1.5 displayed PIP2-dependent shifts in both activation and steady-state inactivation, indicating isoform-specific lipid coupling. Induced-fit docking and molecular dynamics simulations identified a PIP2-interaction interface between the domain IV voltage sensor and pore that contains disease-linked residues. The disease-reported variant R1644C weakened and redistributed the predicted PIP2-contact network, produced elevated basal INa,L, showed enhanced sensitivity to PIP2 depletion, and caused an approximately 30-fold reduction in apparent functional PIP2 sensitivity in excised patches. These findings define a lipid-dependent mechanism that stabilizes NaV1.5 gating and reveal how physiological Gq signaling and inherited channel variants can converge on the channel-PIP2 axis to promote proarrhythmic late sodium current.

Abstract: We have developed an optogenetic tool called InLOV-an insulin receptor fused to a LOV domain-that enables activation of the insulin receptor signaling pathway via light-induced phosphorylation of its intracellular domains. Light activation of InLOV promotes insulin-induced neuronal plasticity in the mouse cerebellum and enhances cerebellar-driven self-motion behavior. Thus, InLOV enables optogenetic modulation of insulin receptor phosphorylation, opening new possibilities for disease modeling and therapeutic strategies for pathological insulin signaling in humans.

Abstract: Photoreceptor sensory histidine kinases (SHKs) couple light absorption to conformational changes regulating two-component signaling. Despite their importance and widespread use in optogenetics, the underlying structural signaling mechanisms remain poorly understood. Here, we engineered dimeric SHKs based on Pseudomonas putida short light-oxygen-voltage (LOV) proteins, determined their crystal structures, and investigated their signaling mechanisms. Regardless of illumination, the structures adopted a light-state like LOV-LOV dimer with symmetric/straight kinase modules. In contrast, small-angle x-ray scattering together with functional assays revealed pronounced light-dependent rearrangements in solution and allowed the assignment of the kinase-ON dark state to an asymmetric/kinked conformation, whereas the light state adopts a symmetric/straight structure. Comparative analyses of natural and engineered SHKs identified conserved motifs linking light-induced LOV domain rotation to kinase activity. The findings highlight the central role of dimer asymmetry and flexibility in SHK signaling, thereby not least informing the engineering of new light-responsive signaling systems.

Abstract: The FKBP-based chemically induced dimerization (CID) technology is a fundamental tool for spatiotemporal precise modulation of protein functions in living cells, widely used in gene editing, protein function regulation, disease therapy, and drug development. However, its widespread application is limited by the inherent drawbacks including issues with immunosuppressive activity, stability, reversibility, and in vivo delivery. Recent years have seen remarkable progress in addressing these challenges: orthogonalization strategies eliminate immunosuppressive effects, fast-dissociating ligands and optogenetic systems enable reversible regulation, and protein engineering optimizes the FKBP/FRB domains to enhance stability and reduce immunogenicity. Moreover, integration with novel delivery technologies broadens its application scope greatly. This review summarizes the key optimization strategies and innovative applications of this technology in the cutting-edge biological research, aiming to reference the development of next-generation chemogenetic tools with higher precision, better safety, and greater application potential.

Abstract: Cancer is increasingly recognized as a disease of the dysregulated epigenome; however, current epi-drugs are blunt, systemically toxic instruments. Catalytically dead CRISPR nucleases (dCas9) linked to chromatin effectors have now made it possible not only to write and erase epigenetic marks at specified loci without double-strand breaks but also to add an element of optogenetics, or reversible and light-encoded control over the timing and localization of the editors. In this review, the technological underpinnings of light-controlled CRISPR-dCas9 epigenome editing, which include architectures of dCas9 scaffold and guide, blue-to-near-infrared photoswitches, and high-gain epigenetic effector designs, are synthesized, and viral, non-viral, and stimuli-responsive delivery platforms, which have to be co-optimized with clinical light interfaces, are discussed. We then outline four functional routes by which opto-epigenome editors may be used therapeutically in cancer: tumor suppressor reactivation; oncogene and super-enhancer repression with metabolic rewiring; control of cancer stem cell differentiation; and immunomodulation of the tumor microenvironment. Lastly, a translational roadmap is defined in terms of preclinical model tiers, biomarker strategies, regulatory and manufacturing factors, and future directions, including NIR and bioluminescent actuation, implantable μLED devices, and AI-guided closed-loop illumination. Together, these aspects constitute design principles for advancing light-addressable epigenome editors toward first-in-human studies and for integrating them into combination regimens as a new class of precision cancer therapeutics.

Abstract: Optogenetics represents a promising frontier in precision cancer therapy by enabling spatiotemporal control over cellular behavior. However, its clinical application is limited by inefficient delivery of optogenetic components and poor tissue penetration of visible light. Recent advances in nanotechnology offer solutions to these challenges. Nanoscale drug delivery systems enhance the targeted delivery of optogenetic tools, while light-conversion nanomaterials enable deep-tissue activation. Besides, the integration of nanotechnology with optogenetics further facilitates the development of engineered living therapeutics, including immune cells and bacteria, allowing programmable and localized antitumor responses. Despite promising preclinical progress, key challenges remain in long-term biosafety, immunogenicity, and precise light dosing. Future progress will depend on interdisciplinary efforts combining biocompatible nanomaterials, protein engineering, and artificial intelligence to advance clinically viable optogenetic therapies and pave the way toward personalized cancer treatment. Collectively, the synergistic integration of optogenetics and nanotechnology holds potential for overcoming longstanding barriers in cancer treatment, paving the way for precision cancer therapeutics.

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: Inducible expression of endogenous and foreign genes has been a pivotal driving force behind a lot many seminal breakthroughs in biotechnology. Synthetic biology, a very promising field, largely relies on transgene expression platforms which facilitate convenient and conditional regulation. Optogenetic approaches that exploit light to steer biological events, e.g., gene expression, with excellent spatiotemporal control, are often more precise compared to chemical induction. Light being an omnipresent environmental stimulus, serves as the ideal cue, and enables high spatiotemporal accuracy with respect to gene expression. In this review, we focus on different elements relevant to light-inducible gene expression - light-responsive promoters, light-regulated transcription factors, and photocaged inducers. Using light as a binary input function, we explore the essence of logic gates towards the development of gene expression circuits - thereby understanding the entanglement between optogenetics and synthetic biology. We primarily focus on prokaryotes, but also draw comparisons with analogous eukaryotic gene expression systems.

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: Synthetic biology seeks to build predictable, programmable biological systems. We developed a blue-light-inducible T7RNAP system with dual-input regulation to enable precise spatiotemporal gene control, which is vital for biomanufacturing, therapy, and microbial engineering. We optimized it by replacing RBS sequences, testing tandem T7 promoters, and evaluating split-T7RNAP variants. Expression and bactericidal efficacy were assessed via fluorescent output and real-time growth curves under blue light. RBS variants caused up to 50-fold differences in expression. Three tandem T7 promoters provided the best balance between yield and fidelity. Integration of a benzoate-responsive module enabled 4.5-fold repression at 3 mM benzoate, demonstrating effective chemical off-switching without compromising light induction. This system combines blue light precision with environmental responsiveness, offering non-invasive, on-demand activation for antimicrobial therapy or spatial bioproduction. The benzoate-triggered off-switch is especially valuable for ecological applications such as biocontainment or bioremediation, where gene expression must shut down upon detection of pollutants, for example, aromatic hydrocarbons. Its orthogonal, modular design supports context-dependent control, making it ideal for environmental biosensors, programmable probiotics, and smart antimicrobial delivery in complex ecosystems.

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: 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: Light-responsive hydrogels enable noninvasive, precise, remote control over material properties with great biocompatibility, yet achieving multicolor addressability and dynamic regulation of mechanical properties remains challenging. Most systems rely on single photoswitches and regulate stiffness primarily through changes in crosslinking density. Here, we present a fully protein-based hydrogel that combines multicolor light responsiveness with optical control of crosslink dynamics. The hydrogel includes two visible light-responsive photoceptors: the cyanobacterial phytochrome Cph1, which enables reversible red/far-red light-controlled crosslinking, and CarH, which introduces irreversible green light-induced gel-sol transitions. Remarkably, Cph1-based hydrogels exhibited an attenuated red light intensity response, forming stiffer networks under low-intensity illumination than under high-intensity light and show a subsequent dark-adaptation with stiffening once red light illumination is stopped. This counterintuitive behavior arises from light-driven bidirectional photoisomerization that modulates crosslink lifetimes without altering the photostationary state composition. Together, these findings establish orthogonally addressable reversible and irreversible crosslinks and photoregulated crosslink dynamics as new design principles for multicolor light-responsive biomaterials.

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.