Curated Optogenetic Publication Database

Abstract: Protein homeostasis, or proteostasis, is essential for cellular proteins to function properly. The buildup of abnormal proteins (such as damaged, misfolded, or aggregated proteins) is associated with many diseases, including cancer. Therefore, maintaining proteostasis is critical for cellular health. Currently, genetic methods for modulating proteostasis, such as RNA interference and CRISPR knockout, lack spatial and temporal precision. They are also not suitable for depleting already-synthesized proteins. Similarly, molecular tools like PROTACs and molecular glue face challenges in drug design and discovery. To directly control targeted protein degradation within cells, we introduce an intrabody-based optogenetic toolbox named Flash-Away. Flash-Away integrates the light-responsive ubiquitination activity of the RING domain of TRIM21 for protein degradation, coupled with specific intrabodies for precise targeting. Upon exposure to blue light, Flash-Away enables rapid and targeted degradation of selected proteins. This versatility is demonstrated through successful application to diverse protein targets, including actin, MLKL, and ALFA-tag fused proteins. This innovative light-inducible protein degradation system offers a powerful approach to investigate the functions of specific proteins within physiological contexts. Moreover, Flash-Away presents potential opportunities for clinical translational research and precise medical interventions, advancing the prospects of precision medicine.

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

Abstract: Regulation of cancer cells by their environment contributes to tumorigenesis and drug response, though the extent to which the oncogenic state can alter a cell's perception of its environment is not clear. Prior studies found that EML4-ALK, a receptor tyrosine kinase (RTK) fusion oncoprotein, suppresses transmembrane receptor signaling through EGFR. Moreover, suppression was reversed with targeted ALK inhibition, thereby promoting survival and drug tolerance. Here we tested whether such modulation of EGFR was common among other RTK fusions, which collectively are found in ∼5% of all cancers. Using live- and fixed-cell microscopy in isogenic and patient-derived cell lines, we found that a wide variety of RTK fusions suppress transmembrane EGFR and sequester essential adaptor proteins in the cytoplasm, as evidenced by the localization of endogenous Grb2. Targeted therapies rapidly released Grb2 from sequestration and potentiated EGFR. Synthetic optogenetic analogs of RTK fusions confirmed that cytoplasmic sequestration of Grb2 was sufficient to suppress perception of extracellular EGF and could do so without driving signaling from the synthetic fusion itself, demonstrating that fusion signaling and suppression of EGFR could be functionally decoupled. Our study uncovers that a large number of RTK fusions simultaneously act as both activators and suppressors of signaling, the mechanisms of which could be exploited for new biomimetic therapies that enhance cell killing and suppress drug tolerance.

Abstract: Intracellular optogenetics represents a rapidly advancing biotechnology that enables precise, reversible control of protein activity, signaling dynamics, and cellular behaviours using genetically encoded, light-responsive systems. Originally pioneered in neuroscience through channelrhodopsins to manipulate neuronal excitability, the field has since expanded into diverse intracellular applications with broad implications for medicine, agriculture, and biomanufacturing. Key to these advances are photoreceptors such as cryptochrome 2 (CRY2), light-oxygen-voltage (LOV) domains, and phytochromes, which undergo conformational changes upon illumination to trigger conditional protein-protein interactions, localization shifts, or phase transitions. Recent engineering breakthroughs-including the creation of red-light responsive systems such as MagRed that exploit endogenous biliverdin-have enhanced tissue penetration, minimized phototoxicity, and expanded applicability to complex biological systems. This review provides an overarching synthesis of the molecular principles underlying intracellular optogenetic actuators, including the photophysical basis of light-induced conformational changes, oligomerization, and signaling control. We highlight strategies that employ domain fusions, rational mutagenesis, and synthetic circuits to extend their utility across biological and industrial contexts. We also critically assess current limitations, such as chromophore dependence, light delivery challenges, and safety considerations, so as to frame realistic paths towards translation. Looking ahead, future opportunities include multi-colour and multiplexed systems, integration with high-throughput omics and artificial intelligence, and development of non-invasive modalities suited for in vivo and industrial applications. Intracellular optogenetics is thus emerging as a versatile platform technology, with the potential to reshape how we interrogate biology and engineer cells for therapeutic, agricultural, and environmental solutions.

Abstract: Epigenome editing has become a leading-edge technology of programmable, heritable and reversible control of gene expression in plants without changing the DNA sequence. CRISPR/dCas9 systems along with transcription activator-like effectors (TALEs) and zinc finger systems have made it possible to manipulate DNA methylation, histone modifications, and RNA epigenetic marks in a precise and locus-specific fashion. These tools have been used on major regulatory genes of flowering time, stress adjustment, and yield maximization in model and crop plants. This review synthesizes the current status of plant epigenome editing advances and highlights mechanistic innovations including SunTag, CRISPRoff/on and RNA m6A editing. It also emphasizes new paradigm shifts in chromatin reprogramming, including transcription-resistive chromatin states, locus-specific H3K27me3 demethylation, and nanobody-mediated chromatin targeting. Furthermore, it considers the consequences of these shifts in the context of trait stability and epigenetic inheritance. Moreover, the relative evaluation of dCas9-, TALE-, and ZFP-based platforms indicated that there are still enduring problems in the performance of delivery, off-target effects, and transgenerational stability. The review concludes with a conceptual framework connecting epigenome editing to climate-smart crop improvement and outlines future research priorities focused on combinatorial multi-omics integration and the development of environmentally responsive editing platforms.

Abstract: Cancer can result from abnormal regulation of cells by their environment, potentially because cancer cells may misperceive environmental cues. However, the magnitude to which the oncogenic state alters cellular information processing has not been quantified. Here, we apply pseudorandom pulsatile optogenetic stimulation, live-cell imaging, and information theory to compare the information capacity of receptor tyrosine kinase (RTK) signaling pathways in EML4-ALK-driven lung cancer (STE-1) and in non-transformed (BEAS-2B) cells. The average information rate through RTK/ERK signaling in STE-1 cells was less than 0.5 bit/hour, compared to 7 bit/hour in BEAS-2B cells, but increased to 3 bit/hour after oncogene inhibition. Information was transmitted by 50-70% of cells, whose channel capacity (maximum information rate) was estimated through in silico protocol optimization. In BEAS-2B cells, channel capacity of the parallel RTK/calcineurin pathway surpassed that of the RTK/ERK pathway. This study highlights information capacity as a sensitive metric for identifying disease-associated dysfunction and evaluating the effects of targeted interventions.

Abstract: The genome folds inside the cell nucleus into hierarchical architectural features, such as chromatin loops and domains. If and how this genome organization influences the regulation of gene expression remains only partially understood. The structure-function relationship of genomes has traditionally been probed by population-wide measurements after mutation of critical DNA elements or by perturbation of chromatin-associated proteins. To circumvent possible pleiotropic effects of such approaches, we have developed OptoLoop, an optogenetic system that allows direct manipulation of chromatin contacts by light in a controlled fashion. OptoLoop is based on the fusion between a nuclease-dead SpCas9 protein and the light-inducible oligomerizing protein CRY2. We demonstrate that OptoLoop can drive the induction of contacts between genomically distant, repetitive DNA loci. As a proof-of-principle application of OptoLoop, we probed the functional role of DNA looping in the regulation of the human telomerase gene TERT by long-range contacts with the telomere. By analyzing the extent of chromatin looping and nascent RNA production at individual alleles, we find evidence for looping-mediated repression of TERT. In sum, OptoLoop represents a novel means for the interrogation of structure-function relationships in the genome at single-allele resolution.

Abstract: Cells constantly encounter environmental and physiological fluctuations that challenge homeostasis and threaten viability. In response to these cues, specific proteins and nucleic acids engage in multivalent interactions and undergo phase separation to form membraneless assemblies known as biomolecular condensates. Nuclear condensates include paraspeckles, nuclear speckles, and Cajal bodies, while cytoplasmic condensates include stress granules, processing bodies, RNA transport granules, U-bodies, and Balbiani bodies. These assemblies regulate transcription, splicing fidelity, RNA stability, translational reprogramming, and integration of signaling pathways, thereby serving as dynamic platforms for metabolic regulation and physiological adaptation. However, dysregulation of these condensates has been increasingly recognized as a central pathogenic mechanism in neurodegenerative diseases, cancers, and viral infections, contributing to toxic protein aggregation, nucleic acid dysregulation, and aberrant cell survival signaling. This review provides a comprehensive synthesis of the molecular mechanisms governing condensation, delineates the diverse types and functions of major biomolecular condensates, and examines therapeutic approaches based on their pathophysiological relevance to disease development and progression. Furthermore, we highlight the cutting-edge technologies, including CRISPR/Cas-based imaging, optogenetic manipulation, and AI-driven phase separation prediction tools, which enable the real-time monitoring and precision targeting of cytoplasmic biomolecular condensates. These insights underscore the emerging potential of biomolecular condensates as both biomarkers and therapeutic targets, paving the way for precision medicine approaches in condensate-associated diseases.

Abstract: Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to reverse, and have undesirable off-target effects. Optogenetic systems use light-responsive proteins to control the activity of regulators such that expression is controlled with the "flip of a switch". This study optimizes a simplified light activated CRISPR effector (2pLACE) system, which provides tunable, reversible, and precise control of mammalian gene expression. The OptoPlate-96 enables high-throughput screening via flow cytometry for single-cell analysis and rapid optimization of 2pLACE. This study demonstrates how to use the 2pLACE system with the OptoPlate-96 in HEK293T cells to identify the optimal component ratios for maximizing dynamic range and to find the blue light intensity response curve. Similar workflows can be developed for other mammalian cells and for other optogenetic systems and wavelengths of light. These advancements enhance the precision, scalability, and adaptability of optogenetic tools for biomanufacturing applications.

Abstract: Microtubules (MTs) form dynamic cytoskeletal scaffolds essential for intracellular transport, organelle positioning, and spatial organization of signaling. Their architecture and function are continuously remodeled through the concerted actions of microtubule-associated proteins (MAPs), post-translational modifications (PTMs), and molecular motors. To precisely interrogate these processes in living systems, we developed a genetically encoded optogenetic toolkit for spatiotemporal control of MT organization and dynamics. By replacing native multimerization motifs with a blue light-responsive oligoermization domain, we have engineered single-component probes, OptoMT and OptoTIP, that reversibly label MT polymers or track plus-ends with tunable kinetics from seconds to minutes. When coupled to enzymatic effectors, these modules enable localized tubulin acetylation or detyrosination, directly linking PTMs to MT stability. We further engineered OptoMotor, a light-activatable kinesin platform that reconstitutes tail-dependent cargo transport along MTs, and OptoSAW, a light-triggered severing actuator for controlled MT disassembly. Using these tools, we reveal how local MT integrity governs lysosomal trafficking and ER-associated signaling dynamics. Collectively, this versatile single-component toolkit bridges molecular design with cytoskeletal function, offering new avenues to illuminate how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.

Abstract: Human topoisomerase IIβ binding protein 1 (TopBP1) is a scaffold protein involved in DNA replication initiation, DNA repair, transcription regulation, and checkpoint activation. TopBP1 forms nuclear condensates that act as a molecular switch to amplify ATR activity and promote the activation of the checkpoint effector kinase Chk1. In cancer cells, ATR activity is crucial to tolerate the intrinsically high level of DNA lesions and obstacles that block replication fork progression. Thus, ATR inhibitors are currently tested in clinical trials, often in combination with chemotherapy drugs. However, resistance and toxicity are still major issues. The weak interactions that hold TopBP1 condensates together are highly sensitive to changes in the cellular milieu, suggesting that small molecules may alter the formation of TopBP1 condensates. Here, we developed a high-throughput screening system to identify TopBP1 condensation modulators. This system allowed us to identify FDA-approved drugs, including thimerosal and quinacrine, that inhibit TopBP1 condensation and block the activation of ATR/Chk1 signaling. Mechanistically, quinacrine impaired TopBP1's ability to associate with chromatin, thereby interfering with its capacity to form condensates. Furthermore, quinacrine enhanced the therapeutic efficacy of 5-fluorouracil and irinotecan, components of the clinically used FOLFIRI regimen in a mouse model of peritoneal carcinomatosis from colorectal cancer.

Abstract: Small GTPases are critical regulators of cellular processes, such as cell migration, and comprise a family of over 167 proteins in the human genome. Importantly, the location-dependent regulation of small GTPase activity is integral to coordinating cellular signaling. Currently, there are no generalizable methods for directly controlling the activity of these signaling enzymes with subcellular precision. To address this issue, we introduce a modular, optogenetic platform for the spatial control of small GTPase activity within living cells, termed spLIT-small GTPases. This platform enabled spatially precise control of cytoskeletal dynamics such as filopodia formation (spLIT-Cdc42) and directed cell migration (spLIT-Rac1). Furthermore, a spLIT-RhoA system uncovered previously unreported long-range RhoA signaling in HeLa cells, resulting in bipolar membrane retraction. These results establish spLIT-small GTPases as a versatile platform for the direct, spatial control of small GTPase signaling and demonstrate the ability to uncover spatially defined aspects of small GTPase signaling.

Abstract: Mitochondrial membrane potential (MMP) is essential for mitochondrial functions, yet current methods for modulating MMP lack precise spatial and temporal control. Here, we present an optogenetic system that enables reversible formation of inter-mitochondrial contacts (mito-contacts) with high spatiotemporal precision. Blue light stimulation induces rapid formation of mito-contacts, which fully dissipate upon cessation of illumination. These light-induced mito-contacts can enhance MMP, leading to increased ATP production under stress conditions. Moreover, in human retinal cells and C. elegans, high MMP induced by mito-contacts alleviates the deleterious effects of prolonged blue light exposure, restoring energy metabolism and extending organismal lifespan. This optogenetic approach provides a powerful tool for modulating MMP and offers potential therapeutic applications for diseases linked to mitochondrial dysfunction.

Abstract: Oligomerization of photoactivatable proteins underlies many optogenetic strategies, yet their assembly states remain difficult to quantify in living cells. Here, we applied photon counting histogram analysis to directly measure the oligomerization of widely used optogenetic modules, Vaucheria frigida Aureochrome light-oxygen-voltage (VfAuLOV) and Arabidopsis thaliana cryptochrome 2 (AtCRY2), in living HEK293T cells. Oligomerization of both photoactivatable protein variants is concentration-dependent in cells. VfAuLOV primarily forms dimers, whereas AtCRY2 transitions into tetramers at concentrations above 1,000 nM, consistent with cryoEM structures. Human CRY2 exhibits light-independent oligomerization, while inactive AtCRY2 mutants (D387A and R439L) remain monomeric in light or darkness. Surprisingly, the constitutively active AtCRY2(W374) mutant still undergoes light-mediated oligomerization. The extent of light-induced lytic cell death correlates with the oligomerization state of these proteins when fused to receptor-interacting serine/threonine protein kinase 3. This study establishes a quantitative framework to resolve protein assembly dynamics in living cells, advancing mechanistic understanding of optogenetic tools and broadening their applications in cell signaling research.

Abstract: Most membrane and secreted proteins are transported from the endoplasmic reticulum (ER) to the Golgi apparatus and subsequently directed to their final destinations in the cell. However, the mechanisms underlying transport and cargo sorting remain unclear. Recent advancements in optical microscopy, combined with synchronized cargo protein release methods, have enabled the direct observation of cargo protein transport. We developed a new optically synchronized cargo release method called retention using the dark state of LOV2 (RudLOV). This innovative technique offers three exceptional control capabilities: spatial, temporal, and quantitative control of cargo release. RudLOV uses illumination to trigger transport and detect cargo. Consequently, the selection of an appropriate laser and filter set for controlling the illumination and/or detection is crucial. The protocol presented here provides step-by-step guidelines for obtaining high-resolution live imaging data using RudLOV, thereby enabling researchers to investigate intracellular cargo transport with unprecedented precision and control. Key features • RudLOV is a new optically synchronized intracellular cargo transport method. • RudLOV enables precise spatial, temporal, and quantitative control of cargo release. • RudLOV allows cargo to be released using a 445 or 488 nm laser with less damage than UV. • RudLOV allows you to hook and release cargo without the use of exogenous chemicals.

Abstract: The response of cell populations to external stimuli plays a central role in biological mechanical processes such as epithelial wound healing and developmental morphogenesis. Wave-like propagation of a signal of ERK MAP kinase has been shown to direct collective migration in one direction; however, the mechanism based on continuum mechanics under a traveling wave is not fully understood. To elucidate how the traveling wave of the ERK kinase signal directs collective migration, we constructed the mechanical model of the epithelial cell monolayer by considering the signal-dependent coordination of contractile stress and cellular orientation. The proposed model was studied by using an optogenetically controlled cell system where we found that local signal activation induces changes in cell density and orientation with the direction of propagation. The net motion of the cell population occurred relative to the wave, and the migration velocity showed a maximum in resonance with the velocity of the ERK signal wave. The presented mechanical model was further validated in an in vitro wound healing process.

Abstract: The proper establishment of cell form, fate, and function during morphogenesis requires precise coordination between cell polarity and developmental cues. To achieve this, cells must establish polarity domains that are stable yet sensitive to guiding cues. Here we show that C. elegans germline blastomeres resolve this trade-off by creating a time-varying polarization landscape. Specifically, coupling the PAR polarity network to the cell-cycle kinase CDK-1 ensures that newborn cells operate in a low-feedback regime that lowers barriers to polarity state switching, allowing spatial cues to induce and orient PAR protein asymmetries. As CDK-1 activity rises at mitotic entry, increasing molecular feedback reinforces cue-induced asymmetries to yield robust and stable patterning of PAR domains. Consistent with this model, optogenetic and chemical perturbations show that low-CDK/low-feedback regimes destabilize PAR domains but are required for both de novo polarization and the reorientation of polarity in response to inductive cues. We propose that mitotic oscillations in cell polarity circuits dynamically optimize the polarization landscape to enable coordination of polarity with morphogenesis. Such temporal control of developmental networks is likely a general mechanism to balance robustness of cellular states with sensitivity to signal-induced state switching.

Abstract: Since its discovery, the cyclic GMP-AMP synthase (cGAS)-stimulator of the interferon gene (STING) signaling pathway has been considered a pivotal component of innate immunity and a promising target for cancer immunotherapy. Beyond its canonical role in pathogen defense, accumulating evidence has demonstrated that the cGAS-STING pathway critically regulates diverse cellular processes, including cellular senescence, autophagy, cell death, and tumor immunosurveillance; therefore, dysregulation of this pathway correlates with the pathogenesis and progression of various human diseases, ranging from autoimmune and inflammatory disorders to cancer. Herein, we reviewed the regulatory mechanisms and cellular functions of the cGAS-STING pathway, highlighting its essential role in maintaining immune homeostasis. We systematically discussed the dual roles of the cGAS-STING pathway in cancer immunity, in which it triggers both antitumor and immunosuppressive effects. Finally, we summarized the recent advances and challenges in therapeutic strategies targeting the cGAS-STING pathway and discussed the next generation of therapies, including nanomaterials, antibody-drug conjugates, engineered bacteria, alternative strategies, optogenetic approaches, and combination strategies. We hope that our efforts will advance the understanding of the fundamental principles of innate immune recognition and response, and provide novel directions for improving the clinical outcomes of cGAS-STING-targeted therapies.

Abstract: Phospholipase C-γ1 (PLC-γ1) signaling is required for mesenchymal chemotaxis, but is it sufficient to bias motility? PLC-γ1 enzyme activity is basally autoinhibited, and light-controlled membrane recruitment of wild-type (WT) PLC-γ1 (OptoPLC-γ1) in Plcg1-null fibroblasts does not trigger lipid hydrolysis, complicating efforts to isolate its contribution. Utilizing cancer-associated mutations to investigate the regulatory logic of PLC-γ1, we demonstrate that the canonical hallmark of enzyme activity, phosphorylated Tyr783 (pTyr783), is not a proxy for activity level, but is rather a marker of dysregulated autoinhibition. Accordingly, OptoPLC-γ1 with a deregulating mutation (P867R, S345F, or D1165H) exhibits elevated phosphorylation, and membrane localization of such is sufficient to activate substrate hydrolysis and concomitant motility responses. In particular, local recruitment of OptoPLC-γ1 S345F polarizes cell motility on demand. This response is spatially dose-sensitive and only partially reduced by blocking canonical PLC-γ1 signaling yet is lipase-dependent. Our findings reframe the interpretation of PLC-γ1 regulation and demonstrate that local activation of PLC-γ1 is sufficient to direct cell motility.

Abstract: Optogenetics, a modern tool to control cellular excitability in a non-invasive way, has widely been used in neuroscience. Recently, optogenetic approaches begin to be applied to studies of other biological phenomena including muscle functions. For these analyses, chicken embryos serve as an excellent model animal since they are highly amenable to site-specific manipulations with genes of optogenetics such as Channelrhodopsins, and its following targeted light irradiation. We here overview recent progresses in optogenetics using chicken embryos with a highlight on the studies of axon pathfinding, gut peristalsis, and feather morphogenesis.

Abstract: In adherent cells, actomyosin contractility is regulated mainly by the RhoA signaling pathway, which can be controlled by optogenetics. To model the mechanochemical coupling in such systems, we introduce a finite element framework based on the discontinuous Galerkin method, which allows us to treat cell doublets, chains of cells, and monolayers within the same conceptual framework. While the adherent cell layer is modeled as an actively contracting viscoelastic solid on an elastic foundation, different models are considered for the Rho pathway, starting with a simple linear chain that can be solved analytically and later including direct feedback that can be solved only numerically. Our model predicts signal propagation as a function of coupling strength and viscoelastic timescales and identifies the conditions for optimal cell responses and wave propagation. In general, it provides a systematic understanding of how biochemistry and mechanics simultaneously contribute to the communication of adherent cells.

Abstract: Modeling and simulating gene regulatory networks (GRNs) is crucial for understanding biological processes, predicting system behavior, interpreting experimental data and guiding the design of synthetic systems. In synthetic biology, GRNs are fundamental to enable the design and control of complex functions. However, GRN simulations can be time-consuming and often require specialized expertise. To address this challenge, we developed GRN_modeler - a user-friendly tool with a graphical user interface that enables users without programming experience to create phenomenological models, while also offering command-line support for advanced users. GRN_modeler supports the analysis of both dynamical behaviors and spatial pattern formation. We demonstrate its versatility through several examples in synthetic biology, including the design of novel oscillator families capable of robust oscillation with an even number of nodes, complementing the classical repressilator family, which requires odd-numbered nodes. Furthermore, we showcase how GRN_modeler allowed us to develop a light-detecting biosensor in Escherichia coli that tracks light intensity over several days and leaves a record in the form of ring patterns in bacterial colonies.

Abstract: Recent advances in protein structure prediction by artificial intelligence have enabled the rational design of engineered enzymes with enhanced activity and precise regulatory features. Here, we report the AlphaFold3-guided enhancement of MagMboI, a photoactivatable restriction enzyme designed for light-controlled top-down genome engineering. MagMboI is derived from the type II restriction enzyme MboI and functions through a split-protein strategy in which its N- and C-terminal fragments are fused to light-inducible dimerization modules. Upon exposure to blue light, these domains heterodimerize, restoring nuclease activity in a controlled manner. Using AlphaFold3, we modeled the structure of the MagMboI-DNA complex and gained structural insights into the interaction between MagMboI and its target DNA recognition sequence (5'-GATC-3') required for Mg2+-dependent DNA cleavage. Comparing neighboring split-site variants, we identified an alternative split that increases the MagMboI-DNA interface area and enhances complex stability relative to the original construct. This redesigned variant (designated MagMboI-plus) preserves α-helical integrity while strengthening protein-DNA contacts. Although MagMboI-plus, when introduced in Saccharomyces cerevisiae cells, exhibited slightly increased DNA-cleavage activity in vivo upon blue light activation, it was found to induce more pronounced genomic rearrangements compared to the original MagMboI construct. These findings demonstrate that AlphaFold3-based prediction can accelerate functional improvements in engineered enzymes, providing a strategy for developing light-controlled genome engineering tools.

Abstract: Macrophage-based adoptive cell therapies hold promise for solid tumors, but spatiotemporally controlling macrophage polarization within the immunosuppressive tumor microenvironment remains challenging. Here, we aimed to validate an optogenetic strategy using the LOV2-STIM1 system to achieve light-induced, sustained M1 polarization of macrophages. Upon blue light stimulation, engineered macrophages robustly exhibited M1 phenotypes, suppressed melanoma cell proliferation, migration, and invasion in vitro, and recapitulated the antitumor functions of M1 macrophages. Notably, combining light-activated engineered macrophages with temozolomide in melanoma models resulted in synergistic inhibition of tumor growth. This synergy is accompanied by a profound remodeling of the tumor immune microenvironment, characterized by M1-driven reversal of chemoresistance and enhanced infiltration of cytotoxic CD8+ T cells. Our findings establish a proof-of-concept for optogenetic regulation of macrophage polarization and demonstrate its feasibility for enhancing antitumor effects and chemosensitivity in melanoma models, providing a promising and controllable platform for macrophage-based immunotherapy.

Abstract: Dynamic regulation of cell-cell adhesion is fundamental to numerous biological processes and is the key to engineering multicellular structures. Optogenetic tools offer precise spatiotemporal control over cell-cell adhesions, but current methods often require the genetic modification of each participating cell type. To address this limitation, we engineered a single-component synthetic cell adhesion molecule based on the blue-light-responsive, plasma membrane-binding protein BcLOV4. We tagged BcLOV4 with a transmembrane domain to display it on the outer plasma membrane (BcLOV4-PM). Under blue light but not in the dark, BcLOV4-PM cells formed both homotypic adhesions with other BcLOV4-PM cells and heterotypic adhesions with a range of unmodified wild-type cells. While these adhesions were not reversed in the dark, they could be efficiently disrupted by increasing the temperature to 37 °C, leveraging BcLOV4's thermosensitivity. Using BcLOV4-PM-based adhesions, we demonstrated light-controlled compaction of spheroids in both monocultures and cocultures with wild-type cells. Altogether, BcLOV4-PM enables promiscuous, modular, light-dependent control of cell-cell adhesions without requiring genetic modification of all cell types involved, offering promising applications in tissue engineering and the study of multicellular process.