Curated Optogenetic Publication Database

Abstract: Endoplasmic reticulum-plasma membrane (ER-PM) contact sites are essential signaling hubs that regulate lipid transport, calcium homeostasis, and spatially organized signal transduction. Emerging evidence indicates that not only the presence but also the dynamics, stability, and geometry of ER-PM contacts critically shape cellular functions; however, tools that enable simultaneous high-fidelity visualization and reversible, quantitative control of these contacts in living cells remain limited. Here, we introduce a modular toolkit for inducible ER-PM contact-site reconstitution based on complementary chemical and optical dimerization strategies. We develop a nontoxic and reversible abscisic acid (ABA)-inducible system using the plant-derived ABIcs/PYLcs pair, and a rapidly reversible optogenetic system based on the iLID/SspB module, both of which allow robust visualization and dose-dependent control over contact-site formation kinetics, increasing contact-site density and total area fraction per cell without altering the size of individual contacts. In contrast, systematic variation of rigid α-helical linker length or inducible tether abundance selectively tunes the lateral growth, stability, and lifetime of individual contact sites, without changing their density. By combining these two orthogonal strategies, we achieve independent control of both individual contact-site size and overall contact-site density, providing complementary mechanisms to adjust total contact area per cell. This versatile platform enables quantitative dissection of ER-PM contact site structure-function relationships and offers broad utility in studies of lipid exchange, calcium signaling, membrane repair, metabolic regulation, and disease-relevant dysregulation.

Abstract: Light-oxygen-voltage (LOV) domain proteins represent a versatile class of photoreceptors capable of regulating a wide range of light-dependent biological functions. While a lot of studies have focused on the photochemistry of LOV domains, the mechanisms of signal generation and propagation in multidomain LOV proteins remain incompletely understood. Here, we investigated two multidomain proteins, using time-resolved infrared spectroscopy. The measurements resolve the entire photocycle dynamics from picoseconds to hours and uncover distinct patterns of local and global structural responses. The two multidomain proteins under study, YF1 and PAL, exhibit nearly identical dynamics during excitation and intersystem crossing on the nanosecond timescale, reflecting conserved local interactions between the chromophore and its highly conserved binding pocket. Multiscale simulations attribute minor spectral differences in this regime to a phenylalanine residue located near the chromophore present only in one of the two LOV domains. The similarities, however, end at the microsecond timescale, where adduct formation already involves global structural adaptations. By experimentally isolating the response of the histidine kinase effector domain in the synthetic photoreceptor YF1, we show that major structural adaptions of the effector domain occur concurrently with cysteine-adduct formation and that the Jα-helix putatively mediates unidirectional communication between domains. In PAL, light-induced opening of the RNA binding site during the adduct formation is additionally followed by a subsequent rearrangement in the distal PAS domain after 3 s. This highlights the pivotal yet distinct roles of the Jα-helix in signal transmission, which depend on the domain topology. Ultimately, our study not only deepens the current understanding of signal transduction in full-length LOV proteins, but also contributes to the fundamental framework for the future application of LOV domains in optogenetic engineering.

Abstract: Notch signalling is a critical regulator of multiple developmental processes through its ability to control gene expression and thereby influence cell fate specification and cell proliferation through direct cell-cell communication. Although Notch signalling has been implicated in myogenesis during late embryogenesis, its role in early mesoderm development has been largely unexplored. Endocytosis of the Notch ligand Delta and the Notch receptor extracellular domain, a critical step in Notch pathway activation, has been extensively observed in the ventral mesoderm of the early Drosophila embryo, indicating a potential for Notch signalling activity in this early germ layer. Here, we present evidence that genes critical to mesoderm development require and are responsive to Notch signalling activity. Using a novel light-inducible Optogenetic variant of the Notch intracellular domain (OptoNotch), which affords precise spatial and temporal control over ectopic activation of Notch signalling, in combination with high-resolution fluorescent RNA in situ hybridization and qPCR, we identified a set of mesodermal genes whose expression is directly regulated by Notch signalling. We also provide evidence that Notch signalling indirectly regulates the dorsal-ventral patterning program mediated by the Toll signalling pathway through the Dorsal/Twist/Snail gene network. Our findings demonstrate that Notch signalling regulates ventral mesoderm patterning and is critical for establishing the mesoderm-mesectoderm-ectoderm boundary by regulating gene expression patterns and providing negative feedback on the upstream patterning network.

Abstract: Various blue-light photoreceptor proteins have photo-responsive domains known as light, oxygen, voltage (LOV) domains, which are extensively distributed in plants, algae, fungi, and bacteria. When exposed to blue light, the flavin chromophore and a highly conserved cysteine residue form a covalent adduct on a microsecond time scale. LOV domains are common photosensory modules that can be applied to optogenetics, regulated synthesis of reactive oxygen species, and fluorescence microscopy. This review explores the photocycle kinetics and applications of various LOV domains, which have been explored for confocal microscopy, two-photon microscopy, and super-resolution microscopy. Many LOV domains have been derived and modulated for use in different types of microscopic applications. Molecular understanding, diversity of LOV domains, and versatile photo-physical characteristics of these proteins have immense potential for the development of useful probes for various microscopy tools. There is a great demand for perspective research on LOV domain proteins for harnessing their possible optobiotechnological applications.

Abstract: Cancer is known to be a disease of altered cellular signaling; however, the relationship between mutation-specific changes to signal transduction and the phenotypic consequences produced remains poorly understood. Here, we investigate two common breast cancer driver mutations, the PIK3CAH1047R mutation and the ErbB2 amplification, both of which activate the PI3K-Akt pathway but paradoxically drive distinct cellular outcomes. Indeed, in nontransformed mammary epithelial cells, PI3KH1047R expression induced features of epithelial-mesenchymal transition (EMT), while ErbB2amp cells exhibited a hyperproliferative phenotype. Characterization of PI3K axis signaling revealed that ErbB2amp cells display prolonged, stimulus-dependent PI3K activation, whereas PI3KH1047R cells show constitutive, ligand-independent signaling. To test whether these distinct dynamics contribute to the phenotypic responses, we employed an iLID-based optogenetic system that enables precise, tunable control of endogenous PI3K activity. Using this tool to mimic the mutation-specific dynamics in MCF10A mammary epithelial cells, we found that PI3K signaling patterns alone were sufficient to reproduce key features of the PIK3CA H1047R-associated EMT phenotype but not the ErbB2-associated proliferative phenotype. These findings suggest that the temporal encoding of pathway activity, not merely its magnitude, can drive some phenotypic changes in oncogenic progression, explain how distinct mutations within a common signaling pathway can produce divergent cellular phenotypes, and provide a workflow for interrogating the functional consequences of changes in signaling dynamics.

Abstract: Crafting synthetic in vitro tissues with mammalian cells faces a shortage of methods to define spatial features. Optogenetic tissue engineering can provide the desired spatial and temporal control but requires stable genomic engineering to support long-term cultivation and high response resolution. Here, we developed BlueGENEs, a set of optimized optogenetic gene switches. BlueGENEs support rapid, stable cell line generation, including precision engineering into the human AAVS1 safe harbor locus. By combining a designer endonuclease and a phage integrase, the approach overcomes gene-disruptive effects of random gene delivery and enables reproducible cell line development. BlueGENEs comprise an optogenetic blue light-responsive gene switch, a synthetic response promoter, and selection strategies serving broad use scenarios. We generated various human cell lines for optical control of apoptotic cell fate, 3D tissue formation, and signals promoting cytoskeletal remodeling. Our results demonstrate the integration of optogenetic cells with bioprinting technologies, illustrating the potential of BlueGENEs in advancing the synthesis of de novo or patient-derived in vitro model systems.

Abstract: Single-cell resolution studies have transformed our understanding of microbial systems, revealing substantial cell-to-cell heterogeneity and complex dynamic behaviors. This review describes recent advances in using optogenetics, where light-sensitive proteins control cellular processes, to investigate microbial behavior at the individual cell level. We discuss studies where optogenetic approaches have enabled high-resolution analysis of properties such as relative cell positioning, subcellular localization, morphology, and gene expression dynamics. In addition, we highlight emerging feedback and event-driven control methods that dynamically modulate cellular states using light signals. By leveraging light's unique capabilities for spatial and temporal manipulation, researchers can now probe cellular characteristics with unprecedented precision. We anticipate significant advances as researchers introduce more sophisticated dynamically patterned light signals for single-cell microbial research.

Abstract: RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs that prevent FUS fibrillization by promoting liquid phases and distinct short RNAs that prevent and reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. We define a short RNA that dissolves cytoplasmic FUS aggregates, restores nuclear FUS, and mitigates FUS toxicity in optogenetic models and ALS patient-derived motor neurons. Another short RNA dissolves cytoplasmic TDP-43 aggregates, restores nuclear TDP-43, and mitigates TDP-43 toxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have utility for ALS/FTD and related disorders.

Abstract: Mitochondria besides being the powerhouse of the cell are also involved in performing a multitude of critical cellular functions. Any failure in maintenance of these organelles is implicated in multiple human pathologies, including neurodegenerative disorders. Over the past two decades, significant efforts have been made to investigate the pharmacodynamic propensity of various potential compounds, which could be engaged as efficient therapeutic approach in modulating mitochondrial dynamics during neuronal dysfunctions.

Abstract: CRISPR/Cas9 is a powerful tool for targeted genome editing experiments. Using CRISPR/Cas9, genes can be deleted or modified by inserting specific DNA sequences, encoding for fluorescent proteins, small peptide tags, or other modifications. Such experiments are essential for detailed gene and protein characterization. However, designing and cloning the corresponding constructs can be repetitive, time-consuming, and laborious. To assist users in CRISPR/Cas9-based genome engineering, we developed CrisprBuildr, an open-source, web-based application for designing modifications to their target genes. CrisprBuildr guides users through creating guide RNAs and repair template vectors to generate cloning maps. The application is designed for the Drosophila melanogaster genome but can serve as a template for other available genomes. We also created new tagging vectors using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments.

Abstract: How do cells maintain robust, yet flexible polarization for directed motion? Recent optogenetic experiments by Town and Weiner on neutrophil-like HL-60 cells strongly point to the essential role of a Rac-inhibitor (downstream of the small GTPase Rac) in shaping requisite negative feedback that allows cells to respond to rapidly changing directional cues. Here we adapt a previous mathematical model for cell polarity to model interactions of Rac, its putative inhibitor, and upstream PIP3 (a product of the optogenetically stimulated PI3K). We fit parameters in our partial differential equation (PDE) model to temporal and spatial experimental data. Cell shapes, motility, and stimulus responses are modeled in 2D simulations, with PDEs solved along the cell edge. We show that the Rac-inhibitor-PIP3 circuit accounts for the optogenetic data (including exotic cell trajectories), that it is the minimal circuit to do so, and that it improves gradient sensing under noisy or dynamic conditions.

Abstract: During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report that synthetic cell-cell signaling using designed ligand-receptor pairs can induce synchronized oscillations in PSM organoids. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; nonoscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock and provides a way to program temporal gene expression in organoids and artificial tissues.

Abstract: Molecular biology has benefited enormously from repurposed tools-many enzymes and antibodies evolved for other functions but are now essential for interrogating biological function by manipulating proteins or nucleic acids. In contrast, lipids have remained technically difficult to visualize or manipulate in cells. This review introduces tools that bring lipid biology into reach for molecular cell biologists, using familiar experimental approaches. We first describe adaptations of immunofluorescence and live-cell imaging of fluorescent molecules to track lipids. Then, we discuss tools for manipulating lipid levels, including pharmacologic inhibitors, synthetic biology platforms for inducible lipid generation or degradation, and optogenetic systems for precise temporal control. While some methods remain technically demanding, most tools are now broadly accessible. Our goal is to offer a practical framework for integrating lipid biology into mainstream cell biology experiments.

Abstract: Synthetic gene circuits consist of engineered networks of genes and proteins. The function of these circuits can be tuned by regulating the expression levels of key circuit components. Light is an ideal signal for controlling expression levels in these circuits; it is cheap and easy to modulate relative to traditional chemical signals. Here, we explore Optogenetic Transcription Factor Localizers (OpTFLos) for tuning expression levels with light. OpTFLos are genetically encoded light-responsive tools for controlling the position in the cell, and therefore the activity, of transcription factors. In this protocol, we discuss how to use the automated optogenetics platform Lustro to systematically characterize OpTFLos to identify light programs that generate desired expression levels. One challenge here with optogenetic tools is that many systems respond to blue light. By using microscopy and Lustro, we describe how to identify light programs that allow for two blue-light-responsive OpTFLos with different kinetic properties to achieve distinct gene expression states with minimal crosstalk, potentially useful for tuning the expression of multiple components within a synthetic gene circuit with a single wavelength of light.

Abstract: Precise modulation of protein levels is essential for investigating the dynamics of signaling pathways. Degradation-based approaches allow rapid reduction of protein abundance, and their combination with optogenetic regulation enables controlled and reversible manipulation. In this chapter, we describe an optogenetic strategy that stabilizes a protein of interest by inhibiting its degradation. As a proof of concept, we demonstrate light-dependent stabilization of MKP3 and constitutively active MEK and examine the resulting changes in ERK signaling. We also provide a step-by-step protocol for measuring protein stabilization kinetics. This method integrates a potent degradation tag with optogenetic control to achieve tunable and reversible regulation of protein levels in living cells.

Abstract: Light exerts a profound influence on plant growth and development, functioning both as a primary energy source and as a critical environmental signal. Red light (RL) and blue light (BL) are the principal spectral regions driving photosynthesis, and consequently promoting autotrophic growth. Compared with RL and BL, green light (GL) has long been considered an inefficient component of the photosynthetically active radiation spectrum in terrestrial plants and has been reported to play a contradictory role in plant development. This review aims to provide a comprehensive understanding of GL's implications for plant developmental processes. Considering that the lack of a specific GL receptor has frustrated the utilization of GL, we discussed the possible photoreceptors that may mediate GL responses in terrestrial plants. Furthermore, we highlight the promising applications of GL-based optogenetics strategies for smart agricultural systems.

Abstract: Primordial germ cells (PGCs) are the first cells specified in the Drosophila embryo and serve as precursors to the germline. Their formation requires suppression of somatic fates, a process achieved by excluding the receptor tyrosine kinase Torso from the posterior pole through degradation mediated by the ubiquitin ligase adaptor Germ Cell-Less (GCL). Although Torso is known to antagonize PGC formation, the underlying mechanism has remained unclear. Here, we combine optogenetic Ras activation and Ras effector loop mutants to show that Ras signaling suppresses PGC formation independently of the canonical Raf/MEK/ERK pathway. We identify an unexpected early role for Torso in activating phosphoinositide 3-kinase (PI3K), generating posterior membrane domains enriched in phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Elevated PI3K activity disrupts PGC formation, while reduced PI3K activity leads to ectopic PGCs. We further demonstrate that GCL remodels the posterior pole membrane by suppressing Torso-dependent PI3K activation. Clearing PIP3 enables Myosin II enrichment, thereby constricting the pole bud for PGC formation. Together, our findings reveal how antagonistic Torso and GCL activities establish the soma-germline boundary by regulating cortical lipid organization.

Abstract: Precise regulation of protein abundance is essential for understanding dynamic cellular processes and for advancing therapeutic development. However, existing approaches lack the spatiotemporal resolution required to these cellular processes. Recent advances in optogenetics have enabled the design of optogenetic targeted protein degradation systems (Opto-TPD) allowing reversible and non-invasive control of protein stability with high spatiotemporal precision. In this review, we systematically summarize the design principles of Opto-TPD tools, including those based on light-oxygen-voltage (LOV)-domain conformational systems, light-inducible dimerization systems, and light-controlled degradation tool expression systems. We further highlight their applications in probing protein function, modulating signaling pathways, and therapeutic translations. By comparing the mechanistic features, performance, and limitations of each platform, we aim to provide a comprehensive resource for guiding future tool optimization. Altogether, these Opto-TPD tools represent a powerful and versatile complement to existing protein manipulation technologies, expanding the toolbox for precise control of protein homeostasis in living systems.

Abstract: The blue light photoreceptor cum transcription factors, aureochromes (Aureos), are present exclusively in photosynthetic stramenopiles. Co-existence of Light-Oxygen-Voltage (LOV) and basic leucine zipper (bZIP) is unique to Aureos - therefore ideal to study light-dependent DNA binding/transcriptional regulation. Further, Aureos' inverse effector-sensor topology, resembling several sensory eukaryotic transcription factors, makes them prototypical optogenetic scaffolds. In absence of 3D data, this study aims for a thorough investigation of the bZIP domains from Aureos and others, and their interaction with substrate DNA using tools from sequence/structural bioinformatics, network theory, molecular dynamics simulation and in vitro experiments. An in-depth comparison of 173 Aureo/plant/opisthokont bZIPs reveals Aureos' uniqueness and evolutionary significance in DNA binding specificity as well as dimer stability. An all-atom network analysis on representative bZIP-DNA co-crystal structures, especially the measurement of eigenvector centrality, further adds importance to hydrophobic interactions in the zipper region to stabilize bZIP dimer and facilitate DNA binding in Aureos and other bZIPs. The most notable finding is the unique presence of histidine at the basic region of Aureos unlike other bZIPs. Histidine not just promotes blue light independent substrate DNA-binding affinity but also serves as a potential switch point in Aureo/bZIP evolution.

Abstract: Outer mitochondrial membranes (OMM) function as dynamic hubs for inter-organelle communication, integrating bidirectional signals, and coordinating organelle behavior in a context-dependent manner. However, tools for mapping mitochondrial surface proteomes with high spatial and temporal resolution remain limited. Here, we introduce an optogenetic proximity labeling strategy using LOV-Turbo, a light-activated biotin ligase, to profile mitochondrial surface proteomes with improved precision, temporal control, and reduced background. By fusing LOV-Turbo to a panel of variants of an OMM-anchored protein, Miro1, we generate spatially distinct baits that resolve modular architectures and regulatory states of the OMM proteomes across diverse conditions, a database we name MitoSurf. Building on this proteomic map, we present RiboLOOM, a platform that defines LOV-Turbo labeled ribosomes and their bound mRNAs at the mitochondrial surface. MitoSurf and RiboLOOM uncover a spatially distinct ribosome pool at the OMM that is maintained by Miro1, enabling local mRNA engagement and translation of mitochondria-related proteins. These findings establish Miro1 as a key organizer of mitochondrial protein biogenesis through spatial confinement of surface-associated ribosomes. Our platform reveals an uncharted layer of mitochondrial surface biology and provides a generalizable strategy to dissect dynamic RNA-protein-organelle interfaces in living cells.

Abstract: Smart microscopy is transforming biological imaging by integrating real-time analysis with adaptive acquisition to enhance imaging efficiency. Whereas many emerging implementations are event-driven and focus on on-demand data acquisition to reduce phototoxicity, we here present 'outcome-driven' microscopy, a framework combining smart microscopy with optogenetics to control cell biological processes and achieve predefined outcomes. We validate this approach using light-based control of cell migration and nucleocytoplasmic transport, demonstrating robust spatiotemporal control of cellular behaviour in single cells and in cell populations.

Abstract: Cellular receptors serve as central hubs that translate external signals into intracellular programs governing cell fate, function and behavior. Achieving precise and reversible control over receptor activity has long been a major challenge in both fundamental biology and translational medicine. Optogenetic receptor engineering provides a transformative solution by integrating photosensitive domains into natural receptor frameworks. This strategy enables light-dependent modulation of signaling with high spatial and temporal precision while maintaining minimal disturbance to endogenous pathways. Unlike chemogenetic systems or classical photoreceptive ion channels, this approach preserves endogenous ligand specificity and avoids slow ligand diffusion/clearance-associated artifacts. Through such systems, researchers can dissect causal relationships in dynamic signaling events, finely manipulate neuromodulatory and immune circuits and program cellular activities involved in development and tissue regeneration. The approach also allows quantitative control of signaling intensity and duration, offering new opportunities for linking molecular design to physiological outcomes. By combining optogenetic principles with advances in materials science and bioelectronics, future designs may achieve improved optical fidelity, enhanced light penetration and better signal amplification within complex biological environments. Integration with AI-guided protein engineering may also accelerate the discovery of optimized photosensory-receptor pairings. Together, these developments point to an emerging field where light-responsive receptors function as programmable interfaces between photonic control and cellular computation. In summary, the engineering of optogenetic receptors establishes a conceptual and technological framework for reversible, accurate and tunable regulation of cellular communication. This review summarizes current progress, outlines key design principles and provides conceptual guidelines for advancing next-generation light-responsive receptors and their biomedical applications. However, key translational challenges-including immunogenicity of non-human photoreceptors, limited gene-delivery efficiency and long-term biosafety-remain to be addressed through nonviral delivery strategies, autologous cell engineering and de-immunized or humanized photoreceptor design.

Abstract: Microtubules are crucial regulators of plant development and are organized by a suite of microtubule-associated proteins (MAPs) that can rapidly remodel the array in response to various cues. This complexity has inspired countless studies into microtubule function from the subcellular to tissue scale, revealing an ever-increasing number of microtubule-dependent processes. Developing a comprehensive understanding of how local microtubule configuration, dynamicity, and remodeling drive developmental progression requires new approaches to capture and alter microtubule behavior. In this review, we will introduce the technological advancements we believe are poised to transform the study of microtubules in plant cells. In particular, we focus on (1) advanced imaging and analysis methods to quantify microtubule organization and behavior, and (2) novel tools to target specific microtubule populations in vivo. By showcasing innovative methodologies developed in non-plant systems, we hope to motivate their increased adoption and raise awareness of possible means of adapting them for studying microtubules in plants.

Abstract: The CRISPR-Cas9 system has been proven to be a powerful tool for gene editing in living cells and shows great potential in genetic disease treatment. Anti-CRISPR (Acr)-based optogenetic tools could spatiotemporally regulate the activity of CRISPR-Cas9, thereby improving the precision and safety of gene editing. However, these tools could only regulate a certain Cas9 protein because of the high specificity of Acr used, limiting their further application. In this study, we developed a new optogenetic tool named CASANOVA-A5 (CRISPR-Cas9 activity switching via a novel optogenetic variant of AcrIIA5) by inserting the blue light sensor AsLOV2 into AcrIIA5 with a broad inhibition spectrum. We proved that the CASANOVA-A5 could regulate the gene editing activity of SpCas9, SaCas9, NmeCas9, and St1Cas9 in a blue light-dependent manner. Additionally, we engineered AcrIIA5-LOV9 by integrating the blue light-dependent degron module LOV9, showing obvious optical regulation for SpCas9. Together, our work demonstrates two feasible methods to engineer the Acrs to potent optogenetic tools and suggests systematic strategies for further optimization.

Abstract: Cellular signaling cascades rely on transfer of information from one protein to another or within a single protein. To facilitate signal integration, specific structural motifs evolved that allow signal processing and also enable modular downstream response integration, facilitating sophisticated regulatory mechanisms. On a structural level, especially coiled-coil helices are frequently observed as signaling motifs. In diguanylate cyclases (DGCs) featuring GGDEF domains, N-terminal coiled-coils frequently activate systems by rearrangements of the interdimer active site. The variety of sensory domains that modulate this structural equilibrium in response to different stimuli highlights the importance of DGCs in bacterial adaptation. One interesting example of sensor DGCs is blue light-activated light-oxygen-voltage (LOV)-GGDEF couples. Here, we describe molecular details of a two-stage mechanism that allows tight dark-state inhibition while enabling high enzymatic activities upon illumination, achieving fold changes exceeding 10,000-fold. Using an in vivo activity assay, we screened amino acid substitutions at the inhibitory interface and the sensor-effector linker region to identify variants that promote enzymatic activity in the dark. In combination with chimeras of LOV and GGDEF domains preventing inhibitory interface formation, we successfully stabilized elongated active-state conformations and confirmed the role of the inhibitory interface between sensor and effector in the tight dark-state inhibition. Interestingly, the initially generated chimeras are still light regulatable as long as the linker sequence is not stabilized in either inhibiting or stimulating coiled-coil register. Our results offer valuable insights for potential optogenetic applications but also demonstrate inherent challenges associated with Methylotenera sp. LOV-activated DGCs.