Curated Optogenetic Publication Database

Abstract: Optogenetic bacterial technology is a cutting-edge approach that combines optogenetics and microbiology, offering a transformative strategy for disease diagnosis and therapy. This synergistic merger transcends the limitations of traditional diagnostic and therapeutic methodologies in a highly controllable, accurate and non-invasive manner. In this review, we introduce the optogenetic systems developed for microbial engineering and summarize fundamental in vitro design principles underlying light-responsive signal transduction in bacteria, as well as the optogenetic regulation of bacterial behaviors. We address multidisciplinary solutions to the challenges in the in vivo applications of light-controlled bacteria, such as limited light excitation, suboptimal delivery and targeting, and difficulties in signal tracking and management. Furthermore, we comprehensively highlight the recent progress in photo-responsive bacteria for disease diagnosis and therapy, and discuss how to accelerate translational applications.

Abstract: Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but it reduced cell stiffness. Mitochondria and endoplasmic reticulum (ER) sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

Abstract: Mitochondrial fission is controlled by dynamin-like proteins, the dysregulation of which is correlated with diverse diseases. Fission dynamin-like proteins are GTP hydrolysis-driven mechanoenzymes that self-oligomerize into helical structures that constrict membranes to achieve fission while also remodeling membranes by inducing negative Gaussian curvature, which is essential for the completion of fission. Despite advances in optical and electron imaging technologies, the underlying mechanics of mitochondrial fission remain unclear due to the multiple times involved in the dynamics of mechanoenzyme activity, oligomer disassembly, and membrane remodeling. Here, we examine how multiscale phenomena in dynamin Drp1 synergistically influence membrane fission using a mechanical model calibrated with small-angle X-ray scattering structural data and informed by a machine learning analysis of the Drp1 sequence, and tested the concept using optogenetic mechanostimulation of mitochondria in live cells. We find that free dynamin-like proteins can trigger a "snap-through instability" that enforces a shape transition from an oligomer-confined cylindrical membrane to a drastically narrower catenoid-shaped neck within the spontaneous hemi-fission regime, in a manner that depends critically on the length of the confined tube. These results indicate how the combination of assembly and paradoxically disassembly of dynamin-like proteins can lead to diverse pathways to scission.

Abstract: Oncolytic viruses (OVs) have the ability to efficiently enter, replicate within, and destroy cancer cells. This capacity to selectively target cancer cells while inducing long-term anti-tumor immune responses, makes OVs a promising tool for next-generation cancer therapy. Immunogenic cell death (ICD) induced by OVs initiates the cancer-immunity cycle (CIC) and plays a critical role in activating and reshaping anti-cancer immunity. Genetic engineering, including arming OVs with cancer cell-specific binders and immunostimulatory molecules, further enhances immune responses at various stages of the CIC, improving the specificity and safety of virotherapy.The aim of this study is to update current knowledge in immunotherapy using OVs and to highlight the remarkable plasticity of viruses in shaping the tumor immune microenvironment, which may facilitate anti-cancer treatment through various approaches.

Abstract: Cells compartmentalize biomolecules in membraneless structures called biomolecular condensates. While their roles in regulating cellular processes are increasingly understood, tools for their synthetic manipulation remain limited. Here, we introduce RELISR (Reversible Light-Induced Store and Release), an optogenetic condensate system that enables reversible storage and release of proteins or mRNAs. RELISR integrates multivalent scaffolds, optogenetic switches, and cargo-binding domains to trap cargo in the dark and release it upon blue-light exposure. We demonstrate its utility in primary neurons and show that light-triggered release of signaling proteins can modulate fibroblast morphology. Furthermore, light-induced release of cargo mRNA results in protein translation both in vitro and in live mice. RELISR thus provides a versatile platform for spatiotemporal control of protein activity and mRNA translation in complex biological systems, with broad potential for research and therapeutic applications.

Abstract: Proper vertebrate development is dependent on tightly regulated expression of genes at the correct time and place. To identify normal but also dysregulated development leading to disease, in vivo interrogation methods with high spatiotemporal resolution are required. Recently, optogenetic tools to manipulate gene expression with spatiotemporal control have emerged, but their in vivo applications remain challenging. Here, we present a transgenic zebrafish strain termed zebrafish for heat-shock-inducible optogenetic recombinase expression (zHORSE) with inducible expression of a light-activatable Cre recombinase. We demonstrate that zHORSE endows robust spatiotemporal control over gene expression down to single-cell level at different developmental stages. We apply zHORSE for lineage tracing to identify caudal fin progenitors and for targeted expression of oncogenes. Surprisingly, one oncogene, EWS::FLI1, can cause ectopic fin formation when induced in permissive environments. zHORSE is compatible with existing loxP zebrafish effector strains and will enable many applications ranging from dissecting and precisely manipulating development to clonal cancer modeling.

Abstract: Plant science has entered a transformative era as genome editing enables precise DNA modifications to address global challenges such as climate adaptation and food security. These modifications are primarily driven by the integration of three modular components-DNA-targeting modules, effector modules, and control modules-that can be selectively activated or suppressed. The field has evolved from protein-based systems (e.g., zinc finger nucleases and transcription activator-like effector nucleases) to RNA-guided systems (e.g., CRISPR-Cas) that can control both genetic and epigenetic states. Modular pairing of DNA-targeting and effector domains, with or without inducible control, enables precise transcriptional regulation and chromatin remodeling. The present review examines these three modules and highlights strategies for their optimization. It also outlines innovative tools, such as optogenetic and receptor-integrated systems, that enable spatiotemporal control over genome editor expression. These modular approaches bypass traditional limitations and allow scientists to create plants with desirable traits, decipher complex gene networks, and promote sustainable agriculture.

Abstract: Neurodegenerative diseases involve toxic protein aggregation. Recent evidence suggests that biomolecular phase separation, a process in which proteins and nucleic acids form dynamic, liquid-like condensates, plays a key role in this aggregation. Optogenetics, originally developed to control neuronal activity with light, has emerged as a powerful tool to investigate phase separation in living systems. This is achieved by fusing disease-associated proteins to light-sensitive oligomerization domains, enabling researchers to induce or reverse condensate formation with precise spatial and temporal control. This review highlights how optogenetic systems such as OptoDroplet are being used to dissect the mechanisms of neurodegenerative disease. We examine how these tools have been applied in models of neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, and Huntington's disease. These studies implicate small oligomeric aggregates as key drivers of toxicity and highlight new opportunities for therapeutic screening. Finally, we discuss advances in light-controlled dissolution of condensates and future directions for applying optogenetics to combat neurodegeneration. By enabling precise, dynamic control of protein phase behavior in living systems, optogenetic approaches provide a powerful framework for elucidating disease mechanisms and informing the development of targeted therapies.

Abstract: Precise temporal and spatial control of gene expression greatly benefits the study of specific cellular circuits and activities. Compared to chemical inducers, light-dependent control of gene expression by optogenetics achieves a higher spatial and temporal resolution. Beyond basic research, this could also prove decisive for manufacturing difficult-to-express proteins in pharmaceutical bioproduction. However, current optogenetic gene-expression systems limit this application in mammalian cells, as expression levels and the degree of induction upon light stimulation are insufficient. To overcome this limitation, we designed a photoswitch by fusing the blue light-activated light-oxygen-voltage receptor EL222 from Erythrobacter litoralis to the three transcriptional activator domains VP64, p65, and Rta in tandem. The resultant photoswitch, dubbed DEL-VPR, allows up to a 570-fold induction of target gene expression by blue light, thereby achieving expression levels of strong constitutive promoters. Here, we used DEL-VPR to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression and increased yield of functional protein complexes. Our approach offers temporally controlled yet strong gene expression and applies to academic and industrial settings.

Abstract: This study explores how the small GTPase RhoA modulates plasma membrane lipid nanodomains, particularly cholesterol-rich ordered membrane domains (OMDs). These nanodomains play a critical role in regulating ion channel activity and neuronal excitability. However, due to their nanoscale dimensions, OMDs remain challenging to visualize using conventional light microscopy. Here, we used fluorescently labeled cholera toxin B (CTxB) and the palmitoylated peptide Lck-10 (L10) as probes to visualize OMDs and quantified their size via confocal fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET). Pharmacological inhibition of RhoA significantly reduced OMD sizes in both human cell lines and dorsal root ganglion (DRG) neurons. To achieve better spatiotemporal control of specific RhoA activation, we employed an improved light-inducible dimerization (iLID) system. Optogenetic activation of RhoA rapidly increased FRET efficiency between CTxB probes, indicating OMD coalescence. Functionally, RhoA inhibition potentiated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity in nociceptive DRG neurons, increasing spontaneous action potential firing. Conversely, in a spared nerve injury rat model, RhoA activation expanded OMDs in nociceptive DRG neurons. Constitutive RhoA activation suppressed HCN channel activity and decreased membrane excitability. These findings support a neuroprotective role for RhoA activation, where it restores OMD size and suppresses pathological hyperexcitability in neuropathic pain.

Abstract: The in vivo continuous evolution system OrthoRep (orthogonal replication) is a powerful strategy for rapid enzyme evolution in Saccharomyces cerevisiae that diversifies genes at a rate exceeding the endogenous genome mutagenesis rate by several orders of magnitude. However, it is difficult to neofunctionalize genes using OrthoRep partly because of the way selection pressures are applied. Here we combine OrthoRep with optogenetics in a selection strategy we call OptoRep, which allows fine-tuning of selection pressure with light. With this capability, we evolved a truncated form of the endogenous monocarboxylate transporter JEN1 (JEN1t) into a de novo mevalonate importer. We demonstrate the functionality of the evolved JEN1t (JEN1tY180C/G) in the production of farnesene, a renewable aviation biofuel, from mevalonate fed to fermentation media or produced by microbial consortia. This study shows that the light-induced complementation of OptoRep may improve the ability to evolve functions not currently accessible for selection, while its fine tunability of selection pressure may allow the continuous evolution of genes whose desired function has a restrictive range between providing effective selection and cellular viability.

Abstract: The cofactor LIM-domain-binding protein 1 (Ldb1) is linked to many processes in gene regulation, including enhancer-promoter communication, interchromosomal interactions, and enhanceosome-cofactor-like activity. However, its functional requirement and molecular role during embryogenesis remain unclear. Here, we used optogenetics (iLEXY) to rapidly deplete Drosophila Ldb1 (Chip) from the nucleus at precise time windows. Remarkably, this pinpointed the essential window of Chip's function to just 1 h of embryogenesis, overlapping zygotic genome activation (ZGA). We show that Zelda, a pioneer factor essential for ZGA, recruits Chip to chromatin, and both factors regulate concordant changes in gene expression, suggesting that Chip is a cofactor of Zelda. Chip does not significantly impact chromatin architecture at these stages, but instead recruits CBP, and is essential for H3K27ac deposition at enhancers and promoters, and for the proper expression of co-regulated genes. These data identify Chip as a functional bridge between Zelda and the coactivator CBP to regulate gene expression in early embryogenesis.

Abstract: Nanobodies, the camelid-derived single-chain variable domain of heavy-chain-only antibodies, are compact in size and exhibit high binding affinity and specificity to their binding partners. As innovative antibody modalities, nanobodies have garnered significant attention in medicine and biological research. To achieve higher spatiotemporal precision, nanobody-based light-controlled systems-such as photobody, optobody, photoactivatable nanobody conjugate inducers of dimerization, and others-have been developed. These systems enable optical control of biological processes while leveraging the advantages of nanobodies as a binding moiety. This concept, summarizes nanobody-based photoregulated systems for investigating biology through light, highlights their advantages and potential limitations, and discusses future directions in this emerging research area.

Abstract: Microbial optogenetic tools can regulate gene expression with high spatial and temporal precision, offering excellent potential for single-cell resolution studies. However, bacterial optogenetic systems have primarily been deployed for population-level experiments. It is not always clear how these tools perform in single cells, where stochastic effects can be substantial. In this study, we focus on optogenetic Cre recombinase and systematically compare the performance of three variants (OptoCre-REDMAP, OptoCre-Vvd, and PA-Cre) for their population-level and single-cell activity. We quantify recombination efficiency, expression variability, and activation dynamics using reporters which produce changes in fluorescence or antibiotic resistance following light-induced Cre activity. Our results indicate that optogenetic recombinase performance can be reporter-dependent, suggesting that this is an important consideration in system design. Further, our single-cell analysis reveals highly heterogeneous activity across cells. Although general trends match expectations for mean levels of light-dependent recombination, we found substantial variation in this behavior across individual cells. In addition, our results show that the timing of recombinase activity is highly variable from cell to cell. These findings suggest critical criteria for selecting appropriate optogenetic recombinase systems and indicate areas for optimization to improve the single-cell capabilities of bacterial optogenetic tools.

Abstract: Light-activated enzymes are an important class of biocatalysts in which light energy is directly converted into biochemical activity. In most cases the light absorbing group is the isoalloxazine ring of an embedded flavin cofactor and in general two types of mechanism are in operation depending on whether the excited chromophore directly participates in catalysis or where photoexcitation triggers conformational changes that modulate the activity of a downstream output partner. This review will summarize studies on DNA photolyase, fatty acid photodecarboxylase (FAP), the monooxygenase PqsL, and flavin-dependent ene-reductases, where flavin radicals generated by excitation are directly used in the reactions catalyzed by these enzymes, and the blue light using FAD (BLUF) and light oxygen voltage (LOV) domain photoreceptors where flavin excitation drives ultrafast structural changes that ultimately result in enzyme activation. Recent advances in methods such as time-resolved spectroscopy and structural imaging have enabled unprecedented insight into the ultrafast dynamics that underly the mechanism of light-activated enzymes, and here we highlight how understanding ultrafast protein dynamics not only provides valuable insights into natural phototransduction processes but also opens new avenues for enzyme engineering and consequent applications in fields such as optogenetics.

Abstract: Komagataella phaffii, also known as Pichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1 promoter. Most K. phaffii bioprocesses for recombinant protein production rely on PAOX1 to achieve dynamic control in two-phase processes. Cells are first grown under conditions that repress PAOX1 (growth phase), followed by methanol-induced recombinant protein expression (production phase). In this study, we propose a methanol-free approach for dynamic metabolic control in K. phaffii using optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light-responsive transcription factor EL222 from Erythrobacter litoralis is used to regulate protein production from the PC120 promoter in K. phaffii with blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab-scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol-inducible bioprocesses using K. phaffii.

Abstract: Optogenetic systems offer precise control over gene expression, but leaky activity in the dark limits their dynamic range and, consequently, their applicability. Here, we enhanced an optogenetic system based on a split T7 RNA polymerase fused to blue-light-inducible Magnets by incorporating a tet-controlled riboregulatory module. This module exploits the photosensitivity of anhydrotetracycline and the designability of synthetic small RNAs to digitize light-controlled gene expression, implementing a repressive action over the translation of a polymerase fragment gene that is relieved with blue light. Our engineered system exhibited 13-fold improvement in dynamic range upon blue light exposure, which even raised to 23-fold improvement when using cells preadapted to chemical induction. As a functional demonstration, we implemented light-controlled antibiotic resistance in bacteria. Such integration of regulatory layers represents a suitable strategy for engineering better circuits for light-based biotechnological applications.

Abstract: Light-inducible regulatory proteins are powerful tools to interrogate fundamental mechanisms driving cellular behavior. In particular, genetically encoded photosensory domains fused to split proteins can tightly modulate protein activity and gene expression. While light-inducible split protein systems have performed well individually, few multichromatic and orthogonal gene regulation systems exist in mammalian cells. The design space for multichromatic circuits is limited by the small number of orthogonally addressable optogenetic switches and the types of effectors that can be actuated by them. We developed a library of red light-inducible recombinases and directed patterned myogenesis in a mesenchymal fibroblast-like cell line. To address the limited number of light-inducible domains (LIDs) responding to unique excitation spectra, we multiplexed light-inducible recombinases with our "Boolean logic and arithmetic through DNA excision" (BLADE) platform. Multiplexed optogenetic tools will be transformative for understanding the role of multiple interacting genes and their spatial context in endogenous signaling networks.

Abstract: Light is a critical environmental factor that governs the growth and development of plants. Plants have specialised photoreceptor proteins, which allow them to sense both quality and quantity of light and drive a wide range of responses critical for optimising growth, resource use and adaptation to changes in environment. Understanding the role of these photoreceptors in plant biology has opened up potential avenues for engineering crops with enhanced productivity by engineering photoreceptor activity and/or action. The ability to manipulate plant genomes through genetic engineering and synthetic biology approaches offers the potential to unlock new agricultural innovations by fine-tuning photoreceptors or photoreceptor pathways that control plant traits of agronomic significance. Additionally, optogenetic tools which allow for precise, light-triggered control of plant responses are emerging as powerful technologies for real-time manipulation of plant cellular responses. As these technologies continue to develop, the integration of photoreceptor engineering and optogenetics into crop breeding programs could potentially revolutionise how plant researchers tackle challenges of plant productivity. Here we provide an overview on the roles of key photoreceptors in regulating agronomically important traits, the current state of plant photoreceptor engineering, the emerging use of optogenetics and synthetic biology, and the practical considerations of applying these approaches to crop improvement. This review seeks to highlight both opportunities and challenges in harnessing photoreceptor engineering approaches for enhancing plant productivity. In this review, we provide an overview on the roles of key photoreceptors in regulating agronomically important traits, the current state of plant photoreceptor engineering, the emerging use of optogenetics and synthetic biology, and the practical considerations of applying these approaches to crop improvement.

Abstract: Bacterial therapy has emerged as a promising approach for disease treatment due to its environmental sensitivity, immunogenicity, and modifiability. However, the clinical application of engineered bacteria is limited by differences of expression levels in patients and possible off-targeting. Optogenetics, which combines optics and genetics, offers key advantages such as remote controllability, non-invasiveness, and precise spatiotemporal control. By utilizing optogenetic tools, the behavior of engineered bacteria can be finely regulated, enabling on-demand control of the dosage and location of their therapeutic products. In this review, we highlight the latest advancements in the optogenetic engineering of bacteria for light-controlled disease theranostics and therapeutic regulation. By constructing a three-dimensional analytical framework of “sense-produce-apply”, we begin by discussing the key components of bacterial optogenetic systems, categorizing them based on their photosensitive protein response to blue, green, and red light. Next, we introduce innovative light-producing tools that extend beyond traditional light sources. Then, special emphasis is placed on the biomedical applications of optogenetically engineered bacteria in treating diseases such as cancer, intestinal inflammation and systemic disease regulation. Finally, we address the challenges and future prospects of bacterial optogenetics, outlining potential directions for enhancing the safety and efficacy of light-controlled bacterial therapies. This review aims to provide insights and strategies for researchers working to advance the application of optogenetically engineered bacteria in drug delivery, precision medicine and therapeutic regulation.

Abstract: Optogenetics is an interdisciplinary field wherein optical and genetic engineering methods are employed together to impart photounresponsive cells (usually of higher animals) the ability to respond to light through expression of light-sensitive proteins sourced generally from algae or bacteria. It enables precise spatiotemporal control of various cellular activities through light stimulation. Recently, emerging as a synthetic biology-based approach for diabetes management, optogenetics can provide user-control of hormonal secretion by photoactivation of a suitably modified cell. For around a decade, studies have been performed on the applicability of various light-sensitive proteins and their incorporation into pancreatic and nonpancreatic cells for photoinduced insulin secretion. Further, in vivo studies demonstrated amelioration of diabetes in mouse models through photoactivation of the implanted engineered cells. Here, we attempt to highlight the various optogenetic approaches explored in terms of influencing the insulin secretion pathway at different points in light of the natural insulin secretion pathway in pancreatic β cells. We also discuss how transgenic cells of both pancreatic as well as nonpancreatic origin are exploited for photoinduced secretion of insulin. Recent advances on integration of “smart” technologies for remote control of light irradiation and thereby insulin secretion from implanted engineered cells in preclinical models are also described. Additionally, the need for further comprehensive studies on irradiation parameters, red-shifted opsins, and host–cell interaction is stressed to realize the full potential of optogenetics as a clinically applicable modality providing user-controlled “on demand” hormonal secretion for better management of diabetes.

Abstract: Lamellipodia are sheet-like protrusions essential for cell migration and endocytosis, but their ultrastructural dynamics remain poorly understood because conventional electron microscopy lacks temporal resolution. Here, we combined optogenetics with cryo-electron tomography (cryo-ET) to visualize the actin cytoskeleton and membrane structures during lamellipodia formation with temporal precision. Using photoactivatable-Rac1 (PA-Rac1) in COS-7 cells, we induced lamellipodia formation with a 2-min blue light irradiation, rapidly vitrified samples, and analyzed their ultrastructure with cryo-ET. We obtained 16 tomograms of lamellipodia at different degrees of extension from three cells. These revealed small protrusions with unbundled actin filaments, "mini filopodia" composed of short, bundled actin filaments at the leading edge, and actin bundles running nearly parallel to the leading edge within inner regions of lamellipodia, suggesting dynamic reorganizations of the actin cytoskeleton. This approach provides a powerful framework for elucidating the ultrastructural dynamics of cellular processes with precise temporal control.

Abstract: Cell signaling regulates a wide range of biological processes including development, homeostasis, and disease. Accessible technologies to precisely manipulate signaling have important applications in basic and translational research. Here, we introduce an optogenetic toolkit comprised of 1) a zebrafish-optimized FGF signaling activator, 2) a single-transcript Nodal signaling activator, and 3) a previously established BMP signaling activator. We thoroughly characterize this suite of tools in zebrafish embryos and show that they provide tunable, light-dependent spatiotemporal control of signaling in vivo. In response to blue light (∼455 nm), receptor kinase domains fused to blue light-dimerizing LOV domains enable robust signaling activation with minimal ectopic activity in the dark or at wavelengths over 495 nm. Optogenetic activation by each tool is pathway-specific and results in increased expression of known target genes. Signaling is activated with rapid on/off kinetics, and activation strength depends on light irradiance. Finally, we demonstrate spatially localized signaling activation with our optimized FGF activator. Together, our results establish this optogenetic toolkit as a potent experimental platform to rapidly, directly, and adjustably activate FGF, BMP, and Nodal signaling in zebrafish embryos.

Abstract: Transcription factors relay information from the external environment to gene regulatory networks that control cell physiology. To confer signalling specificity, robustness and coordination, these signalling networks use temporal communication codes, such as the amplitude, duration or frequency of signals. Although much is known about how temporal information is encoded, a mechanistic understanding of how gene regulatory networks decode signalling dynamics is lacking. Recent advances in our understanding of phase separation of transcriptional condensates provide new biophysical frameworks for both temporal encoding and decoding mechanisms. In this Perspective, we summarize the mechanisms by which transcriptional condensates could enable temporal decoding through signal adaptation, memory and persistence. We further outline methods to probe and manipulate dynamic communication codes of transcription factors and condensates to rationally control gene activation.

Abstract: Biomolecular networks can dynamically encode information, generating time-varying patterns of activity in response to an input. Here we find that dynamic encoding can also be performed by individual proteins. BcLOV4 is an optogenetic protein that uniquely displays pulsatory activation in response to a step input of light, and response dynamics can be shaped by both light and temperature. However, how the BcLOV4 protein generates this step-to-pulse response is not understood. Here we combined live cell imaging and simulations to find that the activity pulse results from an intramolecular incoherent feedforward loop (IFFL) implemented by competitive interactions between protein domains that separately respond to light or temperature. We identified these light- and temperature-sensitive regions and found that they implement the IFFL by competitively caging an activation region. Structural and sequence analysis revealed temperature-responsive regions of BcLOV4 which allowed experimental tuning of activation dynamics and suggested that tuning has also occurred throughout evolution. These findings enabled the generation of more thermostable optogenetic tools and identified a modular thermosensitive domain that endowed thermogenetic control over unrelated proteins. Our findings uncover principles of dynamic and combinatorial signal processing in individual proteins that will fuel development of more sophisticated and compact synthetic systems.