Curated Optogenetic Publication Database

Abstract: Oncolytic virus (OVs) therapy has emerged as a promising modality in cancer immunotherapy, attracting growing attention for its multifaceted mechanisms of tumor elimination. However, its efficacy as a monotherapy remains constrained by physiological barriers, limited delivery routes, and suboptimal immune activation. Phototherapy, an innovative and rapidly advancing cancer treatment technology, can mitigate these limitations when used in conjunction with OVs, enhancing viral delivery, amplifying tumor destruction, and boosting antitumor immune responses. This review provides the first comprehensive analysis of synergistic integration of OVs with both photodynamic therapy (PDT) and photothermal therapy (PTT). It also explores their applications in optical imaging-guided diagnosis and optogenetically controlled delivery. Furthermore, it discusses emerging strategies involving biomimetic virus or viroid-based vectors in conjunction with phototherapy, and delves into the immunomodulatory mechanisms of this combinatorial approach. While promising in preclinical models, these combined strategies are still largely in early-stage research. Challenges such as limited light penetration, delivery efficiency, and safety concerns remain to be addressed for clinical translation. Consequently, the integration of OV therapy and phototherapy represents a compelling strategy in cancer treatment, offering significant promise for advancing precision oncology and next-generation immunotherapies.

Abstract: Organ-on-a-chip platforms have emerged as promising human tissue models for drug screening and mechanistic studies, offering alternatives to traditional animal models. Integration of vascular structures into these platforms is pivotal for creating physiologically faithful models of individual organs and studying interorgan crosstalk. However, most vascular structures grown in vitro do not account for organ-specific endothelial permeability or its modulation in response to disease. Here, we present optoBarrier, an optogenetic organ-on-a-chip platform designed to modulate endothelial barrier permeability through light stimulation. By optically activating RhoA signaling in engineered optogenetic endothelial cells, we demonstrate the formation of stress fibers, disruption of vascular endothelial cadherin (VE-cadherin) and increased barrier permeability. We further show that permeability is tunable in a reversible and dose-dependent manner in response to light. We therefore propose that optoBarrier offers a user-defined, controlled and simple method to manipulate endothelial permeability for in vitro studies of human vasculature.

Abstract: Nuclear factor kappa B (NF-κB) has been extensively investigated for approximately four decades. Throughout this timeframe, significant progress has been accomplished in determining the structure, function, and regulation of NF-κB; however, some nuanced complexities of this fundamental signaling pathway remain underexplored. A notable gap exists in the spatiotemporal regulation and molecular dynamics of NF-κB nucleocytoplasmic shuttling, which significantly impacts the complex function and behavior, yet lacks comprehensive characterization. The nucleocytoplasmic shuttling process is also related to resistance mechanisms that evolved following the application of NF-κB or proteasomal inhibitors. Furthermore, the NF-κB complex has a stochastic variability in its trafficking that contributes to heterogeneous cellular responses at the single-cell level and lacks a well-defined druggable pocket, making its complete suppression in cancer cells challenging and uncertain. Engineering synthetic gene circuits and utilizing optogenetic tools can pave the way for precise control of the NF-κB complex, enabling advanced investigations into NF-κB regulation and post-therapeutic behavior implicated in cancer resistance. This approach also permits tumor microenvironment (TME)-immune modulation by synthetic gene circuits that reactivate immune cells within the TME. In this review, we discussed the structure and function of NF-κB, the molecular dynamics of NF-κB nucleocytoplasmic shuttling based on established findings, NF-κB engineering via synthetic biology tools, and critically deciphered the post-therapeutic behavior of NF-κB in cancer, supported by potential therapeutic targets to abrogate resistance.

Abstract: Optogenetically regulated enzymes offer unprecedented spatiotemporal control over protein activity, intermolecular interactions, and intracellular signaling. Many design strategies have been developed for their fabrication based on the principles of intrinsic allostery, oligomerization or 'split' status, intracellular compartmentalization, and steric hindrance. In addition to employing photosensory domains as part of the traditional optogenetic toolset, the specificity of effector domains has also been leveraged for endogenous applications. Here, we discuss the dynamics of light activation while providing a bird's eye view of the crafting approaches, targets, and impact of optogenetic enzymes in orchestrating cellular functions, as well as the bottlenecks and an outlook into the future.

Abstract: Molecular optogenetics allows the control of molecular signaling pathways in response to light. This enables the analysis of the kinetics of signal activation and propagation in a spatially and temporally resolved manner. A key strategy for such control is the light-inducible clustering of signaling molecules, which leads to their activation and subsequent downstream signaling. In this work, an optogenetic approach is developed for inducing graded clustering of different proteins that are fused to eGFP, a widely used protein tag. To this aim, an eGFP-specific nanobody is fused to Cryptochrome 2 variants engineered for different orders of cluster formation. This is exemplified by clustering eGFP-IKKα and eGFP-IKKβ, thereby achieving potent and reversible activation of NF-κB signaling. It is demonstrated that this approach can activate downstream signaling via the endogenous NF-κB pathway and is thereby capable of activating both an NF-κB-responsive reporter construct as well as endogenous NF-κB-responsive target genes as analyzed by RNA sequencing. The generic design of this system is likely transferable to other signaling pathways to analyze the kinetics of signal activation and propagation.

Abstract: Methods for monitoring physiological changes in cellular Ca2+ levels have been in high demand for their utility in monitoring neuronal signaling. Recently, we introduced SCANR (Split-Tobacco Etch Virus (TEV) protease Calcium-regulated Neuron Recorder), which reports on Ca2+ changes in cells through the binding of calmodulin and M13 to reconstitute an active TEV protease. First-generation SCANR marked all of the Ca2+ spikes that occur throughout the lifetime of the cell, but it did not have a mechanism for controlling the time window in which recording of physiological changes in Ca2+ occurred. Here, we explore both chemical and light-based strategies for controlling the time and place in which Ca2+ recording occurs. We describe the adaptation of six popular chemo- and opto-genetics methods for controlling protein activity and subcellular localization to the SCANR system. We report two successful strategies, one that leverages the LOV-Jα optogenetics system for sterically controlling protein interactions and another that employs chemogenetic manipulation of subcellular protein distribution using the FKBP/FRB rapamycin binding pair.

Abstract: Nucleocytoplasmic transport (NCT) is essential for maintaining cellular homeostasis, and its disruption is involved in various diseases, including neurodegenerative disorders and amyotrophic lateral sclerosis. This underscores the need to develop tools to monitor and quantify NCT. Amongst these tools, the fast and reversible optogenetics probes, LEXY (light-inducible nuclear export system) and LINuS (light-inducible nuclear localization signal), allow the measurement of NCT dynamics in live cells. The original publications describe manual segmentation and quantification of the fluorescent probe signal in the nucleus and cytosol upon transfection of LEXY and LINuS constructs in live-cell imaging. However, both transfection and manual segmentation limit the number of cells that can be analyzed and are subject to imprecision due to potential user-dependent errors. While the high speed and reversibility provided by optogenetics should, in principle, allow for high sensitivity in detecting changes in NCT dynamics, it depends on the acquisition parameters and analysis of a sufficient number of cells. We have therefore established lentiviral vectors expressing LEXY and LINuS to create stable cell lines, tested live imaging markers and control conditions, and implemented a semi-automated image analysis pipeline that allows for the analysis of hundreds of cells. This analysis method uses the open-access software FIJI, is accessible to beginners in bioinformatics, and does not require advanced computer setups. Here we provide a step-by-step protocol to set up LEXY as an example of these optogenetic tools to monitor nuclear export, from preparation of the samples to live-cell imaging acquisition and automated analysis, while demonstrating how to adapt the protocol for other conditions, controls, or models in any lab. All plasmids and cell lines used in this protocol will be made available to the scientific community, therefore further increasing the accessibility of the method.

Abstract: Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, nontranscriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling, whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells, whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that Ras activity is regulated by guanine nucleotide exchange factors generally increase cortical tension while Ras activity is regulated by GTPase-activating proteins decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration.

Abstract: Inositol-requiring enzyme 1 (IRE1) is one of three known sensor proteins that respond to homeostatic perturbations in the metazoan endoplasmic reticulum. The three sensors collectively initiate an intertwined signaling network called the Unfolded Protein Response (UPR). Although IRE1 plays pivotal roles in human health and development, understanding its specific contributions to the UPR remains a challenge due to signaling crosstalk from the other two stress sensors. To overcome this problem, we engineered a light-activatable version of IRE1 and probed the transcriptomic effects of IRE1 activity in isolation from the other branches of the UPR. We demonstrate that 1) oligomerization alone is sufficient to activate IRE1 in human cells, 2) IRE1's transcriptional response evolves substantially under prolonged activation, and 3) the UPR induces major changes in mRNA splice isoform abundance in an IRE1-independent manner. Our data reveal previously unknown targets of IRE1 transcriptional regulation and direct degradation. Additionally, the tools developed here will be broadly applicable for precise dissection of signaling networks in diverse cell types, tissues, and organisms.

Abstract: CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems and provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and CRISPR-Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains, that together respond to six clinically relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and II-C CRISPR-Cas9s, and CRISPR-Cas12a. In sum, this work establishes a versatile platform for the multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology, and with the potential for therapeutic use in the future.

Abstract: Targeted protein degradation is a powerful tool for biological research, cell therapy, and synthetic biology. However, conventional methods often depend on pre-fused degrons or chemical degraders, limiting their wider applications. Here we develop a guided protein labeling and degradation system (GPlad) in Escherichia coli, using de novo designed guide proteins and arginine kinase (McsB) for precise degradation of various proteins, including fluorescent proteins, metabolic enzymes, and human proteins. We expand GPlad into versatile tools such as antiGPlad, OptoGPlad, and GPTAC, enabling reversible inhibition, optogenetic regulation, and biological chimerization. The combination of GPlad and antiGPlad allows for programmable circuit construction, including ON/OFF switches, signal amplifiers, and oscillators. OptoGPlad-mediated degradation of MutH accelerates E. coli evolution under protocatechuic acid stress, reducing the required generations from 220 to 100. GPTAC-mediated degradation of AroE enhanced the titer of 3-dehydroshikimic acid to 92.6 g/L, a 23.8% improvement over the conventional CRISPR interference method. We provide a tunable, plug-and-play strategy for straightforward protein degradation without the need for pre-fusion, with substantial implications for synthetic biology and metabolic engineering.

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) technology represents a landmark advance in the field of gene editing. However, conventional CRISPR/Cas systems are limited by inadequate temporal and spatial control. In recent years, the development of optically controlled CRISPR (Opto-CRISPR) technology has offered a novel solution to this issue. As a combination of optogenetics and the CRISPR technology, the Opto-CRISPR technology enables dynamic space-time-specific gene editing and regulation in cells and organisms. In this review, we concisely introduce the basic principles of Opto-CRISPR, summarize its operational mechanisms, and discuss its applications and recent advances across various research fields. In addition, this review analyzes the limitations of Opto-CRISPR, aiming to provide a reference for the development of this emerging field.

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: The integrated stress response (ISR) is a conserved stress response that maintains homeostasis in eukaryotic cells. Modulating the ISR holds therapeutic potential for diseases including viral infection, cancer, and neurodegeneration, but few known compounds can do so without toxicity. Here, we present an optogenetic platform for the discovery of compounds that selectively modulate the ISR. Optogenetic clustering of PKR induces ISR-mediated cell death, enabling the high-throughput screening of 370,830 compounds. We identify compounds that potentiate cell death without cytotoxicity across diverse cell types and stressors. Mechanistic studies reveal that these compounds upregulate activating transcription factor 4 (ATF4), sensitizing cells to stress and apoptosis, and identify GCN2 as a molecular target. Additionally, these compounds exhibit antiviral activity, and one compound reduced viral titers in a mouse model of herpesvirus infection. Structure-activity and toxicology studies highlight opportunities to optimize therapeutic efficacy. This work demonstrates an optogenetic approach to drug discovery and introduces ISR potentiators with therapeutic potential.

Abstract: Teixeira et al. present UltraID-light-inducible protein aggregation (UltraID-LIPA), a technique that combines optogenetic induction of α-synuclein aggregation with proximity-based proteomics. This system enables high-resolution capture of early aggregation events in live cells and implicates known and novel endolysosomal proteins, offering a robust framework for dissecting early pathogenic mechanisms in synucleinopathies and guiding future innovations.

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: p53 protein, a crucial transcription factor in cellular responses to a wide variety of stress, regulates multiple target genes involved in tumor suppression, senescence induction, and metabolic functions. To characterize the context-dependent roles of p53, it is still needed to develop an experimental system that enables selective activation of p53 in cells and tissues. In this study, we developed an optogenetic tool, Opto-p53, to control p53 signaling by light. Opto-p53 was designed to trigger p53 signaling by reconstituting p53 N-terminal and C-terminal fragments with a light-inducible dimerization (LID) system. Upon light exposure, cells expressing Opto-p53 demonstrated p53 transcriptional activation, resulting in cell death and cell cycle arrest. We further enhanced the efficacy of light-induced p53 activation by introducing specific mutations into Opto-p53 fragments. Our findings unveil the capability of Opto-p53 to serve as a powerful tool for dissecting the complex roles of p53 in cellular processes, thereby contributing to the field of synthetic biology and providing general design principles for optogenetic tools using endogenous transcription factors.Key words: synthetic biology, transcriptional factor, p53, optogenetics.

Abstract: Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components.

Abstract: In cancer cells, lipid droplets (LDs) establish extensive membrane contact sites (MCSs) with mitochondria to facilitate fatty acid transfer and sustain energy production, thus enabling cancer cell survival, in nutrient-deprived tumor microenvironments. However, effective strategies to disrupt these LD-mitochondria interactions remain unavailable. We engineered an optogenetic system to control LD intracellular organization through clustering. Upon blue light stimulation, the system induces LDs to undergo spatial reorganization and form clusters, thereby restricting LD accessibility by reducing the available surface area for mitochondrial interaction. Consequently, this clustering significantly diminishes the number of LD-mitochondria MCSs, suppresses fatty acid transport from LDs to mitochondria during starvation, and ultimately leads to cancer cell death in vitro and tumor growth inhibition in vivo. Collectively, our results demonstrate that optogenetically controlled LD clustering offers a novel approach to impede tumor progression by blocking nutrient flow from LDs to mitochondria.

Abstract: After spinal cord injury (SCI), cyclic adenosine monophosphate (cAMP) levels drop in the spinal cord, cortex and brainstem, unlike in regenerating peripheral neurons. To address SCI recovery, we expressed photoactivatable adenylate cyclase (bPAC) in corticospinal neurons of female rats with dorsal hemisection for on-demand cAMP inductions. bPAC stimulation restored passive and firing properties of corticospinal neurons, promoted early and sustained locomotor recovery and increased corticospinal tract plasticity. Additionally, bPAC enhanced sparing of lumbar-projecting brainstem neurons after SCI, accompanied by activation of cAMP signaling in the raphe-reticular formation and increased excitatory/inhibitory neurotransmitter balance. Accordingly, augmented density of serotonergic tracts was found caudal to the injury in bPAC rats, correlating with enhanced functional performance. Serotonergic implication in motor recovery was further evidenced by selective depletion, resulting in the abrogation of bPAC-mediated recovery. Overall, our findings underscore that cAMP induction in corticospinal neurons enhances locomotion after SCI, through a cortical rerouting pathway via the serotonergic descending tract.

Abstract: Tau pathological aggregation in neurofibrillary tangles is a hallmark of several neurodegenerative diseases, including Alzheimer's disease. Phase separation is a thermodynamic process that plays an important role in biomolecular membrane-less condensate formation, while abnormal phase separation of tau leads to pathological aggregate formation. However, the detailed molecular mechanism underlying tau condensation remains not fully understood. Moreover, whether condensation-based pharmacological intervention will be helpful for the treatment of tau-associated neurodegenerative diseases remains elusive. Here, we used an optogenetic tool (optoDroplets) in combination with cell biology and pharmacology to explore the contribution of different domains for tau condensation in cells, and we found that proline-rich domain (PRD) phosphorylation, which is mainly regulated by glycogen synthase kinase 3 β (GSK3β), plays important roles for tau condensation. Moreover, phosphorylation of tau PRD regulates its mis-localization on nuclear speckle. Interestingly and importantly, we found that pharmacological inhibition of GSK3β can impede abnormal tau condensation to slow down the tau-associated pathological process.

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.