Curated Optogenetic Publication Database

Abstract: Morphogen gradients convey essential spatial information during tissue patterning. Although the concentration and timing of morphogen exposure are both crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homolog of NF-κB, which orchestrates dorsoventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning and characterized the resulting effect on spatiotemporal transcription of target genes in terms of timing, coordination and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL was detectable at the level of single-cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

Abstract: Cortical positioning of the meiotic spindle within an oocyte is required to expel chromosomes into polar bodies to generate a zygote with the correct number of chromosomes. In C. elegans, yolk granules and mitochondria are packed inward, away from the cortex, while the spindle moves outward, both in a kinesin-dependent manner. The kinesin-dependent inward packing of yolk granules suggests the existence of microtubules with minus ends at the cortex and plus ends extending inward, making it unclear how kinesin moves the spindle outward. We hypothesize that the inward packing of organelles might indirectly force the spindle outward by volume exclusion. To test this hypothesis, we generate a strain in which the only kinesin consists of motor domains with no cargo-binding tail optogenetically attached to mitochondria. This mitochondria-only kinesin packs mitochondria into a tight ball and efficiently moves the meiotic spindle to the cortex, supporting the volume exclusion hypothesis.

Abstract: To address the need for methods for tagging and manipulating neuronal ensembles underlying specific behaviors, we present an improved version of FLARE, termed cytoFLARE (cytosol-expressed FLARE). cytoFLARE incorporates cytosolic tethering of a transcription factor and expression of a more sensitive pair of calcium-sensing domains. We show that cytoFLARE captures more calcium- and light-dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further establish cytoFLARE transgenic Drosophila models and apply cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through the cytoFLARE-driven expression of optogenetic actuators, we successfully reactivated and inhibited neurons involved in the larval nociceptive system. Our findings demonstrate the characterization and application of time-gated calcium integrators for both recording and manipulating neuronal activity in Drosophila larvae.

Abstract: There is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats), coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here, we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs, BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa, and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.

Abstract: Disintegration of organelle membranes induces various cellular responses and has pathological consequences, including autoinflammatory diseases and neurodegeneration. Establishing methods to induce membrane rupture of specific organelles is essential to analyze the downstream effects of membrane rupture; however, the spatiotemporal induction of organelle membrane rupture remains challenging. Here, we develop a series of optogenetic tools to induce organelle membrane rupture by using engineered Bcl-2-associated X protein (BAX), which primarily functions to form membrane pores in the outer mitochondrial membrane (OMM) during apoptosis. When BAX is forced to target mitochondria, lysosomes, or the endoplasmic reticulum (ER) by replacing its C-terminal transmembrane domain (TMD) with organelle-targeting sequences, the BAX mutants rupture their targeted membranes. To regulate the activity of organelle-targeted BAX, the photosensitive light-oxygen-voltage-sensing 2 (LOV2) domain is fused to the N-terminus of BAX. The resulting LOV2-BAX fusion protein exhibits blue light-dependent membrane-rupture activity on various organelles, including mitochondria, the ER, and lysosomes. Thus, LOV2-BAX enables spatiotemporal induction of membrane rupture across a broad range of organelles, expanding research opportunities on the consequences of organelle membrane disruption.

Abstract: Lipids are a major class of biological molecules, the primary components of cellular membranes, and critical signaling molecules that regulate cell biology and physiology. Due to their dynamic behavior within membranes, rapid transport between organelles, and complex and often redundant metabolic pathways, lipids have traditionally been considered among the most challenging biological molecules to study. In recent years, a plethora of tools bridging the chemistry-biology interface has emerged for studying different aspects of lipid biology. Here, we provide an overview of these approaches. We discuss methods for lipid detection, including genetically encoded biosensors, synthetic lipid analogs, and metabolic labeling probes. For targeted manipulation of lipids, we describe pharmacological agents and controllable enzymes, termed membrane editors, that harness optogenetics and chemogenetics. To conclude, we survey techniques for elucidating lipid-protein interactions, including photoaffinity labeling and proximity labeling. Collectively, these strategies are revealing new insights into the regulation, dynamics, and functions of lipids in cell biology.

Abstract: Microbial fermentation provides a sustainable method of producing valuable chemicals. Adding dynamic control to fermentations can significantly improve titers, but most systems rely on transcriptional controls of metabolic enzymes, leaving existing intracellular enzymes unregulated. This limits the ability of transcriptional controls to switch off metabolic pathways, especially when metabolic enzymes have long half-lives. We developed a two-layer transcriptional/post-translational control system for yeast fermentations. Specifically, the system uses blue light to transcriptionally activate the major pyruvate decarboxylase PDC1, required for cell growth and concomitant ethanol production. Switching to darkness transcriptionally inactivates PDC1 and instead activates the anti-Pdc1p nanobody, NbJRI, to act as a genetically encoded inhibitor of Pdc1p accumulated during the growth phase. This dual transcriptional/post-translational control improves the production of 2,3-BDO and citramalate by up to 100 and 92% compared to using transcriptional controls alone in dynamic two-phase fermentations. This study establishes the NbJRI nanobody as an effective genetically encoded inhibitor of Pdc1p that can enhance the production of pyruvate-derived chemicals.

Abstract: Keeping condensates in liquid-like states throughout the biosynthesis process in microbial cell factories remains an ongoing challenge. Here, we present a light-controlled phase regulator, which maintains the liquid-like features of synthetic condensates on demand throughout the biosynthesis process upon light induction, as demonstrated by various live cell-imaging techniques. Specifically, the tobacco etch virus (TEV) protease controlled by light cleaves intrinsically disordered proteins (IDPs) to alter their valency and concentration for controlled phase transition and programmable fluidity of cellular condensates. As a proof of concept, we harness this capability to significantly improve the production of squalene and ursolic acid (UA) in engineered Saccharomyces cerevisiae. Our work provides a powerful approach to program the solid-liquid phase transition of biomolecular condensates for improved biosynthesis.

Abstract: Motile cilia perform crucial functions during embryonic development and in adult tissues. They are anchored by an apical actin network that forms microridge-like structures on the surface of multiciliated cells. Using Xenopus as a model system to investigate the mechanisms underlying the formation of these specialized actin structures, we observed stochastic bursts of intracellular calcium concentration in developing multiciliated cells. Through optogenetic manipulation of calcium signaling, we found that individual calcium bursts triggered the fusion and extension of actin structures by activating non-muscle myosin. Repeated cycles of calcium activation promoted assembly and coherence of the maturing apical actin network. Inhibition of the endogenous inositol triphosphate-calcium pathway disrupted the formation of apical actin/microridge-like structures by reducing local centriolar RhoA signaling. This disruption was rescued by transient expression of constitutively active RhoA in multiciliated cells. Our findings identify repetitive calcium bursts as a driving force that promotes the self-organization of the highly specialized actin cytoskeleton of multiciliated cells.

Abstract: Millions of ribosomes are packed within mammalian cells, yet we lack tools to visualize them in toto and characterize their subcellular composition. In this study, we present ribosome expansion microscopy (RiboExM) to visualize individual ribosomes and an optogenetic proximity-labeling technique (ALIBi) to probe their composition. We generated a super-resolution ribosomal map, revealing subcellular translational hotspots and enrichment of 60S subunits near polysomes at the endoplasmic reticulum (ER). We found that Lsg1 tethers 60S to the ER and regulates translation of select proteins. Additionally, we discovered ribosome heterogeneity at mitochondria guiding translation of metabolism-related transcripts. Lastly, we visualized ribosomes in neurons, revealing a dynamic switch between monosomes and polysomes in neuronal translation. Together, these approaches enable exploration of ribosomal localization and composition at unprecedented resolution.

Abstract: The binding of photoswitchable molecules to partners forms the basis of many naturally occurring light-dependent signaling pathways and various photopharmacological and optogenetic tools. A critical parameter affecting the function of these molecules is the thermal half-life of the light state. Reports in the literature indicate that, in some cases, a binding partner can significantly influence the thermal half-life, while in other cases it has no effect. Here, we present a unifying framework for quantitatively analyzing the effects of binding partners on thermal reversion rates. We focus on photoswitchable protein/binder interactions involving LOV domains, photoactive yellow protein, and CBCR GAF domains with partners that bind either the light or the dark state of the photoswitchable domain. We show that the effect of a binding partner depends on the extent to which the transition state for reversion resembles the dark state or the light state. We quantify this resemblance with a ϕswitching value, where ϕswitching = 1 if the conformation of the part of the photoswitchable molecule that interacts with the binding partner closely resembles its dark state conformation and ϕswitching = 0 if it resembles its light state. In addition to providing information on the transition state for switching, this analysis can guide the design of photoswitchable systems that retain useful thermal half-lives in practice. The analysis also provides a basis for the use of simple kinetic measurements to determine effective changes in affinity even in complex milieu.

Abstract: Light-based immunotherapy uses specific wavelengths of light to activate or modulate immune responses. It primarily employs two mechanisms: direct activation of immune cells and indirect modulation of the tumor microenvironment (TME). Several light-based technologies are under investigation or clinical use in immunotherapy, including photodynamic immunotherapy (PDIT) and photothermal therapy (PTT). Optogenetic tools have the potential to precisely control T-cell receptor activation, cytokine release, or the activity of other immune effector cells. Light-based technologies present innovative opportunities within the realm of immunotherapy. The ability to precisely regulate immune cell activation via optogenetics, alongside the improved targeting of cancer cells through photoimmunotherapy, signifies a transformative shift in our strategies for immune modulation. Although many of these technologies remain in the experimental stage for various applications, initial findings are encouraging, especially concerning cancer treatment and immune modulation. Continued research and clinical trials are essential to fully harness the capabilities of light technology in the context of immune cell therapy.

Abstract: Optogenetics has substantially enhanced our understanding of biological processes by enabling high-precision tracking and manipulation of individual cells. It relies on photosensitive proteins to monitor and control cellular activities, thereby paving the way for significant advancements in complex system research. Photosensitive proteins play a vital role in the development of optogenetics, facilitating the establishment of cutting-edge methods. Recent breakthroughs in protein design have opened up opportunities to develop protein-based tools that can precisely manipulate and monitor cellular activities. These advancements will significantly accelerate the development and application of optogenetic tools. This article emphasizes the pivotal role of protein design in the development of optogenetic tools, offering insights into potential future directions. We begin by providing an introduction to the historical development and fundamental principles of optogenetics, followed by an exploration of the operational mechanisms of key photosensitive domains, which includes clarifying the conformational changes they undergo in response to light, such as allosteric modulation and dimerization processes. Building on this foundation, we reveal the development of protein design tools that will enable the creation of even more sophisticated optogenetic techniques.

Abstract: With the development of metabolic engineering, increasing requirements for efficient microbial biosynthesis call for establishment of multi-strain co-culture system. Dynamic regulation of population ratios is crucial for optimizing bioproduction performance. Optogenetic systems with high universality and flexibility have the potential to realize dynamic control of population proportion. In this study, we utilized an optimized chromatic acclimation sensor/regulator (CcaS/R) system and a blue light-activated YF1-FixJ-PhlF system as induction modules. A pair of orthogonal quorum sensing systems and a toxin-antitoxin system were employed as communication module and effector module, respectively. By integrating these modules, we developed a dual light-controlled co-culture system that enables dynamic regulation of population ratios. This co-culture system provides a universal toolkit for applications in metabolic engineering and synthetic biology.

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, non-transcriptional 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 RasGEFs generally increase cortical tension while RasGAPs 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: The wine strains of Saccharomyces cerevisiae transform glucose into ethanol and other byproducts such as glycerol and acetate. The balance of these metabolites is important during the fermentation process, which impacts the organoleptic properties of wines. Ethanol and glycerol productions are mainly controlled by the ADH1 and GPD1 genes, which encode for the alcohol dehydrogenase and glycerol-3-phosphate-dehydrogenase enzymes, respectively. Genetic modification of these genes can thus be used to alter the levels of the corresponding metabolites and to reroute fermentation. In this work, we used an optogenetic system named FUN-LOV (FUNgal-Light Oxygen Voltage) to regulate the expression of ADH1 and GPD1 in a wine yeast strain using light. Initially, we confirmed the light-controlled expression of GPD1 and ADH1 in the engineered strains via RT-qPCR and a translational reporter, respectively. To characterize the generated yeast strains, we performed growth curve assays and laboratory-scale fermentations, observing phenotypic differences between illumination conditions that confirm the optogenetic control of the target genes. We also monitored glucose consumption and ethanol and glycerol productions during a fermentation time course, observing that the optogenetic control of GPD1 increased glycerol production under constant illumination without affecting ethanol production. Interestingly, the optogenetic control of ADH1 showed an inverted phenotype, where glycerol production increased under constant darkness conditions. Altogether, our results highlight the feasibility of using optogenetic tools to control yeast fermentation in a wine yeast strain, which allows changing the balance of metabolic products of interest in a light-dependent manner.

Abstract: Optogenetics is an emerging technology that uses the light-responsive effects of photosensitive proteins to regulate the function of specific cells. This technique combines genetics with optics, allowing for the precise inhibition or activation of cell functions through the introduction of photosensitive proteins into target cells and subsequent light stimulation to activate these proteins. In recent years, numerous basic and clinical studies have demonstrated the unique advantages of this approach in the research and treatment of neurological disorders. This review aims to introduce the fundamental principles and techniques of optogenetics, as well as its applications in the research and treatment of neurological diseases.

Abstract: No organism is an island: organisms of varying taxonomic complexity, including genetic variants of a single species, can coexist in particular niches, cooperating for survival while simultaneously competing for environmental resources. In recent years, synthetic biology strategies have witnessed a surge of efforts focused on creating artificial microbial communities to tackle pressing questions about the complexity of natural systems and the interactions that underpin them. These engineered ecosystems depend on the number and nature of their members, allowing complex cell communication designs to recreate and create diverse interactions of interest. Due to its experimental simplicity, the budding yeast Saccharomyces cerevisiae has been harnessed to establish a mixture of varied cell populations with the potential to explore synthetic ecology, metabolic bioprocessing, biosensing, and pattern formation. Indeed, engineered yeast communities enable advanced molecule detection dynamics and logic operations. Here, we present a concise overview of the state-of-the-art, highlighting examples that exploit optogenetics to manipulate, through light stimulation, key yeast phenotypes at the community level, with unprecedented spatial and temporal regulation. Hence, we envision a bright future where the application of optogenetic approaches in synthetic communities (optoecology) illuminates the intricate dynamics of complex ecosystems and drives innovations in metabolic engineering strategies.

Abstract: The actin cytoskeleton and its nanoscale organization are central to all eukaryotic cells-powering diverse cellular functions including morphology, motility, and cell division-and is dysregulated in multiple diseases. Historically studied largely with purified proteins or in isolated cells, tools to study cell type-specific roles of actin in multicellular contexts are greatly needed. DeActs are recently created, first-in-class genetic tools for perturbing actin nanostructures and dynamics in specific cell types across diverse eukaryotic model organisms. Here, ChiActs are introduced, the next generation of actin-perturbing genetic tools that can be rapidly activated in cells and optogenetically targeted to distinct subcellular locations using light. ChiActs are composed of split halves of DeAct-SpvB, whose potent actin disassembly-promoting activity is restored by chemical-induced dimerization or allosteric switching. It is shown that ChiActs function to rapidly induce actin disassembly in several model cell types and are able to perturb actin-dependent nano-assembly and cellular functions, including inhibiting lamellipodial protrusions and membrane ruffling, remodeling mitochondrial morphology, and reorganizing chromatin by locally constraining actin disassembly to specific subcellular compartments. ChiActs thus expand the toolbox of genetically-encoded tools for perturbing actin in living cells, unlocking studies of the many roles of actin nano-assembly and dynamics in complex multicellular systems.

Abstract: Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.

Abstract: Inducible protein switches are currently limited for use in tissues and organisms because common inducers cannot be controlled with precision in space and time in optically dense settings. Here, we introduce a protein that can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization using temperature) oligomerizes and translocates to the plasma membrane when temperature is lowered. We generated a library of Melt variants with switching temperatures ranging from 30 °C to 40 °C, including two that operate at and above 37 °C. Melt was a highly modular actuator of cell function, permitting thermal control over diverse processes including signaling, proteolysis, nuclear shuttling, cytoskeletal rearrangements and cell death. Finally, Melt permitted thermal control of cell death in a mouse model of human cancer. Melt represents a versatile thermogenetic module for straightforward, non-invasive and spatiotemporally defined control of mammalian cells with broad potential for biotechnology and biomedicine.

Abstract: Photosynthetic cyanobacteria can be utilised in biotechnology as environmentally sustainable cell factories to convert CO2 into a diverse range of biochemicals. However, a lack of molecular tools available for precise and dynamic control of gene expression hinders metabolic engineering and contributes to low product titres. Optogenetic tools enable light-regulated control of gene expression with high tunability and reversibility. To date, their application in cyanobacteria is limited and transferability between species remains unclear. In this study, we expressed the blue light-repressible YF1/FixJ and the green/red light-responsive CcaS/CcaR systems in Synechococcus sp. PCC 7002 and characterised their performance using GFP fluorescence assays and qRT-PCR. The YF1/FixJ system of non-cyanobacterial origin showed poor performance with a maximum dynamic range of 1.5-fold despite several steps to improve this. By contrast, the CcaS/CcaR system originating from the cyanobacterium Synechocystis sp. PCC 6803 responded well to light wavelengths and intensities, with a 6-fold increased protein fluorescence output observed after 30 min of green light. Monitoring GFP transcript levels allowed us to quantify the kinetics of transcriptional activation and deactivation and to test the effect of both multiple green/red and light/dark cycles on system performance. Finally, we increased CcaS/CcaR system activity under green light through targeted genetic modifications to the pCpcG2 output promoter. This study provides a detailed characterisation of the behaviour of the CcaS/CcaR system in Synechococcus sp. PCC 7002, as well as underlining the complexity of transferring optogenetic tools across species.

Abstract: Oscillatory dynamics and their modulation are crucial for cellular decision-making; however, analysing these dynamics remains challenging. Here, we present a tool that combines the light-activated adenylate cyclase mPAC with the cAMP biosensor Pink Flamindo, enabling precise manipulation and real-time monitoring of cAMP oscillation frequencies in Dictyostelium. High-frequency modulation of cAMP oscillations induced cell aggregation and multicellular formation, even at low cell densities, such as a few dozen cells. At the population level, chemotactic aggregation is driven by modulated frequency signals. Additionally, modulation of cAMP frequency significantly reduced the amplitude of the shuttling behaviour of the transcription factor GtaC, demonstrating low-pass filter characteristics capable of converting subtle oscillation changes, such as from 6 min to 4 min, into gene expression. These findings enhance our understanding of frequency-selective cellular decoding and its role in cellular signalling and development.

Abstract: Cerebral organoids pioneered in replicating complex brain tissue architectures in vitro, offering a vast potential for human disease modeling. They enable the in vitro study of human physiological and pathophysiological mechanisms of various neurological diseases and disorders. The trajectory of technological advancements in brain organoid generation and engineering over the past decade indicates that the technology might, in the future, mature into indispensable solutions at the horizon of personalized and regenerative medicine. In this review, we highlight recent advances in the engineering of brain organoids as disease models and discuss some of the challenges and opportunities for future research in this rapidly evolving field.

Abstract: Cells contain thousands of different lipids. Their rapid and redundant metabolism, dynamic movement, and many interactions with other biomolecules have justly earned lipids a reputation as a vexing class of molecules to understand. Further, as the cell’s hydrophobic metabolites, lipids assemble into supramolecular structures─most commonly bilayers, or membranes─from which they carry out myriad biological functions. Motivated by this daunting complexity, researchers across disciplines are bringing order to the seeming chaos of biological lipids and membranes. Here, we formalize these efforts as “synthetic lipid biology”. Inspired by the idea, central to synthetic biology, that our abilities to understand and build biological systems are intimately connected, we organize studies and approaches across numerous fields to create, manipulate, and analyze lipids and biomembranes. These include construction of lipids and membranes from scratch using chemical and chemoenzymatic synthesis, editing of pre-existing membranes using optogenetics and protein engineering, detection of lipid metabolism and transport using bioorthogonal chemistry, and probing of lipid–protein interactions and membrane biophysical properties. What emerges is a portrait of an incipient field where chemists, biologists, physicists, and engineers work together in proximity─like lipids themselves─to build a clearer description of the properties, behaviors, and functions of lipids and membranes.