Curated Optogenetic Publication Database

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: S-Palmitoylation is a reversible post-translational modification that tunes the localization, stability, and function of an impressive array of proteins including ion channels, G-proteins, and synaptic proteins. Indeed, altered protein palmitoylation is linked to various human diseases including cancers, neurodevelopmental and neurodegenerative diseases. As such, strategies to selectively manipulate protein palmitoylation with enhanced temporal and subcellular precision are sought after to both delineate physiological functions and as potential therapeutics. Here, we develop chemogenetically and optogenetically inducible engineered depalmitoylases to manipulate the palmitoylation status of target proteins. We demonstrate that this strategy is programmable allowing selective depalmitoylation in specific organelles, triggered by cell-signaling events, and of individual protein complexes. Application of this methodology revealed bidirectional tuning of neuronal excitability by distinct depalmitoylases. Overall, this strategy represents a versatile and powerful method for manipulating protein palmitoylation in live cells, providing insights into their regulation in distinct physiological contexts.

Abstract: During mating in budding yeast, cells use pheromones to locate each other and fuse. This model system has shaped our current understanding of signal transduction and cell polarization in response to extracellular signals. The cell populations producing extracellular signal landscapes themselves are, however, less well understood, yet crucial for functionally testing quantitative models of cell polarization and for controlling cell behavior through bioengineering approaches. Here we engineered optogenetic control of pheromone landscapes in mating populations of budding yeast, hijacking the mating-pheromone pathway to achieve spatial control of growth, cell morphology, cell-cell fusion, and distance-dependent gene expression in response to light. Using our tool, we were able to spatially control and shape pheromone gradients, allowing the use of a biophysical model to infer the properties of large-scale gradients generated by mating populations in a single, quantitative experimental setup, predicting that the shape of such gradients depends quantitatively on population parameters. Spatial optogenetic control of diffusible signals and their degradation provides a controllable signaling environment for engineering artificial communication and cell-fate systems in gel-embedded cell populations without the need for physical manipulation.

Abstract: Cytokinesis challenges epithelial tissue homeostasis by generating forces that pull on neighboring cells. Junction reinforcement at the furrow in Xenopus epithelia regulates the speed of furrowing, suggesting that cytokinesis is subject to resistive forces from epithelial neighbors. We show that contractility factors accumulate near the furrow in neighboring cells, and increasing neighbor cell stiffness slows furrowing. Optogenetically increasing contractility in one or both neighbor cells slows furrowing or induces cytokinetic failure. Uncoupling mechanotransduction between dividing cells and their neighbors increases the furrow ingression rate, alters topological cell packing following cytokinesis, and impairs barrier function at the furrow. Computational modeling validates our findings and provides additional insights about epithelial mechanics during cytokinesis. We conclude that forces from the cytokinetic array must be carefully balanced with restraining forces generated by neighbor cells to regulate the speed and success of cytokinesis and maintain epithelial homeostasis.

Abstract: Studying the physical properties of sub-cellular components is increasingly important in understanding cell mechanics. This review focuses on the most advanced techniques available for investigating intracellular mechanics. We distinguish methods that act as force generators and those that act as force sensors. We highlight six state-of-the-art techniques, with increased spatial and temporal resolutions: optogenetics, Brillouin microscopy, bacterial cells and nanorobots, optical tweezers, membrane tension probes, and magnetic particles.

Abstract: Chickens serve as an excellent model organism for developmental biology, offering unique opportunities for precise spatiotemporal access to embryos within eggs. Optogenes are light-activated proteins that regulate gene expression, offering a non-invasive method to activate genes at specific locations and developmental stages, advancing developmental biology research. This study employed the Magnet-Cre optogenetic system to control gene expression in developing chicken embryos. Magnet-Cre consists of two light-sensitive protein domains that dimerize upon light activation, each attached to an inactive half of the Cre recombinase enzyme, which becomes active upon dimerization. We developed an all-in-one plasmid containing a green fluorescent protein marker, the Magnet-Cre system, and a light-activated red fluorescent protein gene. This plasmid was electroporated into the neural tube of Hamburger and Hamilton (H&H) stage 14 chicken embryos. Embryo samples were cleared using the CUBIC protocol and imaged with a light sheet microscope to analyze optogenetic activity via red-fluorescent cells. We established a pipeline for Magnet-Cre activation in chicken embryos, demonstrating that a single 3-min exposure to blue light following incubation at 28 °C was sufficient to trigger gene activity within the neural tube, with increased activity upon additional light exposure. Finally, we showed a spatiotemporal control of gene activity using a localized laser light induction. This research lays the groundwork for further advancements in avian developmental biology and poultry research, enabling spatiotemporal control of genes in both embryos and transgenic chickens.

Abstract: Adrenoceptors (ARs) play a vital role in various physiological processes and are key therapeutic targets. The advent of optical control techniques, including optogenetics and photopharmacology, offers the potential to modulate AR signaling with precise temporal and spatial resolution. In this review, we summarize the latest advancements in the optical control of AR signaling, encompassing optogenetics, photocaged compounds, and photoswitchable compounds. We also discuss the limitations of current tools and provide an outlook on the next generation of optogenetic and photopharmacological tools. These emerging optical technologies not only enhance our understanding of AR signaling but also pave the way for potential therapeutic developments.

Abstract: Cancer cell invasion relies on dynamic cell shape changes, which originate from protrusive and contractile intracellular forces. Previous studies revealed that contractile forces are controlled by positive-feedback amplification of the contraction regulator Rho by Lbc GEFs. These GEFs were previously linked to tumor progression, however, the underlying mechanisms are poorly understood. Here, we generated a mouse melanoma model, in which cytosolic levels of the Lbc GEF GEF-H1 are controlled by light. Using this model, we found that increased GEF-H1 levels strongly stimulate cell contraction dynamics. Interestingly, increased contraction dynamics rapidly induced expansion of tumor spheroids via a focal adhesion kinase-dependent mechanism. Furthermore, long-term stimulation led to the escape of individual cells from spheroids. These findings reveal new insights into the oncogenic roles of Lbc GEFs, and how they might promote tumor cell invasion. We propose a mechanism, in which increased cell contraction dynamics results in asymmetric pulling forces at the tumor border, promoting the detachment and escape of individual cells.

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: Cell migration is crucial for development and tissue homeostasis, while its dysregulation leads to severe pathologies. Cell migration is driven by the extension of actin-based lamellipodia protrusions, powered by actin polymerization, which is tightly regulated by signaling pathways, including Rho GTPases and Ca2+ signaling. While the importance of Ca2+ signaling in lamellipodia protrusions has been established, the molecular mechanisms linking Ca2+ to lamellipodia assembly are unknown. Here, we identify a novel Ca2+ signaling axis involving the mechano-gated channel TRPV4, which regulates lamellipodia protrusions in various cell types. Using Ca2+ and FRET imaging, we demonstrate that TRPV4-mediated Ca2+ influx upregulates RhoA activity within lamellipodia, which then facilitates formin-mediated actin assembly. Mechanistically, we identify CaMKII and TEM4 as key mediators relaying the TRPV4-mediated Ca2+ signal to RhoA. These data define a molecular pathway by which Ca2+ influx regulates small GTPase activity within a specific cellular domain – lamellipodia - and demonstrate the critical role in organizing the actin machinery and promoting cell migration in diverse biological contexts.

Abstract: Ferroptosis is a lytic, iron-dependent form of regulated cell death characterized by excessive lipid peroxidation and associated with necrosis spread in diseased tissues through unknown mechanisms. Using a novel optogenetic system for light-driven ferroptosis induction via degradation of the anti-ferroptotic protein GPX4, we show that lipid peroxidation and ferroptotic death can spread to neighboring cells through their closely adjacent plasma membranes. Ferroptosis propagation is dependent on cell distance and completely abolished by disruption of α-catenin-dependent intercellular contacts or by chelation of extracellular iron. Remarkably, bridging cells with a lipid bilayer or increasing contacts between neighboring cells enhances ferroptosis spread. Reconstitution of iron-dependent spread of lipid peroxidation between pure lipid, contacting liposomes provides evidence for the physicochemical mechanism involved. Our findings support a model in which iron-dependent lipid peroxidation propagates across proximal plasma membranes of neighboring cells, thereby promoting the transmission of ferroptotic cell death with consequences for pathological tissue necrosis spread.

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: 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: 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: 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: Chromatin compaction defines genome topology, evolution, and function. The Saccharomycotina subphylum, including the fermenting yeast Saccharomyces cerevisiae have a decompacted genome, possibly because they lost two genes mediating a specific histone lysine methylation and histone binding protein heterochromatin protein 1 (HP1). This decompaction may result in the higher-than-expected mutation and meiotic recombination rates observed in this species. To test this hypothesis, we retro-engineered S. cerevisiae to compact the genome by expressing the HP1 homologue of Schizosaccharomyces pombe SpSwi6 and H3K9 methyltransferase SpClr4. The resulting strain had significantly more compact chromatin and reduced rates of mutation and meiotic recombination. The increased genomic stability significantly prolongs the optogenetic control of an engineered strain designed to grow only in blue light. This result demonstrates the potential of our approach to enhance the stability of strains for metabolic engineering and other synthetic biology applications, which are prone to lose activities due to genetic instability.

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.