Curated Optogenetic Publication Database

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: Near-infrared (NIR) fluorescent proteins and optogenetic tools derived from bacterial phytochromes' photosensory core modules (PCMs) operate within the first (NIR-I) tissue transparency window under single-photon activation. Leveraging two-photon (2P) light in the second transparency window (NIR-II) for photoswitching bacterial phytochromes between Pr and Pfr absorption states offers significant advantages, including enhanced tissue penetration, spatial resolution, and signal-to-noise ratio. However, 2P photoconversion of bacterial phytochromes remains understudied. Here, we study the non-linear Pr to Pfr photoconversion's dependence on irradiation wavelength (1180–1360 nm) and energy fluence (41–339 mJ/cm2) for the PCM of DrBphP bacterial phytochrome. Our findings reveal substantially higher photoconversion efficiency for the engineered monomeric DrBphP-PCM (73%) compared to the natural dimeric DrBphP-PCM (57%). Molecular mechanical calculations, based on experimentally determined 2P absorption cross-section coefficients for the monomer (167 GM) and dimer (170 GM), further verify these results. We demonstrate both short- (SWE) and long-wavelength excitation (LWE) fluorescence of the Soret band using 405 and 810–890 nm laser sources, respectively. Under LWE, fluorescence emission (724 nm) exhibits saturation at a peak power density of 1.5 GW/cm2. For SWE, we observe linear degradation of fluorescence for both DrBphP-PCMs, decreasing by 32% as the temperature rises from 19 to 38°C. Conversely, under LWE, the monomeric DrBphP-PCM's brightness increases up to 182% (at 37°C), surpassing the dimeric form's fluorescence rise by 39%. These findings establish the monomeric DrBphP-PCM as a promising template for developing NIR imaging and optogenetic probes operating under the determined optimal parameters for its 2P photoconversion and LWE fluorescence.

Abstract: Background Cytoplasmic aggregation of TAR DNA binding protein 43 (TDP-43) in neurons is one of the hallmarks of TDP-43 proteinopathy. Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are closely associated with TDP-43 proteinopathy; however, it remains uncertain whether TDP-43 aggregation initiates the pathology or is a consequence of it. Methods To demonstrate the pathology of TDP-43 aggregation, we applied the optoDroplet technique in Caenorhabditis elegans (C. elegans), which allows spatiotemporal modulation of TDP-43 phase separation and assembly. Results We demonstrate that optogenetically induced TDP-43 aggregates exhibited insolubility similar to that observed in TDP-43 proteinopathy. These aggregates increased the severity of neurodegeneration, particularly in GABAergic motor neurons, and exacerbated sensorimotor dysfunction in C. elegans. Conclusions We present an optogenetic C. elegans model of TDP-43 proteinopathy that provides insight into the neuropathological mechanisms of TDP-43 aggregates. Our model serves as a promising tool for identifying therapeutic targets for TDP-43 proteinopathy.

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.

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: Post-translational modifications (PTMs) are indispensable modulators of protein activity. Most cellular behaviors, from cell division to cytoskeletal organization, are controlled by PTMs, their misregulation being associated with a plethora of human diseases. Traditionally, the role of PTMs has been studied employing biochemical techniques. However, these approaches fall short when studying PTM dynamics in vivo. In recent years, functionalized protein binders have allowed the PTM of endogenous proteins by bringing an enzymatic domain in close proximity to the protein they recognize. To date, most of these methods lack the temporal control necessary to understand the complex effects triggered by PTMs. In this study, we have developed a method to phosphorylate endogenous Myosin in a light-inducible manner. The method relies both on nanobody-targeting and light-inducible activation in order to achieve both tight specificity and temporal control. We demonstrate that this technology is able to disrupt cytoskeletal dynamics during Drosophila embryonic development. Together, our results highlight the potential of combining optogenetics and protein binders for the study of the proteome in multicellular systems.

Abstract: G protein-coupled receptors (GPCRs) are the largest class of receptors in the genome and control many signaling cascades essential for survival. GPCR signaling is regulated by β-arrestins, multifunctional adapter proteins that direct receptor desensitization, internalization, and signaling. While at many GPCRs, β-arrestins interact with a wide array of signaling effectors, it is unclear how β-arrestins promote such varied functions. Here we show that β-arrestins undergo liquid-liquid phase separation (LLPS) to form condensates that regulate GPCR function. We demonstrate that β-arrestin oligomerization occurs in proximity to the GPCR and regulates GPCR functions such as internalization and signaling. This model is supported by a cryoEM structure of the adhesion receptor ADGRE1 in a 2:2 complex with β-arrestin 1, with a β-arrestin orientation that can promote oligomerization. Our work provides a paradigm for β-arrestin condensates as regulators of GPCR function, with LLPS serving as an important promoter of signaling compartmentalization at GPCRs.

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: 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: A near-infrared optogenetic system was developed for the controlled expression of therapeutics in engineered oncolytic bacteria, demonstrating significant anti-tumor efficacy in multiple tumor mouse models. This approach offers a non-invasive, customizable method for targeted solid tumor therapy and has broader applications in engineered living therapeutics.

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: 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: While optogenetic tools have recently opened new avenues for controlling and understanding cellular behavior, Suh et al.1 present an effective strategy to regulate tissue densification and outgrowth through optogenetic control of EGFR. Their work ultimately uncovers fundamental principles that pave the way for improved tissue engineering approaches.

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: 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: Precise regulation of protein abundance is critical for cellular homeostasis, whose dysfunction may directly lead to human diseases. Optogenetics allows rapid and reversible control of precisely defined cellular processes, which has the potential to be utilized for regulation of protein dynamics at various scales. Here, we developed a novel optogenetics-based protein degradation system, namely Peptide-mediated OptoTrim-Away (POT) which employs expressed small peptides to effectively target endogenous and unmodified proteins. By engineering the light-induced oligomerization of the E3 ligase TRIM21, POT can rapidly trigger protein degradation via the proteasomal pathway. Our results showed that the developed POT-PI3K and POT-GPX4 modules, which used the iSH2 and FUNDC1 domains to specifically target phosphoinositide 3-kinase (PI3K) and glutathione peroxidase 4 (GPX4) respectively, were able to potently induce the degradation of these endogenous proteins by light. Both live-cell imaging and biochemical experiments validated the potency of these tools in downregulating cancer cell migration, proliferation, and even promotion of cell apoptosis. Therefore, we believe the POT offers an alternative and practical solution for rapid manipulation of endogenous protein levels, and it could potentially be employed to dissect complex signaling pathways in cell and for targeted cellular therapies.