Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 25 of 466 results

Selective induction of programmed cell death using synthetic biology tools.

blue green near-infrared red UV violet BLUF domains Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Semin Cell Dev Biol, 17 Aug 2023 DOI: 10.1016/j.semcdb.2023.07.012 Link to full text
Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Spatiotemporal optical control of Gαq-PLCβ interactions.

blue CRY2/CIB1 iLID HeLa RAW264.7 Signaling cascade control
bioRxiv, 12 Aug 2023 DOI: 10.1101/2023.08.10.552801 Link to full text
Abstract: Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington’s Disease, and Alzheimer’s Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro.

blue CRY2/CIB1 Magnets S. cerevisiae
J Vis Exp, 4 Aug 2023 DOI: 10.3791/65686 Link to full text
Abstract: Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro's components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Optogenetic strategies for optimizing the performance of biosensors of membrane phospholipids in live cells.

blue cpLOV2 CRY2/CIB1 CRY2/CRY2 LOVTRAP HEK293T HeLa Organelle manipulation
bioRxiv, 4 Aug 2023 DOI: 10.1101/2023.08.03.551799 Link to full text
Abstract: High-performance biosensors are crucial for elucidating the spatiotemporal regulatory roles and dynamics of membrane lipids, but there is a lack of improvement strategies for biosensors with low sensitivity and low-content substrates detection. Here we developed universal optogenetic strategies to improve a set of membrane biosensors by trapping them into specific region and further reducing the background signal, or by optically-controlled phase separation for membrane lipids detection and tracking. These improved biosensors were superior to typical tools and light simulation would enhance their detection performance and resolution, which might contribute to the design and optimization of other biosensors.

Design principles for engineering light-controlled antibodies.

blue red Cryptochromes LOV domains Phytochromes Review
Trends Biotechnol, 26 Jul 2023 DOI: 10.1016/j.tibtech.2023.06.006 Link to full text
Abstract: Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.

Lustro: High-Throughput Optogenetic Experiments Enabled by Automation and a Yeast Optogenetic Toolkit.

blue CRY2/CIB1 Magnets S. cerevisiae Transgene expression
ACS Synth Biol, 11 Jul 2023 DOI: 10.1021/acssynbio.3c00215 Link to full text
Abstract: Optogenetic systems use genetically encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light; however, these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to the high-throughput characterization of optogenetic systems across a range of biological systems and applications.

Optogenetic control of Cdc48 for dynamic metabolic engineering in yeast.

blue AsLOV2 CRY2/CIB1 S. cerevisiae Cell cycle control
Metab Eng, 7 Jul 2023 DOI: 10.1016/j.ymben.2023.06.013 Link to full text
Abstract: Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid β-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.

Mechanosensitive dynamics of lysosomes along microtubules regulate leader cell emergence in collective cell migration.

blue CRY2/CIB1 MDCK Control of cytoskeleton / cell motility / cell shape
bioRxiv, 4 Jul 2023 DOI: 10.1101/2022.08.03.502740 Link to full text
Abstract: Collective cell migration during embryonic development, wound healing, and cancer metastasis entails the emergence of leader cells at the migration front. These cells with conspicuous lamellipodial structures provide directional guidance to the collective. Despite their physiological relevance, the mechanisms underlying the emergence of leader cells remain elusive. Here we report that in diverse model systems for wound healing, including cultured epithelial monolayer, Drosophila embryo, and mouse embryonic skin, leader cells display a peripheral accumulation of lysosomes. This accumulation appears essential for leader cell emergence, involves lysosomal movement along microtubules, and depends on the actomyosin contractility-generated cellular forces. Peripheral lysosomes associate with inactive Rac1 molecules to remove them from the leading periphery, which increases local Rac1-activity, triggering actin polymerization and promoting lamellipodium formation. Taken together, we demonstrate that beyond their catabolic role, lysosomes act as the intracellular platform that links mechanical and biochemical signals to control the emergence of leader cells.

Concept and considerations of a medical device: the active noise cancelling incubator.

blue CRY2/CIB1 iLID TULIP D. discoideum HL-60 MCF10A RAW264.7 Control of cytoskeleton / cell motility / cell shape
Front Pediatr, 3 Jul 2023 DOI: 10.3389/fcell.2023.1195806 Link to full text
Abstract: An increasingly 24/7 connected and urbanised world has created a silent pandemic of noise-induced hearing loss. Ensuring survival to children born (extremely) preterm is crucial. The incubator is a closed medical device, modifying the internal climate, and thus providing an environment for the child, as safe, warm, and comfortable as possible. While sound outside the incubator is managed and has decreased over the years, managing the noise inside the incubator is still a challenge.

An optogenetic-phosphoproteomic study reveals dynamic Akt1 signaling profiles in endothelial cells.

blue CRY2/CIB1 EA.Hy926 HeLa HUVEC Signaling cascade control
Nat Commun, 26 Jun 2023 DOI: 10.1038/s41467-023-39514-1 Link to full text
Abstract: The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.

Shining a light on RhoA: Optical control of cell contractility.

blue Cryptochromes LOV domains Review
Int J Biochem Cell Biol, 20 Jun 2023 DOI: 10.1016/j.biocel.2023.106442 Link to full text
Abstract: In addition to biochemical and electrochemical signaling, cells also rely extensively on mechanical signaling to regulate their behavior. While a number of tools have been adapted from physics and engineering to manipulate cell mechanics, they typically require specialized equipment or lack spatiotemporal precision. Alternatively, a recent, more elegant approach is to use light itself to modulate the mechanical equilibrium inside the cell. This approach leverages the power of optogenetics, which can be controlled in a fully reversible manner in both time and space, to tune RhoA signaling, the master regulator of cellular contractility. We review here the fundamentals of this approach, including illustrating the tunability and flexibility that optogenetics offers, and demonstrate how this tool can be used to modulate both internal cytoskeletal flows and contractile force generation. Together these features highlight the advantages that optogenetics offers for investigating mechanical interactions in cells.

Optogenetic engineered umbilical cord MSC-derived exosomes for remodeling of the immune microenvironment in diabetic wounds and the promotion of tissue repair.

blue CRY2/CIB1 hMSCs Control of vesicular transport
J Nanobiotechnology, 2 Jun 2023 DOI: 10.1186/s12951-023-01886-3 Link to full text
Abstract: Angiogenesis and tissue repair in chronic non-healing diabetic wounds remain critical clinical problems. Engineered MSC-derived exosomes have significant potential for the promotion of wound healing. Here, we discuss the effects and mechanisms of eNOS-rich umbilical cord MSC exosomes (UCMSC-exo/eNOS) modified by genetic engineering and optogenetic techniques on diabetic chronic wound repair.

Optogenetic Activation of Ripk3 Reveals a Thresholding Mechanism in Intracellular and Intercellular Necroptosis.

blue CRY2/CIB1 CRY2/CRY2 CRY2clust CRY2olig PtAU1-LOV HEK293T NIH/3T3 Cell death
J Comput Soc Sci, 23 May 2023 DOI: 10.2139/ssrn.4453793 Link to full text
Abstract: Necroptosis is programmed cell death that involves active cytokine production and membrane ruptures. Whereas intracellular necroptosis has been extensively studied, intercellular propagation of necroptosis is much less understood. Pharmacological induction of necroptosis cannot delineate whether a necroptotic cell can propagate the death signal to its neighbor because of the confounding effect from the exogenously administrated death-inducers. To address this challenge, we develop an optogenetic system to enable ligand-free, optical induction of necroptosis at the single-cell level. This system, termed Light-activatable Receptor-Interacting Protein Kinase 3 or La-RIPK3, utilizes CRY2olig, a variant of the photoactivatable protein cryptochrome, to induce oligomerization of RIPK3 under blue light stimulation. Kinetic analysis La-RIPK3-activated cells shows that cytokine production and membrane rupture follows distinct kinetics. Moreover, membrane rupture requires a higher threshold of RIPK3 kinase activity than cytokine production. Intriguingly, intercellular propagation of necroptosis requires at least two proximal necroptotic cells, and a single necroptotic cell rarely induces such propagation. These results imply that RIPK3 acts as a gatekeeper to define the threshold of distinct functional outcomes of intracellular and intercellular necroptosis. Such a thresholding mechanism could allow cells to make informed decisions by evaluating the severity of environmental stress when walking a tightrope between committing an immunogenic suicidal fate and maintaining membrane integrity. This study highlights the role of RIPK3-containing necrosomes in regulating intracellular and intercellular necroptosis and offers an optimized optogenetic tool for investigating RIPK3-dependent necroptotic pathways.

Activity-based directed evolution of a membrane editor in mammalian cells.

blue CRY2/CIB1 HEK293T
Nat Chem, 22 May 2023 DOI: 10.1038/s41557-023-01214-0 Link to full text
Abstract: Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of 'superPLDs' with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

Actuation of single downstream nodes in growth factor network steers immune cell migration.

blue CRY2/CIB1 iLID D. discoideum HL-60 RAW264.7 Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Dev Cell, 22 May 2023 DOI: 10.1016/j.devcel.2023.04.019 Link to full text
Abstract: Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.

Optogenetic Methods in Plant Biology.

blue red UV BLUF domains CarH Cryptochromes Cyanobacteriochromes LOV domains Phytochromes UV receptors Review
Annu Rev Plant Biol, 22 May 2023 DOI: 10.1146/annurev-arplant-071122-094840 Link to full text
Abstract: Optogenetics is a technique employing natural or genetically engineered photoreceptors in transgene organisms to manipulate biological activities with light. Light can be turned on or off, and adjusting its intensity and duration allows optogenetic fine-tuning of cellular processes in a noninvasive and spatiotemporally resolved manner. Since the introduction of Channelrhodopsin-2 and phytochrome-based switches nearly 20 years ago, optogenetic tools have been applied in a variety of model organisms with enormous success, but rarely in plants. For a long time, the dependence of plant growth on light and the absence of retinal, the rhodopsin chromophore, prevented the establishment of plant optogenetics until recent progress overcame these difficulties. We summarize the recent results of work in the field to control plant growth and cellular motion via green light-gated ion channels and present successful applications to light-control gene expression with single or combined photoswitches in plants. Furthermore, we highlight the technical requirements and options for future plant optogenetic research.

Light-responsive nanomedicine for cancer immunotherapy.

blue Cryptochromes LOV domains Review
Acta Pharm Sin B, 19 May 2023 DOI: 10.1016/j.apsb.2023.05.016 Link to full text
Abstract: Immunotherapy emerged as a paradigm shift in cancer treatments, which can effectively inhibit cancer progression by activating the immune system. Remarkable clinical outcomes have been achieved through recent advances in cancer immunotherapy, including checkpoint blockades, adoptive cellular therapy, cancer vaccine, and tumor microenvironment modulation. However, extending the application of immunotherapy in cancer patients has been limited by the low response rate and side effects such as autoimmune toxicities. With great progress being made in nanotechnology, nanomedicine has been exploited to overcome biological barriers for drug delivery. Given the spatiotemporal control, light-responsive nanomedicine is of great interest in designing precise modality for cancer immunotherapy. Herein, we summarized current research utilizing light-responsive nanoplatforms to enhance checkpoint blockade immunotherapy, facilitate targeted delivery of cancer vaccines, activate immune cell functions, and modulate tumor microenvironment. The clinical translation potential of those designs is highlighted and challenges for the next breakthrough in cancer immunotherapy are discussed.

Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development.

blue CRY2/CIB1 zebrafish in vivo Control of cytoskeleton / cell motility / cell shape Developmental processes
PLoS Genet, 15 May 2023 DOI: 10.1371/journal.pgen.1010765 Link to full text
Abstract: An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-β signaling.

blue CRY2/CIB1 human IPSCs Signaling cascade control Cell differentiation
Cell Rep, 12 May 2023 DOI: 10.1016/j.celrep.2023.112509 Link to full text
Abstract: In tissue development and homeostasis, transforming growth factor (TGF)-β signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-β signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-β signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-β gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-β signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.

OptIC Notch reveals mechanism that regulates receptor interactions with CSL.

blue AsLOV2 CRY2/CIB1 D. melanogaster in vivo Signaling cascade control
Development, 12 May 2023 DOI: 10.1242/dev.201785 Link to full text
Abstract: Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes forming a complex with the DNA-binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{ω}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.

Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation.

blue CRY2/CIB1 C. elegans in vivo D. melanogaster in vivo HEK293A rat dorsal root ganglion NSCs zebrafish in vivo Signaling cascade control Developmental processes
bioRxiv, 8 May 2023 DOI: 10.1101/2023.05.06.539674 Link to full text
Abstract: Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, Photo-induced Modulation of Gα protein – Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. We alter the behavior of C. elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gαi signaling.

Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions.

bioRxiv, 6 May 2023 DOI: 10.1101/2022.10.22.513249 Link to full text
Abstract: Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the resolution and accuracy of proximity labeling methods are limited by a lack of control over the enzymatic labeling process. Here, we present a high spatial and temporal resolution technology that can be activated on demand using light, for high accuracy proximity labeling. Our system, called Light Activated BioID (LAB), is generated by fusing the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. Using live cell imaging, immunofluorescence, western blotting, and mass spectrometry, we show that upon exposure to blue light, CRY2 and CIB1 dimerize, reconstitute the split-TurboID enzyme, and biotinylate proximate proteins. Turning off the light halts the biotinylation reaction. We validate LAB in different cell types and demonstrate that it can identify known binding partners of proteins while reducing background labeling and false positives.

The clinical potential of optogenetic interrogation of pathogenesis.

blue cyan green red UV Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Clin Transl Med, May 2023 DOI: 10.1002/ctm2.1243 Link to full text
Abstract: Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye.

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos.

blue CRY2/CIB1 D. melanogaster in vivo
J Vis Exp, 14 Apr 2023 DOI: 10.3791/65314 Link to full text
Abstract: Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at multiple length scales, such as cell division, cell migration, epithelial folding, and branching morphogenesis. An in-depth understanding of the role of actomyosin contractility in morphogenesis requires approaches that allow the rapid inactivation of actomyosin, which is difficult to achieve using conventional genetic or pharmacological approaches. The presented protocol demonstrates the use of a CRY2-CIBN based optogenetic dimerization system, Opto-Rho1DN, to inhibit actomyosin contractility in Drosophila embryos with precise temporal and spatial controls. In this system, CRY2 is fused to the dominant negative form of Rho1 (Rho1DN), whereas CIBN is anchored to the plasma membrane. Blue light-mediated dimerization of CRY2 and CIBN results in rapid translocation of Rho1DN from the cytoplasm to the plasma membrane, where it inactivates actomyosin by inhibiting endogenous Rho1. In addition, this article presents a detailed protocol for coupling Opto-Rho1DN-mediated inactivation of actomyosin with laser ablation to investigate the role of actomyosin in generating epithelial tension during Drosophila ventral furrow formation. This protocol can be applied to many other morphological processes that involve actomyosin contractility in Drosophila embryos with minimal modifications. Overall, this optogenetic tool is a powerful approach to dissect the function of actomyosin contractility in controlling tissue mechanics during dynamic tissue remodeling.

Bioelectricity in Developmental Patterning and Size Control: Evidence and Genetically Encoded Tools in the Zebrafish Model.

blue AsLOV BLUF domains Cryptochromes LOV domains Review
Cells, 13 Apr 2023 DOI: 10.3390/cells12081148 Link to full text
Abstract: Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.
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