Curated Optogenetic Publication Database

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.

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.

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.

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.

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.

Abstract: The concept of phase-separation-mediated formation of biomolecular condensates provides a new framework to understand cellular organization and cooperativity-dependent cellular functions. With growing understanding of how biological systems drive phase separation and how cellular functions are encoded by biomolecular condensates, opportunities have emerged for cellular control through engineering of synthetic biomolecular condensates. In this Review, we discuss how to construct synthetic biomolecular condensates and how they can regulate cellular functions. We first describe the fundamental principles by which biomolecular components can drive phase separation. Next, we discuss the relationship between the properties of condensates and their cellular functions, which informs the design of components to create programmable synthetic condensates. Finally, we describe recent applications of synthetic biomolecular condensates for cellular control and discuss some of the design considerations and prospective applications.

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.

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.

Abstract: The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

Abstract: Biomolecular condensates participate in the regulation of gene transcription, yet the relationship between nuclear condensation and transcriptional activation remains elusive. Here, we devised a biotinylated CRISPR-dCas9-based optogenetic method, light-activated macromolecular phase separation (LAMPS), to enable inducible formation, affinity purification, and multiomic dissection of nuclear condensates at the targeted genomic loci. LAMPS-induced condensation at enhancers and promoters activates endogenous gene transcription by chromatin reconfiguration, causing increased chromatin accessibility and de novo formation of long-range chromosomal loops. Proteomic profiling of light-induced condensates by dCas9-mediated affinity purification uncovers multivalent interaction-dependent remodeling of macromolecular composition, resulting in the selective enrichment of transcriptional coactivators and chromatin structure proteins. Our findings support a model whereby the formation of nuclear condensates at native genomic loci reconfigures chromatin architecture and multiprotein assemblies to modulate gene transcription. Hence, LAMPS facilitates mechanistic interrogation of the relationship between nuclear condensation, genome structure, and gene transcription in living cells.

Abstract: Optogenetic techniques permit non-invasive, spatiotemporal, and reversible modulation of cellular activities. Here, we report a novel optogenetic regulatory system for insulin secretion in human pluripotent stem cell (hPSC)-derived pancreatic islet-like organoids using monSTIM1 (monster-opto-Stromal interaction molecule 1), an ultra-light-sensitive OptoSTIM1 variant. The monSTIM1 transgene was incorporated at the AAVS1 locus in human embryonic stem cells (hESCs) by CRISPR-Cas9-mediated genome editing. Not only were we able to elicit light-induced intracellular Ca2+ concentration ([Ca2+]i) transients from the resulting homozygous monSTIM1+/+-hESCs, but we also successfully differentiated them into pancreatic islet-like organoids (PIOs). Upon light stimulation, the β-cells in these monSTIM1+/+-PIOs displayed reversible and reproducible [Ca2+]i transient dynamics. Furthermore, in response to photoexcitation, they secreted human insulin. Light-responsive insulin secretion was similarly observed in monSTIM1+/+-PIOs produced from neonatal diabetes (ND) patient-derived induced pluripotent stem cells (iPSCs). Under LED illumination, monSTIM1+/+-PIO-transplanted diabetic mice produced human c-peptide. Collectively, we developed a cellular model for the optogenetic control of insulin secretion using hPSCs, with the potential to be applied to the amelioration of hyperglycemic disorders.

Abstract: Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.

Abstract: Barrier tissues such as the epidermis employ complex signal transduction systems to execute morphogenetic programs and to rapidly respond to environmental cues to promote homeostasis. Recent advances in live-imaging techniques and tools allow precise spatial and temporal monitoring and manipulation of intracellular signaling cascades. Leveraging the chemistry of naturally occurring light-sensitive proteins, genetically encoded fluorescent biosensors have emerged as robust tools for visualizing dynamic signaling events. In contrast, optogenetic protein constructs permit laser-mediated control of signal receptors and effectors within live cells, organoids, and even model organisms. In this paper, we review the basic principles underlying novel biosensors and optogenetic tools and highlight how recent studies in cutaneous biology have leveraged these imaging strategies to illuminate the spatiotemporal signals regulating epidermal development, barrier formation, and tissue homeostasis.

Abstract: Prostaglandin E2 (PGE2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors we demonstrate that calcium transients in a single cell cause PGE2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE2 discharge in response to the increasing cytoplasmic Ca2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The ERK MAP kinase activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE2 discharge is regulated temporally by calcium transients and ERK activity.

Abstract: Insulin regulates various cellular metabolic processes by activating specific isoforms of the Akt family of kinases. Here, we elucidated metabolic pathways that are regulated in an Akt2-dependent manner. We constructed a transomics network by quantifying phosphorylated Akt substrates, metabolites, and transcripts in C2C12 skeletal muscle cells with acute, optogenetically induced activation of Akt2. We found that Akt2-specific activation predominantly affected Akt substrate phosphorylation and metabolite regulation rather than transcript regulation. The transomics network revealed that Akt2 regulated the lower glycolysis pathway and nucleotide metabolism and cooperated with Akt2-independent signaling to promote the rate-limiting steps in these processes, such as the first step of glycolysis, glucose uptake, and the activation of the pyrimidine metabolic enzyme CAD. Together, our findings reveal the mechanism of Akt2-dependent metabolic pathway regulation, paving the way for Akt2-targeting therapeutics in diabetes and metabolic disorders.

Abstract: The mechanical properties of biological tissues are key to their physical integrity and function. Although external loading or biochemical treatments allow the estimation of these properties globally, it remains difficult to assess how such external stimuli compare with cell-generated contractions. Here we engineer microtissues composed of optogenetically-modified fibroblasts encapsulated within collagen. Using light to control the activity of RhoA, a major regulator of cellular contractility, we induce local contractions within microtissues, while monitoring microtissue stress and strain. We investigate the regulation of these local contractions and their spatio-temporal distribution. We demonstrate the potential of our technique for quantifying tissue elasticity and strain propagation, before examining the possibility of using light to create and map local anisotropies in mechanically heterogeneous microtissues. Altogether, our results open an avenue to guide the formation of tissues while non-destructively charting their rheology in real time, using their own constituting cells as internal actuators.

Abstract: Bcl-2-associated X protein (BAX) plays a vital role in maintaining tissue homeostasis and participates in the pathogenesis of various diseases. Poor spatiotemporal control remains a challenge in direct pharmacological modulation and genetic perturbation of BAX’s activity. Herein, we developed a near-infrared (NIR) light-inducible BAX (NiBAX) system that enabled remote and spatiotemporal control of BAX-mediated apoptosis. The NiBAX was constructed by integration of two independent modules: blue light-responsive optogenetics BAX plasmids for regulating migration of BAX to mitochondria and upconversion nanoparticles-encapsulated flexible implant for converting tissue-penetrative NIR light into blue light. This NiBAX could readily induce robust BAX-based cellular apoptosis in vitro, and elicit effective apoptosis-mediated oncotherapy in vivo under NIR light. Collectively, the upconversion optogenetic NiBAX system provides an advanced tool for BAX-related cellular behavior control.

Abstract: The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. Here we report that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. We demonstrate this by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. We can thereby engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. Our findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.

Abstract: T cells become activated following one or multiple contacts with antigen-presenting cells. Calcium influx is a key signaling event elicited during these cellular interactions; however, it is unclear whether T cells recall and integrate calcium signals elicited during temporally separated contacts. To study the integration of calcium signals, we designed a programmable, multiplex illumination strategy for temporally patterned optogenetics (TEMPO). We found that a single round of calcium elevation was insufficient to promote nuclear factor of activated T cells (NFAT) activity and cytokine production in a T cell line. However, robust responses were detected after a second identical stimulation even when signals were separated by several hours. Our results suggest the existence of a biochemical memory of calcium signals in T cells that favors signal integration during temporally separated contacts and promote cytokine production. As illustrated here, TEMPO is a versatile approach for dissecting temporal integration in defined signaling pathways.

Abstract: RhoGTPases are well known for being controllers of cell cytoskeleton and share common features in the way they act and are controlled. These include their switch from GDP to GTP states, their regulations by different guanine exchange factors (GEFs), GTPase-activating proteins and guanosine dissociation inhibitors (GDIs), and their similar structure of active sites/membrane anchors. These very similar features often lead to the common consideration that the differences in their biological effects mainly arise from the different types of regulators and specific effectors associated with each GTPase. Focusing on data obtained through biosensors, live cell microscopy and recent optogenetic approaches, we highlight in this review that the regulation of RhoA appears to depart from Cdc42 and Rac1 modes of regulation through its enhanced lability at the plasma membrane. RhoA presents a high dynamic turnover at the membrane that is regulated not only by GDIs but also by GEFs, effectors and a possible soluble conformational state. This peculiarity of RhoA regulation may be important for the specificities of its functions, such as the existence of activity waves or its putative dual role in the initiation of protrusions and contractions.

Abstract: Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

Abstract: Membraneless condensates, such as stress granules (SGs) and processing bodies (P-bodies), have attracted wide attention due to their unique feature of rapid response to stress without first requiring nuclear feedback. In this study, we identify diaphanous-related formin 3 (DIAPH3), an actin nucleator, as a scaffold protein to initiate liquid-liquid phase separation (LLPS) and form abundant cytosolic phase-separated DIAPH3 granules (D-granules) in mammalian cells such as HeLa, HEK293, and fibroblasts under various stress conditions. Neither mRNAs nor known stress-associated condensate markers, such as G3BP1, G3BP2, and TIA1 for SGs and DCP1A for P-bodies, are detected in D-granules. Using overexpression and knockout of DIAPH3, pharmacological interventions, and optogenetics, we further demonstrate that stress-induced D-granules spatially sequester DIAPH3 within the condensation to inhibit the assembly of actin filaments in filopodia. This study reveals that D-granules formed by LLPS act as a regulatory hub for actin cytoskeletal remodeling in response to stress.

Abstract: The initiation of apical-basal (AB) polarity and the process of mitotic cell division are both characterised by the generation of specialised plasma membrane and cortical domains. These are generated using shared mechanisms, such as asymmetric protein accumulation, Rho GTPase signalling, cytoskeletal reorganisation, vesicle trafficking and asymmetric phosphoinositide distribution. In epithelial tissue, the coordination of AB polarity and mitosis in space and time is important both during initial epithelial development and to maintain tissue integrity and ensure appropriate cell differentiation at later stages. Whilst significant progress has been made in understanding the mechanisms underlying cell division and AB polarity, it has so far been challenging to fully unpick the complex interrelationship between polarity, signalling, morphogenesis, and cell division. However, the recent emergence of optogenetic protein localisation techniques is now allowing researchers to reversibly control protein activation, localisation and signalling with high spatiotemporal resolution. This has the potential to revolutionise our understanding of how subcellular processes such as apical-basal polarity are integrated with cell behaviours such as mitosis and how these processes impact whole tissue morphogenesis. So far, these techniques have been used to investigate processes such as cleavage furrow ingression, mitotic spindle positioning, and in vivo epithelial morphogenesis. This review describes some of the key shared mechanisms of cell division and apical-basal polarity establishment, how they are coordinated during development and how the advance of optogenetic techniques is furthering this research field.

Abstract: We describe a protocol for optogenetic inhibition of the small GTPase Rho1 (RhoA) in Drosophila embryos, which allows rapid and spatially confined inactivation of Rho1 and Rho1-mediated actomyosin contractility. We provide step-by-step instruction for optogenetic manipulations of Drosophila embryos using confocal and multiphoton imaging systems. This tool is useful for determining the site- and stage-specific function of Rho1 in Drosophila embryos and for studying the immediate tissue response to acute elimination of cellular contractility. For complete details on the use and execution of this protocol, please refer to Guo et al. (2022).1.

Abstract: The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.