Curated Optogenetic Publication Database

Abstract: Cells commonly communicate with each other through diffusible molecules but nonchemical communication remains elusive. While bioluminescent organisms communicate through light to find prey or attract mates, it is still under debate if signaling through light is possible at the cellular level. Here, we demonstrate that cell to cell signaling through light is possible in artificial cell communities derived from biomimetic vesicles. In our design, artificial sender cells produce an intracellular light signal, which triggers the adhesion to receiver cells. Unlike soluble molecules, the light signal propagates fast, independent of diffusion and without the need for a transporter across membranes. To obtain a predator-prey relationship, the luminescence predator cells is loaded with a secondary diffusible poison, which is transferred to the prey cell upon adhesion and leads to its lysis. This design provides a blueprint for light based intercellular communication, which can be used for programing artificial and natural cell communities.

Abstract: Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Abstract: Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.

Abstract: FG-repeat nucleoporins at the center of the nuclear pore complex (NPC) are highly modified with O-GlcNAc. In this issue, Yoo and Mitchison (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010141) use optogenetic probes to show that O-GlcNAc enhances permeability of the NPC, accelerating transport in both directions.

Abstract: Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

Abstract: Even though transcriptional repressors are studied with ever-increasing molecular resolution, the temporal aspects of gene repression remain poorly understood. Here, we address the dynamics of transcriptional repression by Capicua (Cic), which is essential for normal development and is commonly mutated in human cancers and neurodegenerative diseases.1,2 We report the speed limit for Cic-dependent gene repression based on live imaging and optogenetic perturbations in the early Drosophila embryo, where Cic was originally discovered.3 Our measurements of Cic concentration and intranuclear mobility, along with real-time monitoring of the activity of Cic target genes, reveal remarkably fast transcriptional repression within minutes of removing an optogenetic de-repressive signal. In parallel, quantitative analyses of transcriptional bursting of Cic target genes support a repression mechanism providing a fast-acting brake on burst generation. This work sets quantitative constraints on potential mechanisms for gene regulation by Cic.

Abstract: Cell shape regulation is important but the mechanisms that govern shape are not fully understood, in part due to limited experimental models where cell shape changes and underlying molecular processes can be rapidly and non-invasively monitored in real time. Here, we use an optogenetic tool to activate RhoA in the middle of mononucleated macrophages to induce contraction, resulting in a side with the nucleus that retains its shape and a non-nucleated side which was unable to maintain its shape and collapsed. In cells overexpressing focal adhesion kinase (FAK), the non-nucleated side exhibited a wide flat morphology and was similar in adhesion area to the nucleated side. In cells overexpressing fascin, an actin bundling protein, the non-nucleated side assumed a spherical shape and was similar in height to the nucleated side. This effect of fascin was also observed in fibroblasts even without inducing furrow formation. Based on these results, we conclude that FAK and fascin work together to maintain cell shape by regulating adhesion area and height, respectively, in different cell types.

Abstract: The spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration.

Abstract: Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.

Abstract: Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.

Abstract: Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the relation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here, we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with different optical dimerizers and molecular motors. This method presents a mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.

Abstract: Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.

Abstract: Light-Oxygen-Voltage (LOV) domains are responsible for detecting blue light (BL) and regulating the activities of effector domains in various organisms. Photozipper (PZ), an N-terminally truncated aureochrome-1 protein, contains a LOV domain and a basic leucin zipper (bZIP) domain and plays a role as a light-activatable transcription factor. PZ is monomeric in the dark state and undergoes non-covalent dimerization upon illumination with BL, subsequently increasing its affinity for the target DNA. To clarify the molecular mechanism of aureochromes, we prepared site-directed mutants of PZ and performed quantitative analyses in the dark and light states. Although the amino acid substitutions in the hinge region between the LOV core and A'α helix had minor effects on the dimerization and DNA-binding properties of PZ, the substitutions in the β-sheet region of the LOV core and in the A'α helix significantly affected these properties. We found that light signals are transmitted from the LOV core to the effector bZIP domain via the hydrophobic residues on the β-sheet. The light-induced conformational change possibly deforms the hydrophobic regions of the LOV core and induces the detachment of the A'α helix to expose the dimerization surface, likely activating the bZIP domain in a light-dependent manner.

Abstract: The ability to optically induce biological responses in 3D has been dwarfed by the physical limitations of visible light penetration to trigger photochemical processes. However, many biological systems are relatively transparent to low-energy light, which does not provide sufficient energy to induce photochemistry in 3D. To overcome this challenge, hydrogels that are capable of converting red or near-IR (NIR) light into blue light within the cell-laden 3D scaffolds are developed. The upconverted light can then excite optically active proteins in cells to trigger a photochemical response. The hydrogels operate by triplet–triplet annihilation upconversion. As proof-of-principle, it is found that the hydrogels trigger an optogenetic response by red/NIR irradiation of HeLa cells that have been engineered to express the blue-light sensitive protein Cry2olig. While it is remarkable to photoinduce the clustering of Cry2olig with blanket NIR irradiation in 3D, it is also demonstrated how the hydrogels trigger clustering within a single cell with great specificity and spatiotemporal control. In principle, these hydrogels may allow for photochemical control of cell function within 3D scaffolds, which can lead to a wealth of fundamental studies and biochemical applications.

Abstract: The SpyCatcher/SpyTag protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. The utility of this system could be expanded by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage domain 2 of Avena sativa (AsLOV2) undergoes a dramatic conformational change in its c-terminal Jα-helix in response to blue light. By inserting SpyTag into the different locations of the Jα-helix, we created a blue light inducible SpyTag system (BLISS). In this design, the SpyTag is blocked from reacting with the SpyCatcher in the dark, but upon irradiation with blue light, the Jα-helix of the AsLOV2 undocks to expose the SpyTag. We tested several insertion sites and characterized the kinetics. We found three variants with dynamic ranges over 15, which were active within different concentration ranges. These could be tuned using SpyCatcher variants with different reaction kinetics. Further, the reaction could be instantaneously quenched by removing light. We demonstrated the spatial aspect of this light control mechanism through photopatterning of two fluorescent proteins. This system offers opportunities for many other biofabrication and optogenetics applications.

Abstract: Bacterial pathogens operate by tightly controlling the pathogenicity to facilitate invasion and survival in host. While small molecule inducers can be designed to modulate pathogenicity to perform studies of pathogen-host interaction, these approaches, due to the diffusion property of chemicals, may have unintended, or pleiotropic effects that can impose limitations on their use. By contrast, light provides superior spatial and temporal resolution. Here, using optogenetics we reengineered GacS of the opportunistic pathogen Pseudomonas aeruginosa, signal transduction protein of the global regulatory Gac/Rsm cascade which is of central importance for the regulation of infection factors. The resultant protein (termed YGS24) displayed significant light-dependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in the Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of the Pseudomonas aeruginosa strain PAO1 in a brain-heart infusion and of another strain, PA14, in slow killing media progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined hosts, even specific tissues, to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.

Abstract: Cell death events continuously challenge epithelial barrier function yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of extracellular-signal-regulated kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

Abstract: The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive AsLOV (Avena sativa Light Oxygen Voltage) domain is used to sequester fluorescently-labelled transcriptional regulators YAP1 and TAZ/WWTR1 on the surface of mitochondria and reversibly release them upon blue light illumination. After dissociation, fluorescent signals from mitochondria, cytoplasm and nucleus are extracted with a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlated within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy revealed high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics.

Abstract: Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

Abstract: What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.

Abstract: Ischemia-reperfusion injury is a major cause of acute kidney injury. Recent studies on the pathophysiology of ischemia-reperfusion-induced acute kidney injury showed that immunologic responses significantly affect kidney ischemia-reperfusion injury and repair. Nuclear factor (NF)-ĸB signaling, which controls cytokine production and cell survival, is significantly involved in ischemia-reperfusion-induced acute kidney injury, and its inhibition can ameliorate ischemic acute kidney injury. Using EXPLOR, a novel, optogenetically engineered exosome technology, we successfully delivered the exosomal super-repressor inhibitor of NF-ĸB (Exo-srIĸB) into B6 wild type mice before/after kidney ischemia-reperfusion surgery, and compared outcomes with those of a control exosome (Exo-Naïve)-injected group. Exo-srIĸB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in post-ischemic mice than in the Exo-Naïve treatment group. Systemic delivery of Exo-srIĸB decreased NF-ĸB activity in post-ischemic kidneys and reduced apoptosis. Post-ischemic kidneys showed decreased gene expression of pro-inflammatory cytokines and adhesion molecules with Exo-srIĸB treatment as compared with the control. Intravital imaging confirmed the uptake of exosomes in neutrophils and macrophages. Exo-srIĸB treatment also significantly affected post-ischemic kidney immune cell populations, lowering neutrophil, monocyte/macrophage, and T cell frequencies than those in the control. Thus, modulation of NF-ĸB signaling through exosomal delivery can be used as a novel therapeutic method for ischemia-reperfusion-induced acute kidney injury.

Abstract: Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the synthesis and application of blue-light-generating synthetic cells using bioluminescence, dismissing the need for an external light source. First, the lipid membrane and internal composition of light-producing synthetic cells were optimized to enable high-intensity emission. Next, we show these cells’ capacity for triggering bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride in a quorum-sensing like mechanism. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of activating engineered processes inside tissues.

Abstract: Materials in nature have fascinating properties that serve as a continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization, or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here, we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.

Abstract: For various research questions in metabolism, it is highly desirable to have means available, with which the flux through specific pathways can be perturbed dynamically, in a reversible manner, and at a timescale that is consistent with the fast turnover rates of metabolism. Optogenetics, in principle, offers such possibility. Here, we developed an initial version of a photo-switchable isocitrate dehydrogenase (IDH) aimed at controlling the metabolic flux through the citric acid cycle in budding yeast. By inserting a protein-based light switch (LOV2) into computationally identified active/regulatory-coupled sites of IDH and by using in vivo screening in Saccharomyces cerevisiae, we obtained a number of IDH enzymes whose activity can be switched by light. Subsequent in-vivo characterization and optimization resulted in an initial version of photo-switchable (PS) IDH. While further improvements of the enzyme are necessary, our study demonstrates the efficacy of the overall approach from computational design, via in vivo screening and characterization. It also represents one of the first few examples, where optogenetics were used to control the activity of a metabolic enzyme.

Abstract: Decades of genetic, molecular and biochemical studies in plants have provided foundational knowledge about light sensory proteins and led to their application in synthetic biology. Optogenetic tools take advantage of the light switchable activity of plant photoreceptors to control intracellular signaling pathways. The recent discovery of the UV-B photoreceptor UV RESISTANCE LOCUS 8 in the model plant Arabidopsis thaliana opens up new avenues for light-controllable methodologies. In this review, we discuss current developments in optogenetic control by UV-B light and its signaling components, as well as rational considerations in the design and applications of UV-B-based optogenetic tools.