Curated Optogenetic Publication Database

Abstract: We review fundamental mechanisms and applications of OptoGels: hydrogels with light-programmable properties endowed by photoswitchable proteins ("optoproteins") found in nature. Light, as the primary source of energy on earth, has driven evolution to develop highly-tuned functionalities, such as phototropism and circadian entrainment. These functions are mediated through a growing family of optoproteins that respond to the entire visible spectrum ranging from ultraviolet to infrared by changing their structure to transmit signals inside of cells. In a recent series of articles, engineers and biochemists have incorporated optoproteins into a variety of extracellular systems, endowing them with photocontrollability. While other routes exist for dynamically controlling material properties, light-sensitive proteins have several distinct advantages, including precise spatiotemporal control, reversibility, substrate selectivity, as well as biodegradability and biocompatibility. Available conjugation chemistries endow OptoGels with a combinatorially large design space determined by the set of optoproteins and polymer networks. These combinations result in a variety of tunable material properties. Despite their potential, relatively little of the OptoGel design space has been explored. Here, we aim to summarize innovations in this emerging field and highlight potential future applications of these next generation materials. OptoGels show great promise in applications ranging from mechanobiology, to 3D cell and organoid engineering, and programmable cell eluting materials.

Abstract: Gene and cell therapies are widely recognized as future cancer therapeutics but poor controllability limits their clinical applications. Optogenetics, the use of light-controlled proteins to precisely spatiotemporally regulate the activity of genes and cells, opens up new possibilities for cancer treatment. Light of specific wavelength can activate the immune response, oncolytic activity and modulate cell signaling in tumor cells non-invasively, in dosed manner, with tissue confined action and without side effects of conventional therapies. Here, we review optogenetic approaches in cancer research, their clinical potential and challenges of incorporating optogenetics in cancer therapy. We critically discuss beneficial combinations of optogenetic technologies with therapeutic nanobodies, T-cell activation and CAR-T cell approaches, genome editors and oncolytic viruses. We consider viral vectors and nanoparticles for delivering optogenetic payloads and activating light to tumors. Finally, we highlight herein the prospects for integrating optogenetics into immunotherapy as a novel, fast, reversible and safe approach to cancer treatment.

Abstract: Generating robust, predictable perturbations in cellular protein levels will advance our understanding of protein function and enable the control of physiological outcomes in biotechnology applications. Timed periodic changes in protein levels play a critical role in the cell division cycle, cellular stress response, and development. Here we report the generation of robust protein level oscillations by controlling the protein degradation rate in the yeast Saccharomyces cerevisiae. Using a photo-sensitive degron and red fluorescent proteins as reporters, we show that under constitutive transcriptional induction, repeated triangular protein level oscillations as fast as 5-10 min-scale can be generated by modulating the protein degradation rate. Consistent with oscillations generated though transcriptional control, we observed a continuous decrease in the magnitude of oscillations as the input modulation frequency increased, indicating low-pass filtering of input perturbation. By using two red fluorescent proteins with distinct maturation times, we show that the oscillations in protein level is largely unaffected by delays originating from functional protein formation. Our study demonstrates the potential for repeated control of protein levels by controlling the protein degradation rate without altering the transcription rate.

Abstract: Optogenetics has the advantages of a fast response time, reversibility, and high spatial and temporal resolution, which make it desirable in the metabolic engineering of chassis cells. In this study, a light-induced expression system of Yarrowia lipolytica was constructed, which successfully achieved the synthesis and functional verification of Bleomycin resistance protein (BleoR). The core of the blue light-induced system, the light-responsive element (TF), is constructed based on the blue photosensitive protein EL222 and the transcription activator VP16. The results show that the light-induced sensor based on TF, upstream activation sequence (C120)5, and minimal promoter CYC102 can respond to blue light and initiate the expression of GFPMut3 report gene. With four copies of the responsive promoter and reporter gene assembled, they can produce a 128.5-fold higher fluorescent signal than that under dark conditions after 8 h of induction. The effects of light dose and periodicity on this system were investigated, which proved that the system has good spatial and temporal controllability. On this basis, the light-controlled system was used for the synthesis of BleoR to realize the expression and verification of functional protein. These results demonstrated that this system has the potential for the transcriptional regulation of target genes, construction of large-scale synthetic networks, and overproduction of the desired product.

Abstract: Subcutaneous administration of sustained-release formulations is a common strategy for protein drugs, which avoids first pass effect and has high bioavailability. However, conventional sustained-release strategies can only load a limited amount of drug, leading to insufficient durability. Herein, we developed microcapsules based on engineered bacteria for sustained release of protein drugs. Engineered bacteria were carried in microcapsules for subcutaneous administration, with a production-lysis circuit for sustained protein production and release. Administrated in diabetic rats, engineered bacteria microcapsules was observed to smoothly release Exendin-4 for 2 weeks and reduce blood glucose. In another example, by releasing subunit vaccines with bacterial microcomponents as vehicles, engineered bacterial microcapsules activated specific immunity in mice and achieved tumor prevention. The engineered bacteria microcapsules have potential to durably release protein drugs and show versatility on the size of drugs. It might be a promising design strategy for long-acting in situ drug factory.

Abstract: The plant cytoskeleton regulates fundamental biological processes, including cell division. How to experimentally perturb the cytoskeleton is a key question if one wants to understand the role of both actin filaments (AFs) and microtubules (MTs) in a given biological process. While a myriad of mutants are available, knock-out in cytoskeleton regulators, when nonlethal, often produce little or no phenotypic perturbation because such regulators are often part of a large family, leading to functional redundancy. In this review, alternative techniques to modify the plant cytoskeleton during plant cell division are outlined. The different pharmacological and genetic approaches already developed in cell culture, transient assays, or in whole organisms are presented. Perspectives on the use of optogenetics to perturb the plant cytoskeleton are also discussed.

Abstract: Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.

Abstract: Synthetic biology is a field of research in which molecular parts (mostly nucleic acids and proteins) are de novo created or modified and then used either alone or in combination to achieve new functions that can help solve the problems of our modern society. In synthetic microbiology, microbes are employed rather than other organisms or cell-free systems. Optogenetics, a relatively recently established technology that relies on the use of genetically encoded photosensitive proteins to control biological processes with high spatiotemporal precision, offers the possibility to empower synthetic (micro)biology applications due to the many positive features that light has as an external trigger. In this review, we describe recent synthetic microbiology applications that made use of optogenetics after briefly introducing the molecular mechanism behind some of the most employed optogenetic tools. We highlight the power and versatility of this technique, which opens up new horizons for both research and industry.

Abstract: A comprehensive understanding of signaling mechanisms helps interpret fundamental biological processes and restore cell behavior from pathological conditions. Signaling outcome depends not only on the activity of each signaling component but also on their dynamic interaction in time and space, which remains challenging to probe by biochemical and cell-based assays. Opsin-based optogenetics has transformed neural science research with its spatiotemporal modulation of the activity of excitable cells. Motivated by this advantage, opsin-free optogenetics extends the power of light to a larger spectrum of signaling molecules. This review summarizes commonly used opsin-free optogenetic strategies, presents a historical overview of split protein complementation, and highlights the adaptation of split protein recombination as optogenetic sensors and actuators.

Abstract: The Notch signaling pathway is a crucial regulator of cell differentiation as well as tissue organization, whose deregulation is linked to the pathogenesis of different diseases. NOTCH1 plays a key role in breast cancer progression by increasing proliferation, maintenance of cancer stem cells, and impairment of cell death. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). We circumvent this limitation by regulating Notch activity by light. To achieve this, we have engineered an optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures, although causing distinct cell-type specific migratory phenotypes. Additionally, optoNotch activation induced chemoresistance on the same cell lines. OptoNotch allows the fine-tuning, ligand-independent, regulation of N1ICD activity and thus a better understanding of the spatiotemporal complexity of Notch signaling. Video Abstract.

Abstract: In nature as in biotechnology, light-oxygen-voltage photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.

Abstract: Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.

Abstract: Allosteric regulation of proteins has been utilized to study various aspects of cell signaling, from unicellular events to organism-wide phenotypes. However, traditional methods of allosteric regulation, such as constitutively active mutants and inhibitors, lack tight spatiotemporal control. This often leads to unintended signaling consequences that interfere with data interpretation. To overcome these obstacles, researchers employed protein engineering approaches that enable tight control of protein function through allosteric mechanisms. These methods provide high specificity as well as spatial and temporal precision in regulation of protein activity in vitro and in vivo. In this review, we focus on the recent advancements in engineered allosteric regulation and discuss the various bioengineered allosteric techniques available now, from chimeric GPCRs to chemogenetic and optogenetic switches. We highlight the benefits and pitfalls of each of these techniques as well as areas in which future improvements can be made. Additionally, we provide a brief discussion on implementation of engineered allosteric regulation approaches, demonstrating that these tools can shed light on elusive biological events and have the potential to be utilized in precision medicine.

Abstract: Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

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 design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.

Abstract: Unraveling the transformative power of optogenetics in biology requires sophisticated engineering for the creation and optimization of light-regulatable proteins. In addition, diverse strategies have been used for the tuning of these light-sensitive regulators. This review highlights different protein engineering and synthetic biology approaches, which might aid in the development and optimization of novel optogenetic proteins (Opto-proteins). Focusing on non-neuronal optogenetics, chromophore availability, general strategies for creating light-controllable functions, modification of the photosensitive domains and their fusion to effector domains, as well as tuning concepts for Opto-proteins are discussed. Thus, this review shall not serve as an encyclopedic summary of light-sensitive regulators but aims at discussing important aspects for the engineering of light-controllable proteins through selected examples.

Abstract: Technological advances have driven many recent advances in developmental biology. Light sheet imaging can reveal single-cell dynamics in living three-dimensional tissues, whereas single-cell genomic methods open the door to a complete catalogue of cell types and gene expression states. An equally powerful but complementary set of approaches are also becoming available to define development processes from the bottom up. These synthetic approaches aim to reconstruct the minimal developmental patterns, signaling processes, and gene networks that produce the basic set of developmental operations: spatial polarization, morphogen interpretation, tissue movement, and cellular memory. In this review we discuss recent approaches at the intersection of synthetic biology and development, including synthetic circuits to deliver and record signaling stimuli and synthetic reconstitution of pattern formation on multicellular scales.

Abstract: Engineered, light-sensitive protein switches are used to interrogate a broad variety of biological processes. These switches are typically constructed by genetically fusing naturally occurring light-responsive protein domains with functional domains from other proteins. Protein activity can be controlled using a variety of mechanisms including light-induced colocalization, caging, and allosteric regulation. Protein design efforts have focused on reducing background signaling, maximizing the change in activity upon light stimulation, and perturbing the kinetics of switching. It is common to combine structure-based modeling with experimental screening to identify ideal fusion points between domains and discover point mutations that optimize switching. Here, we introduce commonly used light-sensitive domains and summarize recent progress in using them to regulate protein activity.

Abstract: Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in material's composition and function. Understanding how the spatial confinement in 3D can regulate the behavior of the embedded cells is crucial to design and predict ELM's function, minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and elastic response to deformation of the matrix, a decrease in colony volumes and an increase in their sphericity are observed. Protein production follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that matrix design can be used to control and regulate the composition and function of ELMs containing microorganisms. Interestingly, design parameters for matrices to regulate bacteria behavior show similarities to those elucidated for 3D culture of mammalian cells.

Abstract: Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems. Optogenetics technique can be a solution as it not only enables dynamic control with unprecedented spatiotemporal precision but also is inexpensive and eco-friendlier. The optogenetic technique harnesses natural light-sensing modules that are genetically encodable and re-programmable in various hosts. By further engineering these modules to connect with the microbial regulatory machinery, gene expression and protein activity can be finely tuned simply through light irradiation. Recent works on applying optogenetics to microbial synthetic biology have yielded remarkable achievements. To further expand the usability of optogenetics, more optogenetic tools with greater portability that are compatible with different microbial hosts need to be developed. This review focuses on non-opsin optogenetic systems and the current state of optogenetic advancements in microbes, by showcasing the different designs and functions of optogenetic tools, followed by an insight into the optogenetic approaches used to circumvent challenges in synthetic biology.

Abstract: The importance of pancreatic endocrine cell activity modulation by autonomic innervation has been debated. To investigate this question, we established an in vivo imaging model that also allows chronic and acute neuromodulation with genetic and optogenetic tools. Using the GCaMP6s biosensor together with endocrine cell fluorescent reporters, we imaged calcium dynamics simultaneously in multiple pancreatic islet cell types in live animals in control states and upon changes in innervation. We find that by 4 days post fertilization in zebrafish, a stage when islet architecture is reminiscent of that in adult rodents, prominent activity coupling between beta cells is present in basal glucose conditions. Furthermore, we show that both chronic and acute loss of nerve activity result in diminished beta-beta and alpha-beta activity coupling. Pancreatic nerves are in contact with all islet cell types, but predominantly with beta and delta cells. Surprisingly, a subset of delta cells with detectable peri-islet neural activity coupling had significantly higher homotypic coupling with other delta cells suggesting that some delta cells receive innervation that coordinates their output. Overall, these data show that innervation plays a vital role in the maintenance of homotypic and heterotypic cellular connectivity in pancreatic islets, a process critical for islet function.

Abstract: The discovery of the gut-brain axis has proven that brain functions can be affected by the gut microbiota's metabolites, so there are significant opportunities to explore new tools to regulate gut microbiota and thus work on the brain functions. Meanwhile, engineered bacteria as oral live biotherapeutic agents to regulate the host's healthy homeostasis have attracted much attention in microbial therapy. However, whether this strategy is able to remotely regulate the host's brain function in vivo has not been investigated. Here, we engineered three blue-light-responsive probiotics as oral live biotherapeutic agents. They are spatiotemporally delivered and controlled by the upconversion optogenetic micro-nano system. This micro-nano system promotes the small intestine targeting and production of the exogenous L. lactis in the intestines, which realizes precise manipulation of brain functions including anxiety behavior, Parkinson's disease, and vagal afferent. The noninvasive and real-time probiotic intervention strategy makes the communiation from the gut to the host more controllable, which will enable the potential for engineered microbes accurately and effectively regulating a host's health.

Abstract: Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.

Abstract: By acting both upstream and downstream of biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanoresponsive mTORC2 program in neutrophil polarity and motility. We find that the tension-based inhibition of leading edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the mTORC2 kinase arm is essential for regulation of Rho activity and Myosin II-based contraction at the trailing edge. Cells required mTORC2 for spatial and temporal coordination between the front and back polarity programs and persistent migration under confinement. mTORC2 is in a mechanosensory cascade, but membrane stretch did not suffice to stimulate mTORC2 unless the co-input PIP3 was also present. Our work suggests that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs allowing efficient coordination of neutrophil shape and movement.

Abstract: Cells probe their microenvironment using membrane protrusion-retraction cycles. Spatiotemporal coordination of Rac1 and RhoA GTP-binding activities initiates and reinforces protrusions and retractions, but the control of their finite lifetime remains unclear. We examined the relations of Rac1 and RhoA GTP-binding levels to key protrusion and retraction events, as well as to cell-ECM traction forces at physiologically relevant ECM stiffness. High RhoA-GTP preceded retractions and Rac1-GTP elevation before protrusions. Notable temporal Rac1-GTP nadirs and peaks occurred at the maximal edge velocity of local membrane protrusions and retractions, respectively, followed by declined edge velocity. Moreover, altered local Rac1-GTP consistently preceded similarly altered traction force. Local optogenetic Rac1-GTP perturbations defined a function of Rac1 in restricting protrusions and retractions and in promoting local traction force. Together, we show that Rac1 plays a fundamental role in restricting the size and durability of protrusions and retractions, plausibly in part through controlling traction forces.