Curated Optogenetic Publication Database

Abstract: Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.

Abstract: Uveal melanoma is the most common intraocular primary malignancy in adults and has been considered a fatal disease for decades. Optogenetics is an emerging technique that can control the activation of signaling components via irradiation with visible light. The clinical translation of optogenetics has been limited because of the need for surgical implantation of electrodes and relatively shallow tissue penetration. As visible light easily penetrates the eyes, we hypothesized that an optogenetics approach can be an effective treatment of uveal melanoma without surgery. In this study, we evaluated the feasibility of this strategy by using a genetically encoded optogenetic system based on reversible blue light-induced binding pairs between Fas-CIB1-EGFP and CRY2-mCherry-FADD. Subretinal injection of B16 cells was performed to create a uveal melanoma model. Plasmids pairs were co-transfected into B16 cells. We found that blue light irradiation dynamically controlled the translocation of FADD to Fas on the plasma membrane and induced the apoptosis of B16 cells transfected with the optogenetic nanosystem in vitro. Moreover, the blue light-controlled optogenetic nanosystem suppressed the growth of uveal melanoma in vivo by inducing apoptosis. These results suggest that light-controlled optogenetic therapy can be used as a potential novel therapeutic strategy for uveal melanoma.

Abstract: Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

Abstract: Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control integrated with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization mediated by the proteins CRY2 and CIB1. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae. This article is protected by copyright. All rights reserved.

Abstract: Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell-cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell-cell interactions by pulsed light activation, results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic compact and round multicellular clusters under thermodynamic control form, while otherwise branched and lose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction and diffusion limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell-cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.

Abstract: Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.

Abstract: Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

Abstract: The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.

Abstract: Hydrogel materials have become a versatile platform for in vitro cell culture due to their ability to simulate many aspects of native tissues. However, precise spatiotemporal presentation of peptides and other biomolecules has remained challenging. Here we report the use of light-sensing proteins (LSPs), more commonly used in optogenetics research, as light-activated reversible binding sites within synthetic poly(ethylene glycol) (PEG) hydrogels. We used LOVTRAP, a two component LSP system consisting of LOV2, a protein domain that can cycle reversibly between "light" and "dark" conformations in response to blue light, and a z-affibody, Zdark (Zdk), that binds the dark state of LOV2, to spatiotemporally control the presentation of a recombinant protein within PEG hydrogels. By immobilizing LOV2 within PEG gels, we were able to capture a recombinant fluorescent protein (used as a model biomolecule) containing a Zdk domain, and then release the Zdk fusion protein using blue light. Zdk was removed from LOV2-containing PEG gels using focused blue light, resulting in a 30% reduction of fluorescence compared to unexposed regions of the gel. Additionally, the reversible binding capability of LOVTRAP was observed in our system, enabling our LOV2 gels to capture and release Zdk at least three times. By adding a Zdk domain to a recombinant peptide or protein, dynamic, spatially constrained displays of non-diffusing ligands within a PEG gel could feasibly be achieved using LOV2.

Abstract: Proteins perform an amazingly diverse set of functions in all aspects of life. Critical to the function of many proteins are the highly specific three-dimensional structures they adopt. For this reason, there is strong interest in learning how to rationally design proteins that adopt user-defined structures. Over the last 25-years there has been significant progress in the field of computational protein design as rotamer-based sequence optimization protocols have enabled accurate design of protein tertiary and quaternary structure. In this award article I will summarize how the molecular modeling program Rosetta is used to design new protein structures and describe how we have taken advantage of this capability to create proteins that have important applications in research and medicine.  I will highlight three protein design stories: the use of protein interface design to create therapeutic bispecific antibodies, the engineering of light-inducible proteins that can be used to recruit proteins to specific locations in the cell, and the de novo design of new protein structures from pieces of naturally occurring proteins.

Abstract: In optogenetics, light-sensitive proteins are specifically expressed in target cells and light is used to precisely control the activity of these proteins at high spatiotemporal resolution. Optogenetics initially used naturally occurring photoreceptors to control neural circuits, but has expanded to include carefully designed and engineered photoreceptors. Several optogenetic constructs are based on plant photoreceptors, but their application to plant systems has been limited. Here, we present perspectives on the development of plant optogenetics, considering different levels of design complexity. We discuss how general principles of light-driven signal transduction can be coupled with approaches for engineering protein folding to develop novel optogenetic tools. Finally, we explore how the use of computation, networks, circular permutation, and directed evolution could enrich optogenetics.

Abstract: During embryonic development, spatial and temporal patterns of mechanical forces help to transform unstructured groups of cells into complex, functional tissue architectures. Here, we review emerging approaches to manipulate these patterns of forces to investigate the mechanical mechanisms that shape multicellular tissues, with a focus on recent experimental studies of epithelial tissue sheets in the embryo of the model organism Drosophila melanogaster.

Abstract: Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue- and cell-scale regulatory modes. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.

Abstract: Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.

Abstract: Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

Abstract: Actin waves are filamentous actin (F-actin)-rich structures that initiate in the somato-neuritic area and move toward neurite ends. The upstream cues that initiate actin waves are poorly understood. Here, using an optogenetic approach (Opto-cytTrkB), we found that local activation of the TrkB receptor around the neurite end initiates actin waves and triggers neurite elongation. During actin wave generation, locally activated TrkB signaling in the distal neurite was functionally connected with preferentially localized Rac1 and its signaling pathways in the proximal region. Moreover, TrkB activity changed the location of ankyrinG--the master organizer of the axonal initial segment-and initiated the stimulated neurite to acquire axonal characteristics. Taken together, these findings suggest that local Opto-cytTrkB activation switches the fate from minor to major axonal neurite during neuronal polarization by generating actin waves.

Abstract: Chemokine-guided cell migration is pivotal for many immunological and developmental processes. How chemokine receptor signaling persists to guarantee sustained directional migration despite receptor desensitization and internalization remains poorly understood. Here, we uncover a function for an intracellular pool of the chemokine receptor CCR7 present in human dendritic cells and cellular model systems. We find that CCR7 signaling, initiated at the plasma membrane, is translocated by joint trafficking of β-arrestin and Src kinase to endomembrane-residing CCR7. There, Src tyrosine phosphorylates CCR7, required for the recruitment of Vav1 to form an endomembrane-residing multi-protein signaling complex comprising CCR7, the RhoGEF Vav1, and its effector, Rac1. Interfering with vesicular trafficking affects CCR7-driven cell migration, whereas CCR7:Vav1 interaction at endomembranes is essential for local Rac1 recruitment to CCR7. Photoactivation of Rac1 at endomembranes leads to lamellipodia formation at the cell's leading edge, supporting the role of sustained endomembrane signaling in guiding cell migration.

Abstract: The development of multicellular organisms is controlled by highly dynamic molecular and cellular processes organized in spatially restricted patterns. Recent advances in optogenetics are allowing protein function to be controlled with the precision of a pulse of laser light in vivo, providing a powerful new tool to perturb developmental processes at a wide range of spatiotemporal scales. In this Primer, we describe the most commonly used optogenetic tools, their application in developmental biology and in the nascent field of synthetic morphogenesis.

Abstract: Natural photoreceptors from plants and microorganisms are used for synthetic approaches to control cell behaviour. Light perception by the photoreceptor, often by a cofactor, induces a conformational change, which is transduced to the effector and regulates its activity. Synthetic combinations of photoreceptors and effectors resulted in a wealth of cellular events that are controlled by optogenetic tools. A general approach is to regulate protein abundance controlling either protein stability, protein biosynthesis or both with optogenetic tools.

Abstract: Rationale: Spatial-temporal control of cell fate in vivo is of great importance for regenerative medicine. Currently, there remain no practical strategies to tune cell-fate spatial-temporally. Optogenetics is a biological technique that widely used to control cell activity in genetically defined neurons in a spatiotemporal-specific manner by light. In this study, optogenetics was repurposed for precise bone tissue regeneration. Methods: Lhx8 and BMP2 genes, which are considered as the master genes for mesenchymal stem cell proliferation and differentiation respectively, were recombined into a customized optogenetic control system. In the system, Lhx8 was constitutively expressed, while BMP2 together with shLhx8 expression was driven by blue light. Results: As expected, blue light induced BMP2 expression and inactivated Lhx8 expression in cells infected with the optogenetic control system. Optogenetic control of BMP2 and Lhx8 expression inversely regulates MSC fate in vitro. By animal study, we found that blue light could fine-tune the regeneration in vivo. Blue light illumination significantly promotes bone regeneration when the scaffold was loaded with MSCs infected with adeno-Lhx8, GI-Gal4DBD, LOV-VP16, and BMP2-shLhx8. Conclusions: Together, our study revealed that optogenetic control of the master genes for mesenchymal stem cell proliferation and differentiation would be such a candidate strategy for precise regenerative medicine.

Abstract: Optogenetics has demonstrated great potential in the fields of tissue engineering and regenerative medicine, from basic research to clinical applications. Spatiotemporal encoding during individual development has been widely identified and is considered a novel strategy for regeneration. A as a noninvasive method with high spatiotemporal resolution, optogenetics are suitable for this strategy. In this review, we discuss roles of dynamic signal coding in cell physiology and embryonic development. Several optogenetic systems are introduced as ideal optogenetic tools, and their features are compared. In addition, potential applications of optogenetics for tissue engineering are discussed, including light-controlled genetic engineering and regulation of signaling pathways. Furthermore, we present how emerging biomaterials and photoelectric technologies have greatly promoted the clinical application of optogenetics and inspired new concepts for optically controlled therapies. Our summation of currently available data conclusively demonstrates that optogenetic tools are a promising method for elucidating and simulating developmental processes, thus providing vast prospects for tissue engineering and regenerative medicine applications.

Abstract: Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and β2-adrenergic receptor (β2AR) optobodies suppressed endogenous gelsolin activity and β2AR signaling, respectively.

Abstract: Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light-modulated protein stabilization system (GLIMPSe) to control intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins: mitogen-activated protein kinase phosphatase 3 (MKP3), a negative regulator of the extracellu-lar signal-regulated kinase (ERK) pathway, as well as a constitutively active form of MEK (CA MEK), a positive regulator of the same pathway. Kinetics study showed that light-induced protein stabilization could be achieved within 30 minutes of blue light stimulation. GLIMPSe enables target-independent optogenetic control of protein activities and therefore minimizes the systematic variation embedded within different photoactivatable proteins. Overall, GLIMPSe promises to achieve light-mediated post-translational stabilization of a wide array of target proteins in live cells.

Abstract: While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.

Abstract: The creation of optogenetic switches for specific activation of cell-death pathways can provide insights into apoptosis and could also form a basis for non-invasive, next-generation therapeutic strategies. Previous work has demonstrated that cryptochrome 2 (Cry2)/CIB, a blue light–activated protein–protein dimerization module from the plant Arabidopsis thaliana together with BCL2-associated X apoptosis regulator (BAX), an outer mitochondrial membrane (OMM)-targeting pro-apoptotic protein, can be used for light-mediated initiation of mitochondrial outer-membrane permeabilization (MOMP) and downstream apoptosis. In this work, we further developed the original light-activated Cry2–BAX system (henceforth referred to as OptoBAX) by improving the photophysical properties and light-independent interactions of this optogenetic switch. The resulting optogenetic constructs significantly reduced the frequency of light exposure required for the membrane permeabilization activation and also decreased dark-state cytotoxicity. We used OptoBAX in a series of experiments in Neuro-2a and HEK293T cells to measure the timing of the dramatic morphological and biochemical changes occurring in cells after light-induced MOMP. In these experiments, we used OptoBAX in tandem with fluorescent reporters for imaging key events in early apoptosis, including membrane inversion, caspase cleavage, and actin redistribution. We then used these data to construct a timeline of biochemical and morphological events in early apoptosis, demonstrating a direct link between MOMP-induced redistribution of actin and apoptosis progression. In summary, we have created a next-generation Cry2/CIB–BAX system requiring less frequent light stimulation and established a timeline of critical apoptotic events, providing detailed insights into key steps in early apoptosis.