Curated Optogenetic Publication Database

Abstract: Cells communicate with each other to jointly regulate cellular processes during cellular differentiation and tissue morphogenesis. This multiscale coordination arises through spatiotemporal activity of morphogens to pattern cell signaling and transcriptional factor activity. This coded information controls cell mechanics, proliferation, and differentiation to shape the growth and morphogenesis of organs. While many of the molecular components and physical interactions have been identified in key model developmental systems, there are still many unresolved questions related to the dynamics involved due to challenges in precisely perturbing and quantitatively measuring signaling dynamics. Recently, a broad range of synthetic optogenetic tools have been developed and employed to quantitatively define relationships between signal transduction and downstream cellular responses. These optogenetic tools can control intracellular activities at the single cell or whole tissue scale to direct subsequent biological processes. In this brief review, we highlight a selected set of studies that develop and implement optogenetic tools to unravel quantitative biophysical mechanisms for tissue growth and morphogenesis across a broad range of biological systems through the manipulation of morphogens, signal transduction cascades, and cell mechanics. More generally, we discuss how optogenetic tools have emerged as a powerful platform for probing and controlling multicellular development.

Abstract: The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.

Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Abstract: Regulatory proteins play a crucial role in adaptation to environmental cues. Especially for lifestyle transitions, such as cell proliferation or apoptosis, switch-like characteristics are desirable. While nature frequently uses regulatory circuits to amplify or dampen signals, stand-alone protein switches are interesting for applications like biosensors, diagnostic tools, or optogenetics. However, such stand-alone systems frequently feature limited dynamic and operational ranges and suffer from slow response times. Here, we characterize a LOV-activated diguanylate cyclase (LadC) that offers precise temporal and spatial control of enzymatic activity with an exceptionally high dynamic range over four orders of magnitude. To establish this pronounced activation, the enzyme exhibits a two-stage activation process in which its activity is inhibited in the dark by caging its effector domains and stimulated upon illumination by the formation of an extended coiled-coil. These switch-like characteristics of the LadC system can be used to develop new optogenetic tools with tight regulation.

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

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

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

Abstract: Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 μm) and does not fully exploit the features of the "high spatial resolution" of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII.

Abstract: Optogenetic tools can provide fine spatial and temporal control over many biological processes. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. We demonstrate the approach's utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

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

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

Abstract: The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

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: Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury.

Abstract: Photoreceptor proteins are versatile toolbox for developing biosensors for optogenetic applications. These molecular tools get activated upon illumination of blue light, which in turn offers a non-invasive method for gaining high spatiotemporal resolution and precise control of cellular signal transduction. The Light-Oxygen-Voltage (LOV) domain family of proteins is a well-recognized system for constructing optogenetic devices. Translation of these proteins into efficient cellular sensors is possible by tuning their photochemistry lifetime. However, the bottleneck is the need for more understanding of the relationship between the protein environment and photocycle kinetics. Significantly, the effect of the local environment also modulates the electronic structure of chromophore, which perturbs the electrostatic and hydrophobic interaction within the binding site. This work highlights the critical factors hidden in the protein networks, linking with their experimental photocycle kinetics. It presents an opportunity to quantitatively examine the alternation in chromophore's equilibrium geometry and identify details which have substantial implications in designing synthetic LOV constructs with desirable photocycle efficiency.

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: Genetically encoded calcium indicators (GECIs) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of suboptimal peak signal-to-baseline ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+ transients with peak SBRs around 20-fold larger than the top-of-the-range GCaMP6 series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared with GCaMP6s, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.

Abstract: In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

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: Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.

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: Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVtag, a protein tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVtag by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVtag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVtag system. Finally, we use the LOVtag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVtag system, and introduce a powerful new tool for bacterial optogenetics.

Abstract: Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to exert tight and reversible control over their activity for various biotechnological applications. Here, we apply a rational, computation-guided methodology to design and test a transferase-class enzyme allosterically regulated by Light-oxygen-voltage-sensing domain (LOV2). We utilize computational techniques to determine the intrinsic allosteric networks within N-acyltransferase (Orf11/*Dbv8) and identify potential allosteric sites on the protein's surface. We insert LOV2 at the predicted allosteric site, exerting reversible control over enzymatic activity. We demonstrate blue-light regulation of N-acyltransferase (Orf11/*Dbv8) function. Our study for the first time demonstrates optogenetic regulation of a transferase-class enzyme as a proof-of-concept for controllable transferase design. This successful design opens the door for many future applications in metabolic engineering and cellular programming.

Abstract: Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Abstract: We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.