Curated Optogenetic Publication Database

Abstract: The coactivator p300/CREB-binding protein (CBP) regulates genes by facilitating the assembly of transcriptional machinery and by acetylating histones and other factors. However, it remains mostly unclear how both functions of p300 are dynamically coordinated during gene control. Here, we showed that p300 can orchestrate two functions through the formation of dynamic clusters with certain transcription factors (TFs), which is mediated by the interactions between a TF's transactivation domain (TAD) and the intrinsically disordered regions of p300. Co-condensation can enable spatially defined, all-or-none activation of p300's catalytic activity, priming the recruitment of coactivators, including Brd4. We showed that co-condensation can modulate transcriptional initiation rate and burst duration of target genes, underlying nonlinear gene regulatory functions. Such modulation is consistent with how p300 might shape gene bursting kinetics globally. Altogether, these results suggest an intriguing gene regulation mechanism, in which TF and p300 co-condensation contributes to transcriptional bursting regulation and cooperative gene control.

Abstract: Light has become established as a tool not only to visualize and investigate but also to steer biological systems. This review starts by discussing the unique features that make light such an effective control input in biology. It then gives an overview of how light‐control came to progress, starting with photoactivatable compounds and leading up to current genetic implementations using optogenetic approaches. The review then zooms in on optogenetics, focusing on photosensitive proteins, which form the basis for optogenetic engineering using synthetic biological approaches. As the regulation of transcription provides a highly versatile means for steering diverse biological functions, the focus of this review then shifts to transcriptional light regulators, which are presented in the biotechnologically highly relevant model organism Escherichia coli.

Abstract: Protein phase separation has emerged as a novel paradigm to explain the biogenesis of membraneless organelles and other so-called biomolecular condensates. While the implication of this physical phenomenon within cell biology is providing us with novel ways for understanding how cells compartmentalize biochemical reactions and encode function in such liquid-like assemblies, the newfound appreciation of this process also provides immense opportunities for designing and sculpting biological matter. Here, we propose that understanding the cell's instruction manual of phase separation will enable bioengineers to begin creating novel functionalized biological materials and unprecedented tools for synthetic biology. We present FASE as the synthesis of the existing sticker-spacer framework, which explains the physical driving forces underlying phase separation, with quintessential principles of Scandinavian design. FASE serves both as a designer condensates catalogue and construction manual for the aspiring (membraneless) biomolecular architect. Our approach aims to inspire a new generation of bioengineers to rethink phase separation as an opportunity for creating reactive biomaterials with unconventional properties and to encode novel biological function in living systems. Although still in its infancy, several studies highlight how designer condensates have immediate and widespread potential applications in industry and medicine.

Abstract: ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

Abstract: To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre‐defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.

Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.

Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Abstract: The spatiotemporal organization of oligomeric protein complexes, such as the supramolecular organizing centers (SMOCs) made of MyDDosome and MAVSome, is essential for transcriptional activation of host inflammatory responses and immunometabolism. Light‐inducible assembly of MyDDosome and MAVSome is presented herein to induce activation of nuclear factor‐kB and type‐I interferons. Engineering of SMOCs and the downstream transcription factor permits programmable and customized innate immune operations in a light‐dependent manner. These synthetic molecular tools will likely enable optical and user‐defined modulation of innate immunity at a high spatiotemporal resolution to facilitate mechanistic studies of distinct modes of innate immune activations and potential intervention of immune disorders and cancer.

Abstract: Alzheimer's Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. We developed an optogenetic method to induce Aβ aggregation and tested its biological influence in three model organisms-D. melanogaster, C. elegans and D. rerio. We generated a fluorescently labeled, optogenetic Aβ peptide that oligomerizes rapidly in vivo in the presence of blue light in all organisms. Here, we detail the procedures for expressing this fusion protein in animal models, investigating the effects on the nervous system using time lapse light-sheet microscopy, and performing metabolic assays to measure changes due to intracellular Aβ aggregation. This method, employing optogenetics to study the pathology of AD, allows spatial and temporal control in vivo that cannot be achieved by any other method at present.

Abstract: Stem cells undergo differentiation in complex and dynamic environments wherein instructive signals fluctuate on various timescales. Thus, cells must be equipped to properly respond to the timing of signals, for example, to distinguish sustained signaling from transient noise. However, how stem cells respond to dynamic variations in differentiation cues is not well characterized. Here, we use optogenetic activation of β-catenin signaling to probe the dynamic responses of differentiating adult neural stem cells (NSCs). We discover that, while elevated, sustained β-catenin activation sequentially promotes proliferation and differentiation, transient β-catenin induces apoptosis. Genetic perturbations revealed that the neurogenic/apoptotic fate switch was mediated through cell-cycle regulation by Growth Arrest and DNA Damage 45 gamma (Gadd45γ). Our results thus reveal a role for β-catenin dynamics in NSC fate decisions and may suggest a role for signal timing to minimize cell-fate errors, analogous to kinetic proofreading of stem-cell differentiation.

Abstract: Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.

Abstract: The last two decades have witnessed the emergence of optogenetics; a field that has given researchers the ability to use light to control biological processes at high spatio-temporal and quantitative resolution, in a reversible manner with minimal side effects. Optogenetics has revolutionised the neurosciences, increased our understanding of cellular signalling and metabolic networks and resulted in variety of applications in biotechnology and biomedicine. However, implementing optogenetics in plants has been less straight forward given their dependency on light for their life cycle. Here, we highlight some of the widely used technologies in microorganisms and animal systems derived from plant photoreceptor proteins and discuss strategies recently implemented to overcome the challenges for using optogenetics in plants.

Abstract: Understanding how cells self-organize into functional higher-order structures is of great interest, both towards deciphering animal development, as well as for our ability to predictably build custom tissues to meet research and therapeutic needs. The proper organization of cells across length-scales results from interconnected and dynamic networks of molecules and cells. Optogenetic probes provide dynamic and tunable control over molecular events within cells, and thus represent a powerful approach to both dissect and control collective cell behaviors. Here we emphasize the breadth of the optogenetic toolkit and discuss how these methods have already been used to reverse-engineer the design rules of developing organisms. We also offer our perspective on the rich potential for optogenetics to power forward-engineering of tissue assembly towards the generation of bespoke tissues with user-defined properties.

Abstract: Signaling networks control the flow of information through biological systems and coordinate the chemical processes that constitute cellular life. Optogenetic actuators - genetically encoded proteins that undergo light-induced changes in activity or conformation - are useful tools for probing signaling networks over time and space. They have permitted detailed dissections of cellular proliferation, differentiation, motility, and death, and enabled the assembly of synthetic systems with applications in areas as diverse as photography, chemical synthesis, and medicine. In this review, we provide a brief introduction to optogenetic systems and describe their application to molecular-level analyses of cell signaling. Our discussion highlights important research achievements and speculates on future opportunities to exploit optogenetic systems in the study and assembly of complex biochemical networks.

Abstract: Inhibition of receptor tyrosine kinases (RTKs) by small molecule inhibitors and monoclonal antibodies is used to treat cancer. Conversely, activation of RTKs with their ligands, including growth factors and insulin, is used to treat diabetes and neurodegeneration. However, conventional therapies that rely on injection of RTK inhibitors or activators do not provide spatiotemporal control over RTK signaling, which results in diminished efficiency and side effects. Recently, a number of optogenetic and optochemical approaches have been developed that allow RTK inhibition or activation in cells and in vivo with light. Light irradiation can control RTK signaling non-invasively, in a dosed manner, with high spatio-temporal precision, and without the side effects of conventional treatments. Here we provide an update on the current state of the art of optogenetic and optochemical RTK technologies and the prospects of their use in translational studies and therapy.

Abstract: Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).

Abstract: 'A peculiar severe disease process of the cerebral cortex' are the exact words used by A. Alzheimer in 1906 to describe a patient's increasingly severe condition of memory loss, changes in personality, and sleep disturbance. A century later, this 'peculiar' disease has become widely known as Alzheimer's disease (AD), the world's most common neurodegenerative disease, affecting more than 35 million people globally. At the same time, its pathology remains unclear and no successful treatment exists. Several theories for AD etiology have emerged throughout the past century. In this review, we focus on the metabolic mechanisms that are similar between AD and metabolic diseases, based on the results from genome-wide association studies. We discuss signaling pathways involved in both types of disease and look into new optogenetic methods to study the in vivo mechanisms of AD.

Abstract: Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.

Abstract: Yeast has been a robust platform to manufacture a broad range of biofuels, commodity chemicals, natural products and pharmaceuticals. The membrane-bound organelles in yeast provide us the means to access the specialized metabolism for various biosynthetic applications. The separation and compartmentalization of genetic and metabolic events presents us the opportunity to precisely control and program gene expression for higher order biological functions. To further advance yeast synthetic biology platform, genetically encoded biosensors and actuators haven been engineered for in vivo monitoring and controlling cellular processes with spatiotemporal resolutions. The dynamic response, sensitivity and operational range of these genetically encoded sensors are determined by the regulatory architecture, dynamic assemly and interactions of the related proteins and genetic elements. This review provides an update of the basic design principles underlying the allosteric transcription factors, GPCR and optogenetics-based sensors, aiming to precisely analyze and control yeast cellular processes for various biotechnological applications.

Abstract: Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics affect cell behavior. Here, we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). By patterning human embryonic stem cell (hESC) cultures with varying light intensities, LAVA devices enabled dose-responsive control of optoWnt activation and Brachyury expression. Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models the embryonic presentation of Wnt signals in vitro. LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.

Abstract: T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

Abstract: Brain-derived neurotrophic factor (BDNF), via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB signaling with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB signaling. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2 (CRY2), the light-inducible homo-interaction of the intracellular domain of TrkB (iTrkB) in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB signaling to fulfill customized needs. By comparing all the different strategies, we find that the CRY2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of iTrkB is most efficient in activating TrkB signaling. The optogenetic strategies presented are promising tools to investigate BDNF/TrkB signaling with tight spatial and temporal control.

Abstract: During mitotic cell division, the actomyosin cytoskeleton undergoes several dynamic changes that play key roles in progression through mitosis. Although the regulators of cytokinetic ring formation and contraction are well established, proteins that regulate cortical stability during anaphase and telophase have been understudied. Here, we describe a role for CLIC4 in regulating actin and actin regulators at the cortex and cytokinetic cleavage furrow during cytokinesis. We first describe CLIC4 as a new component of the cytokinetic cleavage furrow that is required for successful completion of mitotic cell division. We also demonstrate that CLIC4 regulates the remodeling of the sub-plasma-membrane actomyosin network within the furrow by recruiting MST4 kinase (also known as STK26) and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in regulating cortex stiffness and blebbing during the late stages of cytokinetic furrowing.

Abstract: Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis CRY2 constitutively active mutant. The structures form dimers and tetramers whose functional importance is examined in vitro and in vivo with Arabidopsis CRY2. Structure-based analysis suggests that blue light may be perceived by CRY to cause conformational changes, whose precise nature remains to be determined, leading to oligomerization that is essential for downstream signaling. This photoactivation mechanism may be widely used by plant CRYs. Our study reveals a molecular mechanism of plant CRY activation and also paves the way for design of CRY as a more efficient optical switch.

Abstract: Cryptochromes (CRYs) are blue-light receptors in plants that harbor FAD as a cofactor and regulate various physiological responses. Photoactivated CRYs undergo oligomerization, which increases the binding affinity to downstream signaling partners. Despite decades of research on the activation of CRYs, little is known about how they are inactivated. Binding of blue-light inhibitors of cryptochromes (BICs) to CRY2 suppresses its photoactivation, but the underlying mechanism remains unknown. Here, we report crystal structures of CRY2N (CRY2 PHR domain) and the BIC2-CRY2N complex with resolutions of 2.7 and 2.5 Å, respectively. In the BIC2-CRY2N complex, BIC2 exhibits an extremely extended structure that sinuously winds around CRY2N. In this way, BIC2 not only restrains the transfer of electrons and protons from CRY2 to FAD during photoreduction but also interacts with the CRY2 oligomer to return it to the monomer form. Uncovering the mechanism of CRY2 inactivation lays a solid foundation for the investigation of cryptochrome protein function.