Curated Optogenetic Publication Database

Abstract: Morphogenesis of multicellular systems is governed by precise spatiotemporal regulation of biochemical reactions and mechanical forces which together with environmental conditions determine the development of complex organisms. Current efforts in the field aim at decoding the system-level principles underlying the regulation of developmental processes. Toward this goal, optogenetics, the science of regulation of protein function with light, is emerging as a powerful new tool to quantitatively perturb protein function in vivo with unprecedented precision in space and time. In this review, we provide an overview of how optogenetics is helping to address system-level questions of multicellular morphogenesis and discuss future directions.

Abstract: Expansions of amino acid repeats occur in >20 inherited human disorders, and many occur in intrinsically disordered regions (IDRs) of transcription factors (TFs). Such diseases are associated with protein aggregation, but the contribution of aggregates to pathology has been controversial. Here, we report that alanine repeat expansions in the HOXD13 TF, which cause hereditary synpolydactyly in humans, alter its phase separation capacity and its capacity to co-condense with transcriptional co-activators. HOXD13 repeat expansions perturb the composition of HOXD13-containing condensates in vitro and in vivo and alter the transcriptional program in a cell-specific manner in a mouse model of synpolydactyly. Disease-associated repeat expansions in other TFs (HOXA13, RUNX2, and TBP) were similarly found to alter their phase separation. These results suggest that unblending of transcriptional condensates may underlie human pathologies. We present a molecular classification of TF IDRs, which provides a framework to dissect TF function in diseases associated with transcriptional dysregulation.

Abstract: Genetically-encoded calcium actuators (GECAs) stemmed from STIM1 have enabled optical activation of endogenous ORAI1 channels in both excitable and non-excitable tissues. These GECAs offer new non-invasive means to probe the structure-function relations of calcium channels and wirelessly control the behavior of awake mice.

Abstract: Activation of Fas (CD95) is observed in various neurological disorders and can lead to both apoptosis and prosurvival outputs, yet how Fas signaling operates dynamically in the hippocampus is poorly understood. The optogenetic dissection of a signaling network can yield molecular-level explanations for cellular responses or fates, including the signaling dysfunctions seen in numerous diseases. Here, we developed an optogenetically activatable Fas that works in a physiologically plausible manner. Fas activation in immature neurons of the dentate gyrus triggered mammalian target of rapamycin (mTOR) activation and subsequent brain-derived neurotrophic factor secretion. Phosphorylation of extracellular signal-regulated kinase (Erk) in neural stem cells was induced under prolonged Fas activation. Repetitive activation of this signaling network yielded proliferation of neural stem cells and a transient increase in spatial working memory in mice. Our results demonstrate a novel Fas signaling network in the dentate gyrus and illuminate its consequences for adult neurogenesis and memory enhancement.

Abstract: The brains of Alzheimer's Disease patients show a decrease in brain mass and a preponderance of extracellular Amyloid-β plaques. These plaques are formed by aggregation of polypeptides that are derived from the Amyloid Precursor Protein (APP). Amyloid-β plaques are thought to play either a direct or an indirect role in disease progression, however the exact role of aggregation and plaque formation in the aetiology of Alzheimer's Disease is subject to debate as the biological effects of soluble and aggregated Amyloid-β peptides are difficult to separate in vivo. To investigate the consequences of formation of Amyloid-β oligomers in living tissues, we developed a fluorescently tagged, optogenetic Amyloid-β peptide that oligomerizes rapidly in the presence of blue light. We applied this system to the crucial question of how intracellular Amyloid-β oligomers underlie the pathologies of Alzheimer's Disease. We use Drosophila, C. elegans and D. rerio to show that, although both expression and induced oligomerization of Amyloid-β were detrimental to lifespan and healthspan, we were able to separate the metabolic and physical damage caused by light-induced Amyloid-β oligomerization from Amyloid-β expression alone. The physical damage caused by Amyloid-β oligomers also recapitulated the catastrophic tissue loss that is a hallmark of late AD. We show that the lifespan deficit induced by Amyloid-β oligomers was reduced with Li+ treatment. Our results present the first model to separate different aspects of disease progression.

Abstract: Homeostasis in adult organs involves replacement of cells from a stem cell pool maintained in specialized niches regulated by extracellular signals. This cell-to-cell communication employs signal transduction pathways allowing cells to respond with a variety of behaviors. To study these cellular behaviors, signaling must be perturbed within tissues in precise patterns, a technique recently made possible by the development of optogenetic tools. We developed tools to study signal transduction in vivo in an adult fly midgut stem cell model where signaling was regulated by the application of light. Activation was achieved by clustering of membrane receptors EGFR and Toll, while inactivation was achieved by clustering the downstream activators ERK/Rolled and NFκB/Dorsal in the cytoplasm, preventing nuclear translocation and transcriptional activation. We show that both pathways contribute to stem and transit amplifying cell numbers and affect the lifespan of adult flies. We further present new approaches to overcome overexpression phenotypes and novel methods for the integration of optogenetics into the already-established genetic toolkit of Drosophila.

Abstract: By offering the possibility to manipulate cellular functions with spatiotemporal control, optogenetics represents an attractive tool for dissecting immune responses. However, applying these approaches to single cells in vivo remains particularly challenging for immune cells that are typically located in scattering tissues. Here, we introduce an improved calcium actuator with sensitivity allowing for two-photon photoactivation. Furthermore, we identify an actuator/reporter combination that permits the simultaneous manipulation and visualization of calcium signals in individual T cells in vivo. With this strategy, we document the consequences of defined patterns of calcium signals on T cell migration, adhesion, and chemokine release. Manipulation of individual immune cells in vivo should open new avenues for establishing the functional contribution of single immune cells engaged in complex reactions.

Abstract: Optogenetic activation of receptors has advantages compared with chemical or ligand treatment because of its high spatial and temporal precision. Especially in the brain, the use of a genetically encoded light-tunable receptor is superior to direct infusion or systemic drug treatment. We applied light activatable TrkB receptor in mouse brain with reduced basal activity by incorporating Cry2PHR mutant, Opto-cytTrkB(E281A). Upon AAV mediated gene delivery, this form was expressed at sufficient levels in the mouse hippocampus (HPC) and medial entorhinal cortex (MEC) retaining normal canonical signal transduction by blue light stimulus, even by delivery of non-invasive LED light on the mouse head. Within target cells, where its expression was driven by a cell type-specific promoter, Opto-cytTrkB(E281A)-mediated TrkB signaling could be controlled by adjusting light-stimulation conditions. We further demonstrated that Opto-cytTrkB(E281A) could locally induce TrkB signaling in axon terminals in the MEC-HPC. In summary, Opto-cytTrkB(E281A) will be useful for elucidating time- and region-specific roles of TrkB signaling ranging from cellular function to neural circuit mechanisms.

Abstract: Intracellular signaling forms complicated networks that involve dynamic alterations of the protein-protein interactions occurring inside a cell. To dissect these complex networks, light-inducible optogenetic technologies have offered a novel approach for modulating the function of intracellular machineries in space and time. Optogenetic approaches combine genetic and optical methods to initiate and control protein functions within live cells. In this review, we provide an overview of the optical strategies that can be used to manipulate intracellular signaling proteins and secondary messengers at the molecular level. We briefly address how an optogenetic actuator can be engineered to enhance homo- or hetero-interactions, survey various optical tools and targeting strategies for controlling cell-signaling pathways, examine their extension to in vivo systems and discuss the future prospects for the field.

Abstract: Optogenetic approaches for controlling Ca2+ channels provide powerful means for modulating diverse Ca2+-specific biological events in space and time. However, blue light-responsive photoreceptors are, in principle, considered inadequate for deep tissue stimulation unless accompanied by optic fiber insertion. Here, we present an ultra-light-sensitive optogenetic Ca2+ modulator, named monSTIM1 encompassing engineered cryptochrome2 for manipulating Ca2+ signaling in the brain of awake mice through non-invasive light delivery. Activation of monSTIM1 in either excitatory neurons or astrocytes of mice brain is able to induce Ca2+-dependent gene expression without any mechanical damage in the brain. Furthermore, we demonstrate that non-invasive Ca2+ modulation in neurons can be sufficiently and effectively translated into changes in behavioral phenotypes of awake mice.

Abstract: In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.

Abstract: Embryogenesis is coordinated by signaling pathways that pattern the developing organism. Many aspects of this process are not fully understood, including how signaling molecules spread through embryonic tissues, how signaling amplitude and dynamics are decoded, and how multiple signaling pathways cooperate to pattern the body plan. Optogenetic approaches can be used to address these questions by providing precise experimental control over a variety of biological processes. Here, we review how these strategies have provided new insights into developmental signaling and discuss how they could contribute to future investigations.

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: 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: 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: 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: Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. The new expansions of the toolbox facilitate control of signal transduction, genome editing, 'painting' patterns of active molecules onto cellular membranes and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches has also seen interesting progress. Here we provide an overview of the optogenetic systems and the emerging chemo-optogenetic systems, and discuss recent applications in tackling complex biological problems.

Abstract: In order to study the role of signaling proteins, such as kinases and GTPases, in brain functions it is necessary to control their activity at the appropriate spatiotemporal resolution and to examine the cellular and behavioral effects of such changes in activity. Reduced spatiotemporal resolution in the regulation of these proteins activity will impede the ability to understand the proteins normal functions as longer modification of their activity in non-normal locations could lead to effects different from their natural functions. To control intracellular signaling proteins at the highest temporal resolution recent innovative optogenetic approaches were developed to allow the control of photoactivable signaling proteins activity by light. These photoactivatable proteins can be activated in selected cell population in brain and in specific subcellular compartments. Minimal-invasive tools are being developed to photoactivate these proteins for study and therapy. Together these techniques afford an unprecedented spatiotemporal control of signaling proteins activity to unveil the function of brain proteins with high accuracy in behaving animals. As dysfunctional signaling proteins are involved in brain diseases, the optogenetic technique has also the potential to be used as a tool to treat brain diseases.

Abstract: Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein-protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.

Abstract: To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

Abstract: Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.

Abstract: Optical induction of intracellular signaling by membrane-associated and integral membrane proteins allows spatiotemporally precise control over second messenger signaling and cytoskeletal rearrangements that are important to cell migration, development, and proliferation. Optogenetic membrane recruitment of a protein-of-interest to control its signaling by altering subcellular localization is a versatile means to these ends. Here, we summarize the signaling characteristics and underlying structure-function of RGS-LOV photoreceptors as single-component membrane recruitment tools that rapidly, reversibly, and efficiently carry protein cargo from the cytoplasm to the plasma membrane by a light-regulated electrostatic interaction with the membrane itself. We place the technology-relevant features of these recently described natural photosensory proteins in context of summarized protein engineering and design strategies for optically controlling membrane protein signaling.

Abstract: TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control. We show that the formation of pathologically relevant inclusions is driven by aberrant interactions between low-complexity domains of TDP-43 that are antagonized by RNA binding. Although stress granules are hypothesized to be a conduit for seeding TDP-43 proteinopathy, we demonstrate pathological inclusions outside these RNA-rich structures. Furthermore, we show that aberrant phase transitions of cytoplasmic TDP-43 are neurotoxic and that treatment with oligonucleotides composed of TDP-43 target sequences prevent inclusions and rescue neurotoxicity. Collectively, these studies provide insight into the mechanisms that underlie TDP-43 proteinopathy and present a potential avenue for therapeutic intervention.

Abstract: Optogenetic dimerizers are modular domains that can be utilized in a variety of versatile ways to modulate cellular biochemistry. Because of their modularity, many applications using these tools can be easily transferred to new targets without extensive engineering. While a number of photodimerizer systems are currently available, the field remains nascent, with new optimizations for existing systems and new approaches to regulating biological function continuing to be introduced at a steady pace.

Abstract: Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, we developed an optogenetic strategy to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, we demonstrate that continued Zelda activity is required throughout genome activation. We show that Zelda binds DNA in the context of nucleosomes and suggest that this allows Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.