Curated Optogenetic Publication Database

Abstract: Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis-regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control.

Abstract: Plant phytochromes enable vital adaptations to red and far-red light. At the molecular level, these responses are mediated by light-regulated interactions between phytochromes and partner proteins, foremost the phytochrome-interacting factors (PIF). Although known for decades, quantitative analyses of these interactions have long been sparse. To address this deficit, we here studied by an integrated fluorescence-spectroscopic approach the equilibrium and kinetics of Arabidopsis thaliana phytochrome B (AtPhyB) binding to a tetramerized PIF6 variant. Several readouts consistently showed the stringently light-regulated interaction to be little affected by PIF tetramerization. Analysis of the binding kinetics allowed the determination of bimolecular association and unimolecular dissociation rate constants as a function of light. Unexpectedly, the stronger affinity of AtPhyB under red light relative to far-red light is entirely due to accelerated association rather than decelerated dissociation. The association reaction under red light is highly efficient and only threefold slower than the diffusion limit. The present findings pertain equally to the analysis of signal transduction in plants and to the biotechnological application of phytochromes.

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: Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.

Abstract: The zebrafish (Danio rerio) is a popular vertebrate model organism to investigate molecular mechanisms driving development and disease. Due to its transparency at embryonic and larval stages, investigations in the living organism are possible with subcellular resolution using intravital microscopy. The beneficial optical characteristics of zebrafish not only allow for passive observation, but also active manipulation of proteins and cells by light using optogenetic tools. Initially, photosensitive ion channels have been applied for neurobiological studies in zebrafish to dissect complex behaviors on a cellular level. More recently, exciting non-neural optogenetic tools have been established to control gene expression or protein localization and activity, allowing for unprecedented non-invasive and precise manipulation of various aspects of cellular physiology. Zebrafish will likely be a vertebrate model organism at the forefront of in vivo application of non-neural optogenetic tools and pioneering work has already been performed. In this review, we provide an overview of non-neuromodulatory optogenetic tools successfully applied in zebrafish to control gene expression, protein localization, cell signaling, migration and cell ablation.

Abstract: cAMP is a positive regulator tightly involved in certain types of synaptic plasticity and related memory functions. However, its spatiotemporal roles at the synaptic and neural circuit levels remain elusive. Using a combination of a cAMP optogenetics approach and voltage-sensitive dye (VSD) imaging with electrophysiological recording, we define a novel capacity of postsynaptic cAMP in enabling dentate gyrus long-term potentiation (LTP) and depolarization in acutely prepared murine hippocampal slices. To manipulate cAMP levels at medial perforant path to granule neuron (MPP-DG) synapses by light, we generated transgenic (Tg) mice expressing photoactivatable adenylyl cyclase (PAC) in DG granule neurons. Using these Tg(CMV-Camk2a-RFP/bPAC)3Koka mice, we recorded field excitatory postsynaptic potentials (fEPSPs) from MPP-DG synapses and found that photoactivation of PAC during tetanic stimulation enabled synaptic potentiation that persisted for at least 30 min. This form of LTP was induced without the need for GABA receptor blockade that is typically required for inducing DG plasticity. The paired-pulse ratio (PPR) remained unchanged, indicating the cAMP-dependent LTP was likely postsynaptic. By employing fast fluorescent voltage-sensitive dye (VSD: di-4-ANEPPS) and fluorescence imaging, we found that photoactivation of the PAC actuator enhanced the intensity and extent of dentate gyrus depolarization triggered following tetanic stimulation. These results demonstrate that the elevation of cAMP in granule neurons is capable of rapidly enhancing synaptic strength and neuronal depolarization. The powerful actions of cAMP are consistent with this second messenger having a critical role in the regulation of synaptic function.

Abstract: During collective migration of epithelial cells, the migration direction is aligned over a tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here, we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and ERK activation triggers cell contraction. The contraction of the activated cell pulls neighboring cells, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear polarization guarantees unidirectional propagation of ERK activation, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration.

Abstract: Syntheses of many commodities that are produced using microorganisms require cofactors such as ATP and NAD(P)H. Thus, optimization of the flux distribution in central carbon metabolism, which plays a key role in cofactor regeneration, is critical for enhancing the production of the target compounds. Since the intracellular and extracellular conditions change over time in the fermentation process, dynamic control of the metabolic system for maintaining the cellular state appropriately is necessary. Here, we review techniques for detecting the intracellular metabolic state with fluorescent sensors and controlling the flux of central carbon metabolism with optogenetic tools, as well as present a prospect of bio-production processes for fine-tuning the flux distribution.

Abstract: Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

Abstract: The discovery of naturally occurring B12-depedent photoreceptors has allowed for applications of cobalamins (Cbls) in optogenetics and synthetic biology to emerge. However, theoretical investigations of the complex mechanisms of these systems have been lacking. Adenosylcobalamin (AdoCbl)-dependent photoreceptor, CarH, is one example and it relies on daylight to perform its catalytic function. Typically, in enzymes employing AdoCbl as their cofactor, the Co-C5' bond activation and cleavage is triggered by substrate binding. The cleavage of the Co-C5' bond is homolytic resulting in radical pair formation. However, in CarH, this bond is instead activated by light. To explore this peculiarity, the ground and first excited state potential energy surfaces (PESs) were constructed using the quantum mechanics/molecular mechanics (QM/MM) framework and compared with other AdoCbl-dependent enzymes. QM/MM results indicate that CarH is photolytically active as a result of the AdoCbl dual role, acting as a radical generator and as a substrate. Photo-cleavage of the Co-C5' bond and subsequent H-atom abstraction is possible because of the specific orientation of the H-C4' bond with respect to the Co(II) center. Comparison with other AdoCbl-dependent enzymes indicate that the protein environment in the CarH active center alters the photochemistry of AdoCbl by controlling the stereochemistry of the ribose moiety.

Abstract: Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are important for SG formation. Super-resolution imaging further reveals that YTHDF proteins appear to be in a super-saturated state, forming clusters that often reside in the periphery of or at the junctions between SG core clusters, and potentially promote SG formation by reducing the activation energy barrier and critical size for SG condensate formation. Our results suggest a new function of the m6A-binding YTHDF proteins in regulating SG formation.

Abstract: To perceive fluctuations in light quality, quantity, and timing, higher plants have evolved diverse photoreceptors including UVR8 (a UV-B photoreceptor), cryptochromes, phototropins, and phytochromes (Phys). In contrast to plants, prokaryotic oxygen-evolving photosynthetic organisms, cyanobacteria, rely mostly on bilin-based photoreceptors, namely, cyanobacterial phytochromes (Cphs) and cyanobacteriochromes (CBCRs), which exhibit structural and functional differences compared with plant Phys. CBCRs comprise varying numbers of light sensing domains with diverse color-tuning mechanisms and signal transmission pathways, allowing cyanobacteria to respond to UV-A, visible, and far-red lights. Recent genomic surveys of filamentous cyanobacteria revealed novel CBCRs with broader chromophore-binding specificity and photocycle protochromicity. Furthermore, a novel Cph lineage has been identified that absorbs blue-violet/yellow-orange light. In this minireview, we briefly discuss the diversity in color sensing and signal transmission mechanisms of Cphs and CBCRs, along with their potential utility in the field of optogenetics.

Abstract: The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species (ROS), and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as amyloid β (Aβ) fibrils and Tau tangles in Alzheimer's disease (AD) are accessible only via invasive cerebrospinal fluid assays, and ROS can be fleeting and challenging to monitor in vivo. Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early-stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the AD brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the "CofActor," we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied, depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early-stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.

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: 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: 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: Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.

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: 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.

Abstract: Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm-1 and acetoin titer to 67.2 g·L-1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L-1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories.

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: Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.

Abstract: Abstract not available.

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.