Curated Optogenetic Publication Database

Abstract: Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.

Abstract: Protein-based sensors and switches provide attractive tools for the real-time monitoring and control of molecular processes in complex biological environments. Fluorescent sensor proteins have been developed for a wide variety of small molecules, but the construction of genetically encoded light-responsive ligand binding proteins remains mostly unexplored. Here we present a generic approach to reengineer a previously developed FRET-based Zn(2+) sensor into a light-activatable Zn(2+) binding protein using a design strategy based on mutually exclusive domain interactions. These so-called VividZn proteins consist of two light-responsive Vivid domains that homodimerize upon illumination with blue light, thus preventing the binding of Zn(2+) between two Zn(2+) binding domains, Atox1 and WD4. Following optimization of the linker between WD4 and the N-terminus of one of the Vivid domains, VividZn variants were obtained that show a 9- to 55-fold decrease in Zn(2+) affinity upon illumination, which is fully reversible following dark adaptation. The Zn(2+) affinities of the switch could be rationally tuned between 1 pM and 2 nM by systematic variation of linker length and mutation of one of the Zn(2+) binding residues. Similarly, introduction of mutations in the Vivid domains allowed tuning of the switching kinetics between 10 min and 7 h. Low expression levels in mammalian cells precluded the demonstration of light-induced perturbation of cytosolic Zn(2+) levels. Nonetheless, our results firmly establish the use of intramolecular Vivid dimerization as an attractive light-sensitive input module to rationally engineer light-responsive protein switches based on mutually exclusive domain interactions.

Abstract: Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2-CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2-CIB system by identifying versions of CRY2-CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13-α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.

Abstract: Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems.

Abstract: Growth cones of extending axons navigate to correct targets by sensing a guidance cue gradient via membrane protein receptors. Although most signaling mechanisms have been clarified using an in vitro approach, it is still difficult to investigate the growth cone behavior in complicated extracellular environment of living animals due to the lack of tools. We develop a system for the light-dependent activation of a guidance receptor, Deleted in Colorectal Cancer (DCC), using Arabidopsis thaliana Cryptochrome 2, which oligomerizes upon blue-light absorption. Blue-light illumination transiently activates DCC via its oligomerization, which initiates downstream signaling in the illuminated subcellular region. The extending axons are attracted by illumination in cultured chick dorsal root ganglion neurons. Moreover, light-mediated navigation of the growth cones is achieved in living Caenorhabditis elegans. The photo-manipulation system is applicable to investigate the relationship between the growth cone behavior and its surrounding environment in living tissue.

Abstract: Light-mediated release of signaling ligands, such as chemoattractants, growth factors, and cytokines is an attractive strategy for investigation and therapeutic targeting of leukocyte communication and immune responses. We introduce a versatile optogenetic method to control ligand secretion, combining UV-conditioned endoplasmic reticulum-to-Golgi trafficking and a furin-processing step. As proof of principle, we achieved light-triggered chemokine secretion and demonstrated that a brief pulse of chemokine release can mediate a rapid flux of leukocyte contacts with target cells in vitro and in vivo. This approach opens new possibilities for dynamic investigation of leukocyte communication in vivo and may confer the potential to control the local release of soluble mediators in the context of immune cell therapies.

Abstract: Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.

Abstract: Dronpa is a novel photochromic fluorescent protein that exhibits fast response to light. The present article is the first report of the resonance and preresonance Raman spectra of Dronpa. We used the intensity and frequency of Raman bands to determine the structure of the Dronpa chromophore in two thermally stable photochromic states. The acid-base equilibrium in one photochromic state was observed by spectroscopic pH titration. The Raman spectra revealed that the chromophore in this state shows a protonation/deprotonation transition with a pKa of 5.2 ± 0.3 and maintains the cis configuration. The observed resonance Raman bands showed that the other photochromic state of the chromophore is in a trans configuration. The results demonstrate that Raman bands selectively enhanced for the chromophore yield valuable information on the molecular structure of the chromophore in photochromic fluorescent proteins after careful elimination of the fluorescence background.

Abstract: Signaling proteins comprise interaction and effector modules connected by linkers. Throughout evolution, these recurring modules have multiply been recombined to produce the present-day plethora of signaling proteins. Likewise, modular recombination lends itself to the engineering of hybrid signal receptors, whose functionality hinges on linker topology, sequence, and length. Often, numerous linkers must be assessed to obtain functional receptors. To expedite linker optimization, we devised the PATCHY strategy (primer-aided truncation for the creation of hybrid proteins) for the facile construction of hybrid gene libraries with defined linker distributions. Empowered by PATCHY, we engineered photoreceptors whose signal response was governed by linker length: whereas blue-light-repressed variants possessed linkers of 7n or 7n+5 residues, variants with 7n+1 residues were blue-light-activated. Related natural receptors predominantly displayed linker lengths of 7n and 7n+5 residues but rarely of 7n+1 residues. PATCHY efficiently explores linker sequence space to yield functional hybrid proteins including variants transcending the natural repertoire of signaling proteins.

Abstract: Optogenetic tools to control gene expression have many advantages over the classical chemically inducible systems, overcoming intrinsic limitations of chemical inducers such as solubility, diffusion, and cell toxicity. They offer an unmatched spatiotemporal resolution and permit quantitative and noninvasive control of the gene expression. Here we describe a protocol of a synthetic light-inducible system for the targeted control of gene expression in plants based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). The synthetic toggle switch system is in the ON state when plant protoplasts are illuminated with red light (660 nm) and can be returned to the OFF state by subsequent illumination with far-red light (760 nm). In this protocol, the implementation of a red light-inducible expression system in plants using Light-Emitting Diode (LED) illumination boxes is described, including the isolation and transient transformation of plant protoplasts from Arabidopsis thaliana and Nicotiana tabacum.

Abstract: Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light).

Abstract: Abstract not available.

Abstract: Methods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells.

Abstract: Migratory immune cells use intracellular signaling networks to generate and orient spatially polarized responses to extracellular cues. The monomeric G protein Cdc42 is believed to play an important role in controlling the polarized responses, but it has been difficult to determine directly the consequences of localized Cdc42 activation within an immune cell. Here we used subcellular optogenetics to determine how Cdc42 activation at one side of a cell affects both cell behavior and dynamic molecular responses throughout the cell. We found that localized Cdc42 activation is sufficient to generate polarized signaling and directional cell migration. The optically activated region becomes the leading edge of the cell, with Cdc42 activating Rac and generating membrane protrusions driven by the actin cytoskeleton. Cdc42 also exerts long-range effects that cause myosin accumulation at the opposite side of the cell and actomyosin-mediated retraction of the cell rear. This process requires the RhoA-activated kinase ROCK, suggesting that Cdc42 activation at one side of a cell triggers increased RhoA signaling at the opposite side. Our results demonstrate how dynamic, subcellular perturbation of an individual signaling protein can help to determine its role in controlling polarized cellular responses.

Abstract: Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.

Abstract: To establish and maintain their complex morphology and function, neurons and other polarized cells exploit cytoskeletal motor proteins to distribute cargoes to specific compartments. Recent studies in cultured cells have used inducible motor protein recruitment to explore how different motors contribute to polarized transport and to control the subcellular positioning of organelles. Such approaches also seem promising avenues for studying motor activity and organelle positioning within more complex cellular assemblies, but their applicability to multicellular in vivo systems has so far remained unexplored. Here, we report the development of an optogenetic organelle transport strategy in the in vivo model system Caenorhabditis elegans. We demonstrate that movement and pausing of various organelles can be achieved by recruiting the proper cytoskeletal motor protein with light. In neurons, we find that kinesin and dynein exclusively target the axon and dendrite, respectively, revealing the basic principles for polarized transport. In vivo control of motor attachment and organelle distributions will be widely useful in exploring the mechanisms that govern the dynamic morphogenesis of cells and tissues, within the context of a developing animal.

Abstract: Synthetic biology aims at manipulating biological systems by rationally designed and genetically introduced components. Efforts in photoactuator engineering resulted in microorganisms reacting to extracellular light-cues with various cellular responses. Some of them lead to the formation of macroscopically observable outputs, which can be used to generate images made of living matter. Several methods have been developed to convert colorless compounds into visible pigments by an enzymatic conversion. This has been exploited as a showcase for successful creation of an optogenetic tool; examples for basic light-controlled biological processes that have been coupled to this biophotography comprise regulation of transcription, protein stability, and second messenger synthesis. Moreover, biological reproduction of images is used as means to facilitate quantitative characterization of optogenetic switches as well as a technique to investigate complex cellular signaling circuits. Here, we will compare the different techniques for biological image generation, introduce experimental approaches, and provide future-perspectives for biophotography.

Abstract: Recent advances in the understanding of photosensing in biological systems have enabled the use of photoreceptors as novel genetic tools. Exploiting various photoreceptors that cyanobacteria possess, a green light-inducible gene expression system was previously developed for the regulation of gene expression in cyanobacteria. However, the applications of cyanobacterial photoreceptors are not limited to these bacteria but are also available for non-photosynthetic microorganisms by the coexpression of a cyanobacterial chromophore with a cyanobacteria-derived photosensing system. An Escherichia coli-derived self-aggregation system based on Antigen 43 (Ag43) has been shown to induce cell self-aggregation of various bacteria by exogenous introduction of the Ag43 gene.

Abstract: The genetically encoded photosensitizer miniSOG (mini Singlet Oxygen Generator) can be used to kill cells in C. elegans. miniSOG generates the reactive oxygen species (ROS) singlet oxygen after illumination with blue light. Illumination of neurons expressing miniSOG targeted to the outer mitochondrial membrane (mito-miniSOG) causes neuronal death. To enhance miniSOG's efficiency as an ablation tool in multiple cell types we tested alternative targeting signals. We find that membrane targeted miniSOG allows highly efficient cell killing. When combined with a point mutation that increases miniSOG's ROS generation, membrane targeted miniSOG can ablate neurons in less than one tenth the time of mito-miniSOG. We extend the miniSOG ablation technique to non-neuronal tissues, revealing an essential role for the epidermis in locomotion. These improvements expand the utility and throughput of optogenetic cell ablation in C. elegans.

Abstract: Biological phenomena, such as cellular differentiation and phagocytosis, are fundamental processes that enable cells to fulfill important physiological roles in multicellular organisms. In the field of synthetic biology, the study of these behaviors relies on the use of a broad range of molecular tools that enable the real-time manipulation and measurement of key components in the underlying signaling pathways. This Review will focus on a subset of synthetic biology tools known as bottom-up techniques, which use technologies such as optogenetics and chemically induced dimerization to reconstitute cellular behavior in cells. These techniques have been crucial not only in revealing causal relationships within signaling networks but also in identifying the minimal signaling components that are necessary for a given cellular function. We discuss studies that used these systems in a broad range of cellular and molecular phenomena, including the time-dependent modulation of protein activity in cellular proliferation and differentiation, the reconstitution of phagocytosis, the reconstitution of chemotaxis, and the regulation of actin reorganization. Finally, we discuss the potential contribution of synthetic biology to medicine.

Abstract: The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

Abstract: Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology.

Abstract: The design of synthetic optogenetic tools that allow precise spatiotemporal control of biological processes previously inaccessible to optogenetic control has developed rapidly over the last years. Rational design of such tools requires detailed knowledge of allosteric light signaling in natural photoreceptors. To understand allosteric communication between sensor and effector domains, characterization of all relevant signaling states is required. Here, we describe the mechanism of light-dependent DNA binding of the light-oxygen-voltage (LOV) transcription factor Aureochrome 1a from Phaeodactylum tricornutum (PtAu1a) and present crystal structures of a dark state LOV monomer and a fully light-adapted LOV dimer. In combination with hydrogen/deuterium-exchange, solution scattering data and DNA-binding experiments, our studies reveal a light-sensitive interaction between the LOV and basic region leucine zipper DNA-binding domain that together with LOV dimerization results in modulation of the DNA affinity of PtAu1a. We discuss the implications of these results for the design of synthetic LOV-based photosensors with application in optogenetics.

Abstract: We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms.

Abstract: Rhythmicity is prevalent in the cortical dynamics of diverse single and multicellular systems. Current models of cortical oscillations focus primarily on cytoskeleton-based feedbacks, but information on signals upstream of the actin cytoskeleton is limited. In addition, inhibitory mechanisms--especially local inhibitory mechanisms, which ensure proper spatial and kinetic controls of activation--are not well understood. Here, we identified two phosphoinositide phosphatases, synaptojanin 2 and SHIP1, that function in periodic traveling waves of rat basophilic leukemia (RBL) mast cells. The local, phase-shifted activation of lipid phosphatases generates sequential waves of phosphoinositides. By acutely perturbing phosphoinositide composition using optogenetic methods, we showed that pulses of PtdIns(4,5)P2 regulate the amplitude of cyclic membrane waves while PtdIns(3,4)P2 sets the frequency. Collectively, these data suggest that the spatiotemporal dynamics of lipid metabolism have a key role in governing cortical oscillations and reveal how phosphatidylinositol 3-kinases (PI3K) activity could be frequency-encoded by a phosphatase-dependent inhibitory reaction.