Curated Optogenetic Publication Database

Abstract: The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

Abstract: Light perception is indispensable for plants to respond adequately to external cues and is linked to proteolysis of key transcriptional regulators. To provide synthetic light control of protein stability, we developed a generic photosensitive degron (psd) module combining the light-reactive LOV2 domain of Arabidopsis thaliana phot1 with the murine ornithine decarboxylase-like degradation sequence cODC1. Functionality of the psd module was demonstrated in the model organism Saccharomyces cerevisiae. Generation of conditional mutants, light regulation of cyclin-dependent kinase activity, light-based patterning of cell growth, and yeast photography exemplified its versatility. In silico modeling of psd module behavior increased understanding of its characteristics. This engineered degron module transfers the principle of light-regulated degradation to nonplant organisms. It will be highly beneficial to control protein levels in biotechnological or biomedical applications and offers the potential to render a plethora of biological processes light-switchable.

Abstract: In this issue of Chemistry & Biology, Renicke et al. report a photosensitive degron (psd) consisting of the LOV2 domain fused to a protein degradation sequence. This design enabled light-dependent protein degradation in yeast. When psd was fused to cell-cycle-dependent proteins, it controlled cell cycle by light with spatiotemporal precision.

Abstract: Light is fundamental to life on earth. Therefore, nature has evolved a multitude of photoreceptors that sense light across all kingdoms. This natural resource provides synthetic biology with a vast pool of light-sensing components with distinct spectral properties that can be harnessed to engineer novel optogenetic tools. These devices enable control over gene expression, cell morphology and signaling pathways with superior spatiotemporal resolution and are maturing towards elaborate applications in basic research, in the production of biopharmaceuticals and in biomedicine. This article provides a summary of the recent advances in optogenetics that use light for the precise control of biological functions in mammalian cells.

Abstract: Formins are potent activators of actin filament assembly in the cytoplasm. In turn, cytoplasmic actin polymerization can promote release of actin from megakaryocytic acute leukemia (MAL) protein for serum response factor (SRF) transcriptional activity. We found that formins polymerized actin inside the mammalian nucleus to drive serum-dependent MAL-SRF activity. Serum stimulated rapid assembly of actin filaments within the nucleus in a formin-dependent manner. The endogenous formin mDia was regulated with an optogenetic tool, which allowed for photoreactive release of nuclear formin autoinhibition. Activated mDia promoted rapid and reversible nuclear actin network assembly, subsequent MAL nuclear accumulation, and SRF activity. Thus, a dynamic polymeric actin structure within the nucleus is part of the serum response.

Abstract: Optogenetics arises from the innovative application of microbial opsins in mammalian neurons and has since been a powerful technology that fuels the advance of our knowledge in neuroscience. In recent years, there has been growing interest in designing optogenetic tools extendable to broader cell types and biochemical signals. To date, a variety of photoactivatable proteins (refers to induction of protein activity in contrast to fluorescence) have been developed based on the understanding of plant and microbial photoreceptors including phototropins, blue light sensors using flavin adenine dinucleotide proteins, cryptochromes, and phytochromes. Such tools offered researchers reversible, quantitative, and precise spatiotemporal control of enzymatic activity, protein-protein interaction, protein translocation, as well as gene transcription in cells and in whole animals. In this review, we will briefly introduce these photosensory proteins, describe recent developments in optogenetics, and compare and contrast different methods based on their advantages and limitations.

Abstract: Near-infrared light is favourable for imaging in mammalian tissues due to low absorbance of hemoglobin, melanin, and water. Therefore, fluorescent proteins, biosensors and optogenetic constructs for optimal imaging, optical readout and light manipulation in mammals should have fluorescence and action spectra within the near-infrared window. Interestingly, natural Bacterial Phytochrome Photoreceptors (BphPs) utilize the low molecular weight biliverdin, found in most mammalian tissues, as a photoreactive chromophore. Due to their near-infrared absorbance BphPs are preferred templates for designing optical molecular tools for applications in mammals. Moreover, BphPs spectrally complement existing genetically-encoded probes. Several BphPs were already developed into the near-infrared fluorescent variants. Based on the analysis of the photochemistry and structure of BphPs we suggest a variety of possible BphP-based fluorescent proteins, biosensors, and optogenetic tools. Putative design strategies and experimental considerations for such probes are discussed.

Abstract: Light-oxygen-voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of anti-sigma factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV domain protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N- and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light- and dark-state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin-cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single-residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb the oligomeric state. Size exclusion chromatography, multiangle light scattering, small-angle X-ray scattering, and cross-linking studies indicate that RsLOV dimerizes in the dark but, upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove to be useful for engineering molecular associations in controlled cellular settings.

Abstract: Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometre precision within cells. While light-actuated chemicals such as photolabile 'caged' compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.

Abstract: Light-oxygen-voltage (LOV) domains serve as the photosensory modules for a wide range of plant and bacterial proteins, conferring blue light-dependent regulation to effector activities as diverse as enzymes and DNA binding. LOV domains can also be engineered into a variety of exogenous targets, allowing similar regulation for new protein-based reagents. Common to these proteins is the ability for LOV domains to reversibly form a photochemical adduct between an internal flavin chromophore and the surrounding protein, using this to trigger conformational changes that affect output activity. Using the Erythrobacter litoralis protein EL222 model system that links LOV regulation to a helix-turn-helix (HTH) DNA binding domain, we demonstrated that the LOV domain binds and inhibits the HTH domain in the dark, releasing these interactions upon illumination [Nash, A. I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 9449-9454]. Here we combine genomic and in vitro selection approaches to identify optimal DNA binding sites for EL222. Within the bacterial host, we observe binding at several genomic sites using a 12 bp sequence consensus that is also found by in vitro selection methods. Sequence-specific alterations in the DNA consensus reduce EL222 binding affinity in a manner consistent with the expected binding mode, a protein dimer binding to two repeats. Finally, we demonstrate the light-dependent activation of transcription of two genes adjacent to an EL222 binding site. Taken together, these results shed light on the native function of EL222 and provide useful reagents for further basic and applications research of this versatile protein.

Abstract: Filamentous fungi are important model systems for understanding eukaryotic cellular processes, including the study of protein expression. A salient feature of fungi is the ability of the protein-processing machinery to perform all of the extensive posttranslational modifications needed in the complex world of eukaryotic organisms, making them great hosts for production of eukaryotic proteins. In the model organism Neurospora crassa, several regulatable promoters have been used for heterologous gene expression but all suffer from leaky expression absent stimuli or an inability to induce protein expression at levels greater than those seen in vivo. To increase and better control in vivo protein expression in Neurospora, we have harnessed the light-induced vvd promoter. vvd promoter-driven mRNA expression is dependent upon light, shows a graded response, and is rapidly shut off when returned to the dark. The vvd promoter is a highly tunable and regulatable system, which could be a useful instrument for those interested in efficient and controllable gene expression.

Abstract: Advanced gene regulatory systems are necessary for scientific research, synthetic biology, and gene-based medicine. An ideal system would allow facile spatiotemporal manipulation of gene expression within a cell population that is tunable, reversible, repeatable, and can be targeted to diverse DNA sequences. To meet these criteria, a gene regulation system was engineered that combines light-sensitive proteins and programmable zinc finger transcription factors. This system, light-inducible transcription using engineered zinc finger proteins (LITEZ), uses two light-inducible dimerizing proteins from Arabidopsis thaliana, GIGANTEA and the LOV domain of FKF1, to control synthetic zinc finger transcription factor activity in human cells. Activation of gene expression in human cells engineered with LITEZ was reversible and repeatable by modulating the duration of illumination. The level of gene expression could also be controlled by modulating light intensity. Finally, gene expression could be activated in a spatially defined pattern by illuminating the human cell culture through a photomask of arbitrary geometry. LITEZ enables new approaches for precisely regulating gene expression in biotechnology and medicine, as well as studying gene function, cell-cell interactions, and tissue morphogenesis.

Abstract: Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels.

Abstract: Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.

Abstract: Highly complex synthetic gene circuits have been engineered in living organisms to develop systems with new biological properties. A precise trigger to activate or deactivate these complex systems is desired in order to tightly control different parts of a synthetic or natural network. Light represents an excellent tool to achieve this goal as it can be regulated in timing, location, intensity, and wavelength, which allows for precise spatiotemporal control over genetic circuits. Recently, light has been used as a trigger to control the biological function of small molecules, oligonucleotides, and proteins involved as parts in gene circuits. Light activation has enabled the construction of unique systems in living organisms such as band-pass filters and edge-detectors in bacterial cells. Additionally, light also allows for the regulation of intermediate steps of complex dynamic pathways in mammalian cells such as those involved in kinase networks. Herein we describe recent advancements in the area of light-controlled synthetic networks.

Abstract: A functional coupling of photosensory domains derived from photoreceptors to effector proteins is a promising strategy for engineering novel photoresponsive proteins in optogenetics. Here, we have fused the light-sensitive LOV2 domain from Avena sativa phototropin1 to the restriction enzyme PvuII to generate a genetically encoded, light-controllable endonuclease. By analyzing several LOV-PvuII fusion enzymes, variants were obtained that show a 3-fold difference in DNA cleavage activity, when illuminated with blue light or kept in the dark. The effect is fully reversible over multiple photocycles. Depending on the particular fusion interface, the LOV-PvuII variants obtained had a bidirectional polarity in photoactivation; i.e., increased DNA cleavage activity was observed either in the dark state, with a compact folded LOV domain, or in the blue light photoexcitation state, when the LOV domain is partially unfolded.

Abstract: We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mito-miniSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.

Abstract: Photocontrol of functional peptides is a powerful tool for spatial and temporal control of cell signaling events. We show that the genetically encoded light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2) can be used to reversibly photomodulate the affinity of peptides for their binding partners. Sequence analysis and molecular modeling were used to embed two peptides into the Jα helix of the AsLOV2 domain while maintaining AsLOV2 structure in the dark but allowing for binding to effector proteins when the Jα helix unfolds in the light. Caged versions of the ipaA and SsrA peptides, LOV-ipaA and LOV-SsrA, bind their targets with 49- and 8-fold enhanced affinity in the light, respectively. These switches can be used as general tools for light-dependent colocalization, which we demonstrate with photo-activable gene transcription in yeast.

Abstract: Aureochrome1, a signaling photoreceptor from a eukaryotic photosynthetic stramenopile, confers blue-light-regulated DNA binding on the organism. Its topology, in which a C-terminal LOV sensor domain is linked to an N-terminal DNA-binding bZIP effector domain, contrasts with the reverse sensor-effector topology in most other known LOV-photoreceptors. How, then, is signal transmitted in Aureochrome1? The dark- and light-state crystal structures of Aureochrome1 LOV domain (AuLOV) show that its helical N- and C-terminal flanking regions are packed against the external surface of the core β sheet, opposite to the FMN chromophore on the internal surface. Light-induced conformational changes occur in the quaternary structure of the AuLOV dimer and in Phe298 of the Hβ strand in the core. The properties of AuLOV extend the applicability of LOV domains as versatile design modules that permit fusion to effector domains via either the N- or C-termini to confer blue-light sensitivity.

Abstract: Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.

Abstract: Plants constantly survey the surrounding environment using several sets of photoreceptors. They can sense changes in the quantity (=intensity) and quality (=wavelength) of light and use this information to adjust their physiological responses, growth, and developmental patterns. In addition to the classical photoreceptors, such as phytochromes, cryptochromes, and phototropins, ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering. The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains: a blue-light-absorbing LOV (Light, Oxygen, or Voltage) domain along with domains involved in protein degradation. Here, we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins. We summarize the distinct photochemical properties of their LOV domains and discuss the molecular mechanisms by which the ZTL/FKF1/LKP2 proteins regulate the circadian clock and photoperiodic flowering by controlling blue-light-dependent protein degradation.

Abstract: The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avenasativa phototropin 1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains a flavin mononucleotide chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy-terminal Jα helix, the biological output signal. Using mutational analysis, circular dichroism, and NMR, we find that the largely ignored amino-terminal helix is a control element in AsLOV2's light-activated conformational change. We further identify a direct amino-to-carboxy-terminal "input-output" signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones.

Abstract: Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

Abstract: Molecular switches are the fundamental building blocks in the field of synthetic biology. The majority of these switches is based on protein-protein, protein-DNA or protein-RNA interactions that are responsive towards endogenous metabolites or external stimuli like small molecules or light. By the rational and predictive reassembling of multiple compatible molecular switches, complex synthetic signaling networks can be engineered. Here we review how these switches were used for the regulation of important cellular processes at every level of the signaling cascade. In the second part we review how these switches can be assembled to open- and closed-loop control signaling networks and how these networks can be applied to facilitate cattle reproduction, to treat diabetes or to autonomously detect and cure disease states like gouty arthritis or cancer.

Abstract: Tools for optical control of proteins offer an unprecedented level of spatiotemporal control over biological processes, adding a new layer of experimental opportunity. While use of light-activated cation channels and anion pumps has already revolutionized neurobiology, an emerging class of more general optogenetic tools may have similar transformative effects. These tools consist of light-dependent protein interaction modules that allow control of target protein interactions and localization with light. Such tools are modular and can be applied to regulate a wide variety of biological activities. This chapter reviews the different properties of light-induced dimerization systems, based on plant phytochromes, cryptochromes, and light-oxygen-voltage domain proteins, exploring advantages and limitations of the different systems and practical considerations related to their use. Potential applications of these tools within the neurobiology field, including light control of various signaling pathways, neuronal activity, and DNA recombination and transcription, are discussed.