Curated Optogenetic Publication Database

Abstract: Targeted endogenous gene activation is necessary for understanding complex gene networks and has great potential in medical and industrial applications. The CRISPR-Cas system offers simple and powerful tools for this purpose. However, these CRISPR-Cas-based tools for activating user-defined genes are unable to offer precise temporal control of gene expression, despite the fact that many biological phenomena are regulated by highly dynamic patterns of gene expression. Here we created a light-inducible, user-defined, endogenous gene activation system based on CRISPR-Cas9. We demonstrated that this CRISPR-Cas9-based transcription system can allow rapid and reversible targeted gene activation by light. In addition, using this system, we have exemplified photoactivation of multiple user-defined endogenous genes in mammalian cells. The present CRISPR-Cas9-based transcription system offers simple and versatile approaches for precise endogenous gene activation in basic biological research and biotechnology applications.

Abstract: Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.

Abstract: Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

Abstract: Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.

Abstract: Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction.

Abstract: The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

Abstract: Protein switches are ubiquitous in biological signal transduction systems, enabling cells to sense and respond to a variety of molecular queues in a rapid, specific, and integrated fashion. Analogously, tailor-engineered protein switches with custom input and output functions have become invaluable research tools for reporting on distinct physiological states and actuating molecular functions in real time and in situ. Here, we analyze recent progress in constructing protein-based switches while assessing their potential in the assembly of defined signaling motifs. We anticipate such systems will ultimately pave the way towards a new generation of molecular diagnostics and facilitate the construction of artificial signaling systems that operate in parallel to the signaling machinery of a host cell for applications in synthetic biology.

Abstract: Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, although useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways and discuss future prospects for the field, including integration of new genetic approaches into optogenetics.

Abstract: Plants depend on the surrounding light environment to direct their growth. Blue light (300-500 nm) in particular acts to promote a wide variety of photomorphogenic responses including seedling establishment, phototropism and circadian clock regulation. Several different classes of flavin-based photoreceptors have been identified that mediate the effects of blue light in the dicotyledonous genetic model Arabidopsis thaliana. These include the cryptochromes, the phototropins and members of the Zeitlupe family. In this review, we discuss recent advances, which contribute to our understanding of how these photosensory systems are activated by blue light and how they initiate signaling to regulate diverse aspects of plant development.

Abstract: The superfamily of phytochrome (Phy) photoreceptors regulates a wide array of light responses in plants and microorganisms through their unique ability to reversibly switch between stable dark-adapted and photoactivated end states. Whereas the downstream signaling cascades and biological consequences have been described, the initial events that underpin photochemistry of the coupled bilin chromophore and the ensuing conformational changes needed to propagate the light signal are only now being understood. Especially informative has been the rapidly expanding collection of 3D models developed by x-ray crystallographic, NMR, and single-particle electron microscopic methods from a remarkably diverse array of bacterial Phys. These structures have revealed how the modular architecture of these dimeric photoreceptors engages the buried chromophore through distinctive knot, hairpin, and helical spine features. When collectively viewed, these 3D structures reveal complex structural alterations whereby photoisomerization of the bilin drives nanometer-scale movements within the Phy dimer through bilin sliding, hairpin reconfiguration, and spine deformation that ultimately impinge upon the paired signal output domains. When integrated with the recently described structure of the photosensory module from Arabidopsis thaliana PhyB, new opportunities emerge for the rational redesign of plant Phys with novel photochemistries and signaling properties potentially beneficial to agriculture and their exploitation as optogenetic reagents.

Abstract: Variation in signaling activity across a cell plays a crucial role in processes such as cell migration. Signaling activity specific to organelles within a cell also likely plays a key role in regulating cellular functions. To understand how such spatially confined signaling within a cell regulates cell behavior, tools that exert experimental control over subcellular signaling activity are required. Here, we discuss the advantages of using optogenetic approaches to achieve this control. We focus on a set of optical triggers that allow subcellular control over signaling through the activation of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases and downstream signaling proteins, as well as those that inhibit endogenous signaling proteins. We also discuss the specific insights with regard to signaling and cell behavior that these subcellular optogenetic approaches can provide.

Abstract: Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus.

Abstract: Optical control of cellular processes is an emerging approach for studying biological systems, affording control with high spatial and temporal resolution. Specifically designed artificial photoswitches add an interesting extension to naturally occurring light-regulated functionalities. However, despite a great deal of structural information, the generation of new tools cannot be based fully on rational design yet; in many cases design is limited by our understanding of molecular details of light activation and signal transduction. Our biochemical and biophysical studies on the established optogenetic tool PA-Rac1, the photoactivatable small GTPase Rac1, reveal how unexpected details of the sensor-effector interface, such as metal coordination, significantly affect functionally important structural elements of this photoswitch. Together with solution scattering experiments, our results favor differences in the population of pre-existing conformations as the underlying allosteric activation mechanism of PA-Rac1, rather than the assumed release of the Rac1 domain from the caging photoreceptor domain. These results have implications for the design of new optogenetic tools and highlight the importance of including molecular details of the sensor-effector interface, which is however difficult to assess during the initial design of novel artificial photoswitches.

Abstract: The ability to perturb living systems is essential to understand how cells sense, integrate, and exchange information, to comprehend how pathologic changes in these processes relate to disease, and to provide insights into therapeutic points of intervention. Several molecular technologies based on natural photoreceptor systems have been pioneered that allow distinct cellular signaling pathways to be modulated with light in a temporally and spatially precise manner. In this review, we describe and discuss the underlying design principles of natural photoreceptors that have emerged as fundamental for the rational design and implementation of synthetic light-controlled signaling systems. Furthermore, we examine the unique challenges that synthetic protein technologies face when applied to the study of neural dynamics at the cellular and network level.

Abstract: Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.

Abstract: Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interact as a red-light-sensing system. In this study, size-exclusion chromatography and light-scattering techniques are combined with UV-vis spectroscopy to investigate light-induced changes in dimeric Deinococcus radiodurans bacterial phytochrome (DrBphP) and its subdomains. The photosensory unit (DrCBD-PHY) shows an unusually stable Pfr state with minimal dark reversion, whereas the histidine kinase (HK) domain facilitates dark reversion to the resting state. Size-exclusion chromatography reveals that all phytochrome fragments remain as dimers in the illuminated state and dark state. Still, the elution profiles of all phytochrome fragments differ between the illuminated and dark states. The differences are observed reliably only when the whole UV-vis spectrum is characterized along the elution profile and show more Pfr-state characteristics at later elution volumes in DrBphP and DrCBD-PHY fragments. This implies that the PHY domain has an important role in amplifying and relaying light-induced conformational changes to the HK domain. In the illuminated state, the HK domain appears partially unfolded and prone to form oligomers. The oligomerization of DrBphP can be diminished by converting the molecule back to the resting Pr state by using far-red light.

Abstract: Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein-protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast.

Abstract: As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial-mesenchymal transition, and angiogenic sprouting-cell behaviors central to cancer progression.

Abstract: Optogenetic gene switches allow gene expression control at an unprecedented spatiotemporal resolution. Recently, light-responsive transgene expression systems that are activated by UV-B, blue, or red light have been developed. These systems perform well on their own, but their integration into genetic networks has been hampered by the overlapping absorbance spectra of the photoreceptors. We identified a lack of orthogonality between UV-B and blue light-controlled gene expression as the bottleneck and employed a model-based approach that identified the need for a blue light-responsive gene switch that is insensitive to low-intensity light. Based on this prediction, we developed a blue light-responsive and rapidly reversible expression system. Finally, we employed this expression system to demonstrate orthogonality between UV-B, blue, and red/far-red light-responsive gene switches in a single mammalian cell culture. We expect this approach to enable the spatiotemporal control of gene networks and to expand the applications of optogenetics in synthetic biology.

Abstract: Light-switchable proteins enable unparalleled control of molecular biological processes in live organisms. Previously, we have engineered red/far-red and green/red photoreversible two-component signal transduction systems (TCSs) with transcriptional outputs in E. coli and used them to characterize and control synthetic gene circuits with exceptional quantitative, temporal, and spatial precision. However, the broad utility of these light sensors is limited by bulky DNA encoding, incompatibility with commonly used ligand-responsive transcription factors, leaky output in deactivating light, and less than 10-fold dynamic range. Here, we compress the four genes required for each TCS onto two streamlined plasmids and replace all chemically inducible and evolved promoters with constitutive, engineered versions. Additionally, we systematically optimize the expression of each sensor histidine kinase and response regulator, and redesign both pathway output promoters, resulting in low leakiness and 72- and 117-fold dynamic range, respectively. These second-generation light sensors can be used to program the expression of more genes over a wider range and can be more easily combined with additional plasmids or moved to different host strains. This work demonstrates that bacterial TCSs can be optimized to function as high-performance sensors for scientific and engineering applications.

Abstract: Biological clocks play key roles in organismal development, homeostasis and function. In recent years, much work has focused on circadian clocks, but emerging studies have highlighted the existence of ultradian oscillators - those with a much shorter periodicity than 24 h. Accumulating evidence, together with recently developed optogenetic approaches, suggests that such ultradian oscillators play important roles during cell fate decisions, and analyzing the functional links between ultradian oscillation and cell fate determination will contribute to a deeper understanding of the design principle of developing embryos. In this Review, we discuss the mechanisms of ultradian oscillatory dynamics and introduce examples of ultradian oscillators in various biological contexts. We also discuss how optogenetic technology has been used to elucidate the biological significance of ultradian oscillations.

Abstract: The Arabidopsis photoreceptor cryptochrome 2 (CRY2) was previously used as an optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we report the development of a new CRY2-derived optogenetic module, 'CRY2olig', which induces rapid, robust, and reversible protein oligomerization in response to light. Using this module, we developed a novel protein interaction assay, Light-Induced Co-clustering, that can be used to interrogate protein interaction dynamics in live cells. In addition to use probing protein interactions, CRY2olig can also be used to induce and reversibly control diverse cellular processes with spatial and temporal resolution. Here we demonstrate disrupting clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with light. These new CRY2-based approaches expand the growing arsenal of optogenetic strategies to probe cellular function.

Abstract: Three major classes of flavin photosensors, light oxygen voltage (LOV) domains, blue light sensor using FAD (BLUF) proteins and cryptochromes (CRYs), regulate diverse biological activities in response to blue light. Recent studies of structure, spectroscopy and chemical mechanism have provided unprecedented insight into how each family operates at the molecular level. In general, the photoexcitation of the flavin cofactor leads to changes in redox and protonation states that ultimately remodel protein conformation and molecular interactions. For LOV domains, issues remain regarding early photochemical events, but common themes in conformational propagation have emerged across a diverse family of proteins. For BLUF proteins, photoinduced electron transfer reactions critical to light conversion are defined, but the subsequent rearrangement of hydrogen bonding networks key for signaling remains highly controversial. For CRYs, the relevant photocycles are actively debated, but mechanistic and functional studies are converging. Despite these challenges, our current understanding has enabled the engineering of flavoprotein photosensors for control of signaling processes within cells.

Abstract: Light-dependent gene regulation systems are advantageous as they allow for precise spatio-temporal control of target gene expression. In this paper, we present a novel UV-A and blue-light-inducible gene control system that is based on the light-dependent heterodimerization of the CRY2 and C1BN domains. Upon their interaction, a transcription factor is released from the cell membrane and initiates target gene expression. Capitalizing on that, sun cream UV-A protection properties were measured intracellularly.

Abstract: Optogenetics, the use of genetically encoded tools to control protein function with light, can generate localized changes in signaling within living cells and animals. For years it has been focused on channel proteins for neurobiology, but has recently expanded to cover many different types of proteins, using a broad array of different protein engineering approaches. These methods have largely been directed at proteins involved in motility, cytoskeletal regulation and gene expression. This review provides a survey of non-channel proteins that have been engineered for optogenetics. Existing molecules are used to illustrate the advantages and disadvantages of the many imaginative new approaches that the reader can use to create light-controlled proteins.