Curated Optogenetic Publication Database

Abstract: Light-mediated control of protein-protein interactions to regulate cellular pathways is an important application of optogenetics. Here, we report an optogenetic system based on the reversible light-induced binding between the bacterial phytochrome BphP1 and its natural partner PpsR2 from Rhodopseudomonas palustris bacteria. We extensively characterized the BphP1-PpsR2 interaction both in vitro and in mammalian cells and then used this interaction to translocate target proteins to specific cellular compartments, such as the plasma membrane and the nucleus. We showed light-inducible control of cell morphology that resulted in a substantial increase of the cell area. We demonstrated light-dependent gene expression with 40-fold contrast in cultured cells, 32-fold in subcutaneous mouse tissue, and 5.7-fold in deep tissues in mice. Characteristics of the BphP1-PpsR2 optogenetic system include its sensitivity to 740- to 780-nm near-infrared light, its ability to utilize an endogenous biliverdin chromophore in eukaryotes (including mammals), and its spectral compatibility with blue-light-driven optogenetic systems.

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: Photoreceptors are found in all kingdoms of life and mediate crucial responses to environmental challenges. Nature has evolved various types of photoresponsive protein structures with different chromophores and signaling concepts for their given purpose. The abundance of these signaling proteins as found nowadays by (meta-)genomic screens enriched the palette of optogenetic tools significantly. In addition, molecular insights into signal transduction mechanisms and design principles from biophysical studies and from structural and mechanistic comparison of homologous proteins opened seemingly unlimited possibilities for customizing the naturally occurring proteins for a given optogenetic task. Here, a brief overview on the photoreceptor concepts already established as optogenetic tools in natural or engineered form, their photochemistry and their signaling/design principles is given. Finally, so far not regarded photosensitive modules and protein architectures with potential for optogenetic application are described.

Abstract: Photoacoustic tomography (PAT) of genetically encoded probes allows for imaging of targeted biological processes deep in tissues with high spatial resolution; however, high background signals from blood can limit the achievable detection sensitivity. Here we describe a reversibly switchable nonfluorescent bacterial phytochrome for use in multiscale photoacoustic imaging, BphP1, with the most red-shifted absorption among genetically encoded probes. BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution. We monitored tumor growth and metastasis with ∼ 100-μm resolution at depths approaching 10 mm using photoacoustic computed tomography, and we imaged individual cancer cells with a suboptical-diffraction resolution of ∼ 140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at several scales.

Abstract: We developed a light-mediated system based on synthetic light-switchable transactivators. The transactivators bind promoter upon blue-light exposure and rapidly initiate transcription of target transgenes in filamentous fungus Trichoderma reesei. Light is inexpensive to apply, easily delivered, and instantly removed, and thus has significant advantages over chemical inducers.

Abstract: Biological imaging based on light microscopy comes at the core of the methods that let us understanding morphology and its dynamics in synergy to the spatiotemporal distribution of cellular and molecular activities as the organism develops and becomes functional. Non-linear optical tools and superesolution methodologies are under constant development and their applications to live imaging of whole organisms keep improving as we speak. Genetically coded biosensors, multicolor clonal methods and optogenetics in different organisms and, in particular, in Drosophila follow equivalent paths. We anticipate a brilliant future for live imaging providing the roots for the holistic understanding, rather than for individual parts, of development and function at the whole-organism level.

Abstract: We present a versatile platform to inactivate proteins in living cells using light, light-activated reversible inhibition by assembled trap (LARIAT), which sequesters target proteins into complexes formed by multimeric proteins and a blue light-mediated heterodimerization module. Using LARIAT, we inhibited diverse proteins that modulate cytoskeleton, lipid signaling and cell cycle with high spatiotemporal resolution. Use of single-domain antibodies extends the method to target proteins containing specific epitopes, including GFP.

Abstract: The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.

Abstract: Gene circuits are dynamical systems that regulate cellular behaviors, often using protein signals as inputs and outputs. Here we have developed an optogenetic 'function generator' method for programming tailor-made gene expression signals in live bacterial cells. We designed precomputed light sequences based on experimentally calibrated mathematical models of light-switchable two-component systems and used them to drive intracellular protein levels to match user-defined reference time courses. We used this approach to generate accelerated and linearized dynamics, sinusoidal oscillations with desired amplitudes and periods, and a complex waveform, all with unprecedented accuracy and precision. We also combined the function generator with a dual fluorescent protein reporter system, analogous to a dual-channel oscilloscope, to reveal that a synthetic repressible promoter linearly transforms repressor signals with an approximate 7-min delay. Our approach will enable a new generation of dynamical analyses of synthetic and natural gene circuits, providing an essential step toward the predictive design and rigorous understanding of biological systems.

Abstract: We report an optogenetic method based on Arabidopsis thaliana cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the β-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.

Abstract: A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light/cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems.

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: We developed a light-switchable transgene system based on a synthetic, genetically encoded light-switchable transactivator. The transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. This transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation.

Abstract: The ability to apply precise inputs to signaling species in live cells would be transformative for interrogating and understanding complex cell-signaling systems. Here we report an 'optogenetic' method for applying custom signaling inputs using feedback control of a light-gated protein-protein interaction. We applied this strategy to perturb protein localization and phosphoinositide 3-kinase activity, generating time-varying signals and clamping signals to buffer against cell-to-cell variability or changes in pathway activity.

Abstract: Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase-mediated DNA recombination using light.

Abstract: Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice. Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.

Abstract: Spatiotemporal regulation of protein function is a key feature of living systems; experimental tools that provide such control are of great utility. Here we report a genetically encoded system for controlling a post-translational process, protein splicing, with light. Studies in Saccharomyces cerevisiae demonstrate that fusion of a photodimerization system from Arabidopsis thaliana to an artificially split intein permits rapid activation of protein splicing to yield a new protein product.

Abstract: The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACalpha and PACbeta. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.