Curated Optogenetic Publication Database

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: 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: 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: BLUF (sensor of blue light using FAD) domain-containing proteins are one of three types of flavin-binding, blue-light-sensing proteins found in many bacteria and some algae. The other types of blue-light-sensing proteins are the cryptochromes and the light, oxygen, voltage (LOV) domain-containing proteins. BLUF proteins control a wide variety of light-dependent physiological activities including photosystem synthesis, biofilm formation and the photoavoidance response. The BLUF domain photochemical reaction is unique in that only small chromophore structural changes are involved in the light activation process, because the rigid flavin moiety is involved, rather than an isomerizable chromophore (e.g. phytochromobilin in phytochromes and retinal in rhodopsins). Recent spectroscopic, biochemical and structural studies have begun to elucidate how BLUF domains transmit the light-induced signal and identify related, subsequent changes in the domain structures. Herein, I review progress made to date concerning the physiological functions and the phototransduction mechanism of BLUF proteins.

Abstract: The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects.

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: 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: 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.

Abstract: Could combating incurable diseases lie in something as simple as light? This scenario might not be too farfetched due to groundbreaking research in optogenetics. This novel scientific area, where genetically encoded photosensors transform light energy into specifically engineered biological processes, has shown enormous potential. Cell morphology can be changed, signaling pathways can be reprogrammed, and gene expression can be regulated all by the control of light. In biomedical applications where precise cell targeting is essential, non-invasive light has shown great promise. This article provides a summary of the recent advances that utilize light in genetic programming and precise control of engineered biological functions.

Abstract: Photoreceptor flavoproteins of the LOV, BLUF, and cryptochrome families are ubiquitous among the three domains of life and are configured as UVA/blue-light systems not only in plants-their original arena-but also in prokaryotes and microscopic algae. Here, we review these proteins' structure and function, their biological roles, and their evolution and impact in the living world, and underline their growing application in biotechnologies. We present novel developments such as the interplay of light and redox stimuli, emerging enzymatic and biological functions, lessons on evolution from picoalgae, metagenomics analysis, and optogenetics applications.

Abstract: Cryptochromes (CRY) are blue-light receptors that mediate various light responses in plants. The photoexcited CRY molecules undergo several biophysical and biochemical changes, including electron transfer, phosphorylation and ubiquitination, resulting in conformational changes to propagate light signals. Two modes of CRY signal transduction have recently been discovered: the cryptochrome-interacting basic-helix-loop-helix 1 (CIB)-dependent CRY2 regulation of transcription; and the SUPPRESSOR OF PHYA1/CONSTITUTIVELY PHOTOMORPHOGENIC1 (SPA1/COP1)-dependent cryptochrome regulation of proteolysis. Both CRY signaling pathways rely on blue light-dependent interactions between the CRY photoreceptor and its signaling proteins to modulate gene expression changes in response to blue light, leading to altered developmental programs in plants.

Abstract: Synthetic biology aims at the rational design and construction of devices, systems and organisms with desired functionality based on modular well-characterized biological building blocks. Based on first proof-of-concept studies in bacteria a decade ago, synthetic biology strategies have rapidly entered mammalian cell technology providing novel therapeutic solutions. Here we review how biological building blocks can be rewired to interactive regulatory genetic networks in mammalian cells and how these networks can be transformed into open- and closed-loop control configurations for autonomously managing disease phenotypes. In the second part of this tutorial review we describe how the regulatory biological sensors and switches can be transferred from mammalian cell synthetic biology to materials sciences in order to develop interactive biohybrid materials with similar (therapeutic) functionality as their synthetic biological archetypes. We develop a perspective of how the convergence of synthetic biology with materials sciences might contribute to the development of truly interactive and adaptive materials for autonomous operation in a complex environment.

Abstract: Phytochromes are red (R)/far-red (FR) light photoreceptors that play fundamental roles in photoperception of the light environment and the subsequent adaptation of plant growth and development. There are five distinct phytochromes in Arabidopsis thaliana, designated phytochrome A (phyA) to phyE. phyA is light-labile and is the primary photoreceptor responsible for mediating photomorphogenic responses in FR light, whereas phyB-phyE are light stable, and phyB is the predominant phytochrome regulating de-etiolation responses in R light. Phytochromes are synthesized in the cytosol in their inactive Pr form. Upon light irradiation, phytochromes are converted to the biologically active Pfr form, and translocate into the nucleus. phyB can enter the nucleus by itself in response to R light, whereas phyA nuclear import depends on two small plant-specific proteins FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL). Phytochromes may function as light-regulated serine/threonine kinases, and can phosphorylate several substrates, including themselves in vitro. Phytochromes are phosphoproteins, and can be dephosphorylated by a few protein phosphatases. Photoactivated phytochromes rapidly change the expression of light-responsive genes by repressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), an E3 ubiquitin ligase targeting several photomorphogenesis-promoting transcription factors for degradation, and by inducing rapid phosphorylation and degradation of Phytochrome-Interacting Factors (PIFs), a group of bHLH transcription factors repressing photomorphogenesis. Phytochromes are targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway.

Abstract: LOV (light, oxygen or voltage) domains are protein photosensors that are conserved in bacteria, archaea, plants and fungi, and detect blue light via a flavin cofactor. LOV domains are present in both chemotrophic and phototrophic bacterial species, in which they are found amino-terminally of signalling and regulatory domains such as sensor histidine kinases, diguanylate cyclases-phosphodiesterases, DNA-binding domains and regulators of RNA polymerase σ-factors. In this Review, we describe the current state of knowledge about the function of bacterial LOV proteins, the structural basis of LOV domain-mediated signal transduction, and the use of LOV domains as genetically encoded photoswitches in synthetic biology.

Abstract: The absorption of light by bound or diffusible chromophores causes conformational rearrangements in natural and artificial photoreceptor proteins. These rearrangements are coupled to the opening or closing of ion transport pathways, the association or dissociation of binding partners, the enhancement or suppression of catalytic activity, or the transcription or repression of genetic information. Illumination of cells, tissues, or organisms engineered genetically to express photoreceptor proteins can thus be used to perturb biochemical and electrical signaling with exquisite cellular and molecular specificity. First demonstrated in 2002, this principle of optogenetic control has had a profound impact on neuroscience, where it provides a direct and stringent means of probing the organization of neural circuits and of identifying the neural substrates of behavior. The impact of optogenetic control is also beginning to be felt in other areas of cell and organismal biology.

Abstract: Light of different wavelengths can serve as a transient, noninvasive means of regulating gene expression for biotechnological purposes. Implementation of advanced gene regulatory circuits will require orthogonal transcriptional systems that can be simultaneously controlled and that can produce several different control states. Fully genetically encoded light sensors take advantage of the favorable characteristics of light, do not need the supplementation of any chemical inducers or co-factors, and have been demonstrated to control gene expression in Escherichia coli. Herein, we review engineered light-sensor systems with potential for in vivo regulation of gene expression in bacteria, and highlight different means of extending the range of available light input and transcriptional output signals. Furthermore, we discuss advances in multiplexing different light sensors for achieving multichromatic control of gene expression and indicate developments that could facilitate the construction of efficient systems for light-regulated, multistate control of gene expression.

Abstract: Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Abstract: Signaling networks in living systems are coordinated through subcellular compartmentalization and precise timing of activation. These spatiotemporal aspects ensure the fidelity of signaling while contributing to the diversity and specificity of downstream events. This is studied through development of molecular tools that generate localized and precisely timed protein activity in living systems. To study the molecular events responsible for cytoskeletal changes in real time, we generated versions of Rho family GTPases whose interactions with downstream effectors is controlled by light. GTPases were grafted to the phototropin LOV (light, oxygen, or voltage) domain (Huala, E., Oeller, P. W., Liscum, E., Han, I., Larsen, E., and Briggs, W. R. (1997). Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science278, 2120-2123.) via an alpha helix on the LOV C-terminus (Wu, Y. I., Frey, D., Lungu, O. I., Jaehrig, A., Schlichting, I., Kuhlman, B., and Hahn, K. M. (2009). A genetically encoded photoactivatable Rac controls the motility of living cells. Nature461, 104-108.). The LOV domain sterically blocked the GTPase active site until it was irradiated. Exposure to 400-500nm light caused unwinding of the helix linking the LOV domain to the GTPase, relieving steric inhibition. The change was reversible and repeatable, and the protein could be returned to its inactive state simply by turning off the light. The LOV domain incorporates a flavin as the active chromophore. This naturally occurring molecule is incorporated simply upon expression of the LOV fusion in cells or animals, permitting ready control of GTPase function in different systems. In cultured single cells, light-activated Rac leads to membrane ruffling, protrusion, and migration. In collectively migrating border cells in the Drosophila ovary, focal activation of photoactivatable Rac (PA-Rac) in a single cell is sufficient to redirect the entire group. PA-Rac in a single cell also rescues the phenotype caused by loss of endogenous guidance receptor signaling in the whole group. These findings demonstrate that cells within the border cell cluster communicate and are guided collectively. Here, we describe optimization and application of PA-Rac using detailed examples that we hope will help others apply the approach to different proteins and in a variety of different cells, tissues, and organisms.

Abstract: The knowledge on the mechanisms by which blue light (BL) is sensed by diverse and numerous organisms, and of the physiological responses elicited by the BL photoreceptors, has grown remarkably during the last two decades. The basis for this "blue revival" was set by the identification and molecular characterization of long sought plant BL sensors, employing flavins as chromophores, chiefly cryptochromes and phototropins. The latter photosensors are the foundation members of the so-called light, oxygen, voltage (LOV)-protein family, largely spread among archaea, bacteria, fungi and plants. The accumulation of sequenced microbial genomes during the last years has added the BLUF (Blue Light sensing Using FAD) family to the BL photoreceptors and yielded the opportunity for intense "genome mining," which has presented to us the intriguing wealth of BL sensing in prokaryotes. In this contribution we provide an update of flavin-based BL sensors of the LOV and BLUF type, from prokaryotic microorganisms, with special emphasis to their light-activation pathways and molecular signal-transduction mechanisms. Rather than being a fully comprehensive review, this research collects the most recent discoveries and aims to unveil and compare signaling pathways and mechanisms of BL sensors.

Abstract: Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed.

Abstract: Blue-light photoreceptors play a pivotal role in detecting the quality and quantity of light in the environment, controlling a wide range of biological responses. Several families of blue-light photoreceptors have been characterized in detail using biophysics and biochemistry, beginning with photon absorption, through intervening signal transduction, to regulation of biological activities. Here we review the light oxygen voltage, cryptochrome, and sensors of blue light using FAD families, three different groups of proteins that offer distinctly different modes of photochemical activation and signal transduction yet play similar roles in a vast array of biological responses. We cover mechanisms of light activation and propagation of conformational responses that modulate protein-protein interactions involved in biological signaling. Discovery and characterization of these processes in natural proteins are now allowing the design of photoregulatable engineered proteins, facilitating the generation of novel reagents for biochemical and cell biological research.