Curated Optogenetic Publication Database

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: 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: Light, oxygen, or voltage (LOV) protein domains are present in many signaling proteins in bacteria, archaea, protists, plants, and fungi. The LOV protein VIVID (VVD) of the filamentous fungus Neurospora crassa enables the organism to adapt to constant or increasing amounts of light and facilitates proper entrainment of circadian rhythms. Here, we determined the crystal structure of the fully light-adapted VVD dimer and reveal the mechanism by which light-driven conformational change alters the oligomeric state of the protein. Light-induced formation of a cysteinyl-flavin adduct generated a new hydrogen bond network that released the amino (N) terminus from the protein core and restructured an acceptor pocket for binding of the N terminus on the opposite subunit of the dimer. Substitution of residues critical for the switch between the monomeric and the dimeric states of the protein had profound effects on light adaptation in Neurospora. The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions.

Abstract: Ca(2+) signals regulate diverse physiological processes through tightly regulated fluxes varying in location, time, frequency, and amplitude. Here, we developed LOVS1K, a genetically encoded and photoactivated synthetic protein to generate local or global Ca(2+) signals. With 300 ms blue light exposure, LOVS1K translocated to Orai1, a plasma membrane Ca(2+) channel, within seconds, generating a local Ca(2+) signal on the plasma membrane, and returning to the cytoplasm after tens of seconds. With repeated photoactivation, global Ca(2+) signals in the cytoplasm were generated to modulate engineered Ca(2+)-inducible proteins. Although Orai1 is typically associated with global store-operated Ca(2+) entry, we demonstrate that Orai1 can also generate local Ca(2+) influx on the plasma membrane. Our photoactivation system can be used to generate spatially and temporally precise Ca(2+) signals and to engineer synthetic proteins that respond to specific Ca(2+) signals.

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: Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.

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: Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

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.

Abstract: Understanding the complexity of neuronal biology requires the manipulation of cellular processes with high specificity and spatio-temporal precision. The recent development of synthetic photo-activatable proteins designed using the light-oxygen-voltage and phytochrome domains provides a new set of tools for genetically targeted optical control of cell signaling. Their modular design, functional diversity, precisely controlled activity and in vivo applicability offer many advantages for investigating neuronal function. Although designing these proteins is still a considerable challenge, future advances in rational protein design and a deeper understanding of their photoactivation mechanisms will allow the development of the next generation of optogenetic techniques.

Abstract: Biological processes are regulated with a high level of spatial and temporal resolution. To understand and manipulate these processes, scientists need to be able to regulate them with Nature's level of precision. In this context, light is a unique regulatory element because it can be precisely controlled in terms of location, timing and amplitude. Moreover, most biological laboratories have a wide range of light sources as standard equipment. This review article summarizes the most recent advances in light-mediated regulation of protein function and its application in a cellular context. Specifically, the photocaging of small-molecule modulators of protein function and of specific amino acid residues in proteins is discussed. In addition, examples of the photochemical control of protein function through the application of genetically engineered natural-light receptors are presented.

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: The small GTPase Rac induces actin polymerization, membrane ruffling and focal contact formation in cultured single cells but can either repress or stimulate motility in epithelial cells depending on the conditions. The role of Rac in collective epithelial cell movements in vivo, which are important for both morphogenesis and metastasis, is therefore difficult to predict. Recently, photoactivatable analogues of Rac (PA-Rac) have been developed, allowing rapid and reversible activation or inactivation of Rac using light. In cultured single cells, light-activated Rac leads to focal membrane ruffling, protrusion and migration. Here we show that focal activation of Rac is also sufficient to polarize an entire group of cells in vivo, specifically the border cells of the Drosophila ovary. Moreover, activation or inactivation of Rac in one cell of the cluster caused a dramatic response in the other cells, suggesting that the cells sense direction as a group according to relative levels of Rac activity. Communication between cells of the cluster required Jun amino-terminal kinase (JNK) but not guidance receptor signalling. These studies further show that photoactivatable proteins are effective tools in vivo.

Abstract: Signaling photoreceptors use the information contained in the absorption of a photon to modulate biological activity in plants and a wide range of organisms. The fundamental-and as yet imperfectly answered-question is, how is this achieved at the molecular level? We adopt the perspective of biophysicists interested in light-dependent signal transduction in nature and the three-dimensional structures that underpin signaling. Six classes of photoreceptors are known: light-oxygen-voltage (LOV) sensors, xanthopsins, phytochromes, blue-light sensors using flavin adenine dinucleotide (BLUF), cryptochromes, and rhodopsins. All are water-soluble proteins except rhodopsins, which are integral membrane proteins; all are based on a modular architecture except cryptochromes and rhodopsins; and each displays a distinct, light-dependent chemical process based on the photochemistry of their nonprotein chromophore, such as isomerization about a double bond (xanthopsins, phytochromes, and rhodopsins), formation or rupture of a covalent bond (LOV sensors), or electron transfer (BLUF sensors and cryptochromes).

Abstract: Recently, several advances have been made in the activation and deactivation of gene expression using light. These developments are based on the application of small molecule inducers of gene expression, antisense- or RNA interference-mediated gene silencing, and the photochemical control of proteins regulating gene function. The majority of the examples employ a classical 'caging technology', through the chemical installation of a light-removable protecting group on the biological molecule (small molecule, oligonucleotide, or protein) of interest and rendering it inactive. UV light irradiation then removes the caging group and activates the molecule, enabling control over gene activity with high spatial and temporal resolution.

Abstract: Protein-protein interactions are essential for many cellular processes. We have developed a technology called light-activated dimerization (LAD) to artificially induce protein hetero- and homodimerization in live cells using light. Using the FKF1 and GIGANTEA (GI) proteins of Arabidopsis thaliana, we have generated protein tags whose interaction is controlled by blue light. We demonstrated the utility of this system with LAD constructs that can recruit the small G-protein Rac1 to the plasma membrane and induce the local formation of lamellipodia in response to focal illumination. We also generated a light-activated transcription factor by fusing domains of GI and FKF1 to the DNA binding domain of Gal4 and the transactivation domain of VP16, respectively, showing that this technology is easily adapted to other systems. These studies set the stage for the development of light-regulated signaling molecules for controlling receptor activation, synapse formation and other signaling events in organisms.

Abstract: Phototropin-like LOV domains form a cysteinyl-flavin adduct in response to blue light but show considerable variation in output signal and the lifetime of the photo-adduct signaling state. Mechanistic studies of the slow-cycling fungal LOV photoreceptor Vivid (VVD) reveal the importance of reactive cysteine conformation, flavin electronic environment and solvent accessibility for adduct scission and thermal reversion. Proton inventory, pH effects, base catalysis and structural studies implicate flavin N(5) deprotonation as rate-determining for recovery. Substitutions of active site residues Ile74, Ile85, Met135 and Met165 alter photoadduct lifetimes by over four orders of magnitude in VVD, and similar changes in other LOV proteins show analogous effects. Adduct state decay rates also correlate with changes in conformational and oligomeric properties of the protein necessary for signaling. These findings link natural sequence variation of LOV domains to function and provide a means to design broadly reactive light-sensitive probes.

Abstract: Light control over enzyme function represents a novel and exciting field of biocatalysis research. Blue-light photoreceptors of the Light, Oxygen, Voltage (LOV) family have recently been investigated for their applicability as photoactive switches. We discuss here the primary photochemical events leading to light activation of LOV domains as well as the proposed signal propagation mechanism to the respective effector domain. Furthermore, we describe the construction of LOV fusions to different effector domains, namely a dihydrofolate reductase from Escherichia coli and a lipase from Bacillus subtilis. Both fusion partners retained functionality, and alteration of enzyme activity by light was also demonstrated. Hence, it appears that fusion of LOV photoreceptors to functional enzyme target sites via appropriate linker structures may represent a straightforward strategy to design light controllable biocatalysts.

Abstract: The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.

Abstract: Oligomeric structures of the four LOV domains in Arabidopsis phototropin1 (phot1) and 2 (phot2) were studied using crosslinking. Both LOV1 domains of phot1 and phot2 form a dimer independently on the light conditions, suggesting that the LOV1 domain can be a stable dimerization site of phot in vivo. In contrast, phot1-LOV2 is in a monomer-dimer equilibrium and phot2-LOV2 exists as a monomer in the dark. Blue light-induced a slight increase in the monomer population in phot1-LOV2, suggesting a possible blue light-inducible dissociation of dimers. Furthermore, blue light caused a band shift of the phot2-LOV2 monomer. CD spectra revealed the unfolding of helices and the formation of strand structures. Both light-induced changes were reversible in the dark.

Abstract: Light is a key stimulus for plant biological functions, several of which are controlled by light-activated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, light-oxygen-voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internally bound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jalpha helix and, ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue [Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain] switches its hydrogen bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the Ibeta strand that interacts with the Jalpha helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jalpha helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudodark state (Q513L) or a pseudolit state (Q513N). Further, both mutations changed the photochemical properties of this receptor, in particular the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from the core of the LOV domain through the Ibeta strand to the peripheral Jalpha helix.

Abstract: Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by approximately 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40 degrees -60 degrees rotational movement within an alpha-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by alpha-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.

Abstract: Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites.