Curated Optogenetic Publication Database

Abstract: Two-component systems (TCS) constitute the predominant means by which prokaryotes read out and adapt to their environment. Canonical TCSs comprise a sensor histidine kinase (SHK), usually a transmembrane receptor, and a response regulator (RR). In signal-dependent manner, the SHK autophosphorylates and in turn transfers the phosphoryl group to the RR which then elicits downstream responses, often in form of altered gene expression. SHKs also catalyze the hydrolysis of the phospho-RR, hence, tightly adjusting the overall degree of RR phosphorylation. Photoreceptor histidine kinases are a subset of mostly soluble, cytosolic SHKs that sense light in the near-ultraviolet to near-infrared spectral range. Owing to their experimental tractability, photoreceptor histidine kinases serve as paradigms and provide unusually detailed molecular insight into signal detection, decoding, and regulation of SHK activity. The synthesis of recent results on receptors with light-oxygen-voltage, bacteriophytochrome and microbial rhodopsin sensor units identifies recurring, joint signaling strategies. Light signals are initially absorbed by the sensor module and converted into subtle rearrangements of α helices, mostly through pivoting and rotation. These conformational transitions propagate through parallel coiled-coil linkers to the effector unit as changes in left-handed superhelical winding. Within the effector, subtle conformations are triggered that modulate the solvent accessibility of residues engaged in the kinase and phosphatase activities. Taken together, a consistent view of the entire trajectory from signal detection to regulation of output emerges. The underlying allosteric mechanisms could widely apply to TCS signaling in general.

Abstract: Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. The new expansions of the toolbox facilitate control of signal transduction, genome editing, 'painting' patterns of active molecules onto cellular membranes and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches has also seen interesting progress. Here we provide an overview of the optogenetic systems and the emerging chemo-optogenetic systems, and discuss recent applications in tackling complex biological problems.

Abstract: Sleep is evolutionarily conserved, thus studying simple invertebrates such as Caenorhabditis elegans can provide mechanistic insight into sleep with single cell resolution. A conserved pathway regulating sleep across phylogeny involves cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that functions in neurons by activating protein kinase A (PKA). C. elegans sleep in response to cellular stress caused by environmental insults (stress-induced sleep (SIS)), a model for studying sleep during sickness. SIS is controlled by simple neural circuitry, thus allows for cellular dissection of cAMP signaling during sleep. We employed a red light activated adenylyl cyclase (AC), IlaC22, to identify cells involved in SIS regulation. We find that pan-neuronal activation of IlaC22 disrupts SIS through mechanisms independent of the cAMP response element binding protein (CREB). Activating IlaC22 in the single DVA interneuron, the paired RIF interneurons, and in the CEPsh glia identified these cells as wake-promoting. Using a cAMP biosensor, epac1-camps, we found that cAMP is decreased in the RIF and DVA interneurons by neuropeptidergic signaling from the ALA neuron. Ectopic over expression of sleep-promoting neuropeptides coded by flp-13 and flp-24, released from the ALA, reduced cAMP in the DVA and RIFs, respectively. Over expression of the wake-promoting neuropeptides coded by pdf-1 increased cAMP levels in the RIFs. Using a combination of optogenetic manipulation and in vivo imaging of cAMP we have identified wake-promoting neurons downstream of the neuropeptidergic output of the ALA. Our data suggest that sleep- and wake-promoting neuropeptides signal to reduce and heighten cAMP levels during sleep, respectively.

Abstract: As diffusible second messengers, cyclic nucleoside monophosphates (cNMPs) relay and amplify molecular signals in myriad cellular pathways. The triggering of downstream physiological responses often requires defined cNMP gradients in time and space, generated through the concerted action of nucleotidyl cyclases and phosphodiesterases (PDEs). In an approach denoted optogenetics, sensory photoreceptors serve as genetically encoded, light-responsive actuators to enable the noninvasive, reversible, and spatiotemporally precise control of manifold cellular processes, including cNMP metabolism. Although nature provides efficient photoactivated nucleotidyl cyclases, light-responsive PDEs are scarce. Through modular recombination of a bacteriophytochrome photosensor and the effector of human PDE2A, we previously generated the light-activated, cNMP-specific PDE LAPD. By pursuing parallel design strategies, we here report a suite of derivative PDEs with enhanced amplitude and reversibility of photoactivation. Opposite to LAPD, far-red light completely reverts prior activation by red light in several PDEs. These improved PDEs thus complement photoactivated nucleotidyl cyclases and extend the sensitivity of optogenetics to red and far-red light. More generally, our study informs future efforts directed at designing bacteriophytochrome photoreceptors.

Abstract: Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Abstract: Optogenetic probes can be powerful tools for dissecting complexity in cell biology, but there is a lack of instrumentation to exploit their potential for automated, high-information-content experiments. This protocol describes the construction and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments that allows simultaneous manipulation of common red- and blue-light-sensitive optogenetic probes. The optoPlate-96 enables illumination of individual wells in 96-well microwell plates or in groups of wells in 384-well plates. Its design ensures that there will be no cross-illumination between microwells in 96-well plates, and an active cooling system minimizes sample heating during light-intensive experiments. This protocol details the steps to assemble, test, and use the optoPlate-96. The device can be fully assembled without specialized equipment beyond a 3D printer and a laser cutter, starting from open-source design files and commercially available components. We then describe how to perform a typical optogenetics experiment using the optoPlate-96 to stimulate adherent mammalian cells. Although optoPlate-96 experiments are compatible with any plate-based readout, we describe analysis using quantitative single-cell immunofluorescence. This workflow thus allows complex optogenetics experiments (independent control of stimulation colors, intensity, dynamics, and time points) with high-dimensional outputs at single-cell resolution. Starting from 3D-printed and laser-cut components, assembly and testing of the optoPlate-96 can be accomplished in 3-4 h, at a cost of ~$600. A full optoPlate-96 experiment with immunofluorescence analysis can be performed within ~24 h, but this estimate is variable depending on the cell type and experimental parameters.

Abstract: The development of optical nano-bio interfaces is a fundamental step toward connecting biological networks and traditional electronic computing systems. Compared to conventional chemical and electrical nano-bio interfaces, the use of light as a mediator enables new type of interfaces with unprecedented spatial and temporal resolutions. In this paper, the state of the art and future research directions in optogenomic interfaces are discussed. Optogenomic interfaces are light-mediated nano-bio interfaces that allow the control of the genome, i.e., the genes and their interactions in the cell nucleus (and, thus, of all the cell functionalities) with (sub) cellular resolution and high temporal accuracy. Given its fundamental role in the process of cell development, the study is focused on the interactions with the fibroblast growth factor receptor 1 (FGFR1) gene and the integrative nuclear FGFR1 signaling (INFS) module in stem cells and in neuronal cells, whose control opens the door to transformative applications, including reconstructive medicine and cancer therapy. Three stages of optogenomic interfaces are described, ranging from already experimentally validated interfaces activating broad cellular responses and expressing individual genes to more advanced interfaces able to regulate and correct DNA topology, chromatin structure, and cellular development.

Abstract: In order to study the role of signaling proteins, such as kinases and GTPases, in brain functions it is necessary to control their activity at the appropriate spatiotemporal resolution and to examine the cellular and behavioral effects of such changes in activity. Reduced spatiotemporal resolution in the regulation of these proteins activity will impede the ability to understand the proteins normal functions as longer modification of their activity in non-normal locations could lead to effects different from their natural functions. To control intracellular signaling proteins at the highest temporal resolution recent innovative optogenetic approaches were developed to allow the control of photoactivable signaling proteins activity by light. These photoactivatable proteins can be activated in selected cell population in brain and in specific subcellular compartments. Minimal-invasive tools are being developed to photoactivate these proteins for study and therapy. Together these techniques afford an unprecedented spatiotemporal control of signaling proteins activity to unveil the function of brain proteins with high accuracy in behaving animals. As dysfunctional signaling proteins are involved in brain diseases, the optogenetic technique has also the potential to be used as a tool to treat brain diseases.

Abstract: Light in the near-infrared optical window (NIRW) penetrates deep through mammalian tissues, including the skull and brain tissue. Here we engineered an adenylate cyclase (AC) activated by NIRW light (NIRW-AC) and suitable for mammalian applications. To accomplish this goal, we constructed fusions of several bacteriophytochrome photosensory and bacterial AC modules using guidelines for designing chimeric homodimeric bacteriophytochromes. One engineered NIRW-AC, designated IlaM5, has significantly higher activity at 37 °C, is better expressed in mammalian cells, and can mediate cAMP-dependent photoactivation of gene expression in mammalian cells, in favorable contrast to the NIRW-ACs engineered earlier. The ilaM5 gene expressed from an AAV vector was delivered into the ventral basal thalamus region of the mouse brain, resulting in the light-controlled suppression of the cAMP-dependent wave pattern of the sleeping brain known as spindle oscillations. Reversible spindle oscillation suppression was observed in sleeping mice exposed to light from an external light source. This study confirms the robustness of principles of homodimeric bacteriophytochrome engineering, describes a NIRW-AC suitable for mammalian optogenetic applications, and demonstrates the feasibility of controlling brain activity via NIRW-ACs using transcranial irradiation.

Abstract: Living cells can impart materials with advanced functions, such as sense-and-respond, chemical production, toxin remediation, energy generation and storage, self-destruction, and self-healing. Here, an approach is presented to use light to pattern Escherichia coli onto diverse materials by controlling the expression of curli fibers that anchor the formation of a biofilm. Different colors of light are used to express variants of the structural protein CsgA fused to different peptide tags. By projecting color images onto the material containing bacteria, this system can be used to pattern the growth of composite materials, including layers of protein and gold nanoparticles. This is used to pattern cells onto materials used for 3D printing, plastics (polystyrene), and textiles (cotton). Further, the adhered cells are demonstrated to respond to sensory information, including small molecules (IPTG and DAPG) and light from light-emitting diodes. This work advances the capacity to engineer responsive living materials in which cells provide diverse functionality.

Abstract: The ability of living systems to self-sort different cells into separate assemblies and the ability to independently regulate different structures are one ingredient that gives rise to their spatiotemporal complexity. Here, this self-sorting behavior is replicated in a synthetic system with two types of colloidal particles; where each particle type independently self-assembles either under blue or red light into distinct clusters, known as narcissistic self-sorting. For this purpose, each particle type is functionalized either with the light-switchable protein VVDHigh or Cph1, which homodimerize under blue and red light, respectively. The response to different wavelengths of light and the high specificity of the protein interactions allows for the independent self-assembly of each particle type with blue or red light and narcissistic self-sorting. Moreover, as both of the photoswitchable protein interactions are reversible in the dark; also, the self-sorting is reversible and dynamic. Overall, the independent blue and red light controlled self-sorting in a synthetic system opens new possibilities to assemble adaptable, smart, and advanced materials similar to the complexity observed in tissues.

Abstract: Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways and valuable sensors for synthetic biology. However, most TCSs remain uncharacterized or difficult to harness for applications. Major challenges are that many TCS output promoters are unknown, subject to cross-regulation, or silent in heterologous hosts. Here, we demonstrate that the two largest families of response regulator DNA-binding domains can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. We exploit this plasticity to eliminate cross-regulation, un-silence a gram-negative TCS in a gram-positive host, and engineer a system with over 1,300-fold activation. Finally, we apply DNA-binding domain swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a previously uncharacterized pH sensor. This work should accelerate fundamental TCS studies and enable the engineering of a large family of genetically encoded sensors with diverse applications.

Abstract: Living organisms exhibit a wide range of intrinsic adaptive responses to incident light. Likewise, in optogenetics, biological systems are tailored to initiate predetermined cellular processes upon light exposure. As genetically encoded, light-gated actuators, sensory photoreceptors are at the heart of these responses in both the natural and engineered scenarios. Upon light absorption, photoreceptors enter a series of generally rapid photochemical reactions leading to population of the light-adapted signaling state of the receptor. Notably, this state persists for a while before thermally reverting to the original dark-adapted resting state. As a corollary, the inactivation of photosensitive biological circuits upon light withdrawal can exhibit substantial inertia. Intermittent illumination of suitable pulse frequency can hence maintain the photoreceptor in its light-adapted state while greatly reducing overall light dose, thereby mitigating adverse side effects. Moreover, several photoreceptor systems may be actuated sequentially with a single light color if they sufficiently differ in their inactivation kinetics. Here, we detail the construction of programmable illumination devices for the rapid and parallelized testing of biological responses to diverse lighting regimes. As the technology is based on open electronics and readily available, inexpensive components, it can be adopted by most laboratories at moderate expenditure. As we exemplify for two use cases, the programmable devices enable the facile interrogation of diverse illumination paradigms and their application in optogenetics and photobiology.

Abstract: The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.

Abstract: Bacteriophytochromes are a subfamily of the diverse light responsive phytochrome photoreceptors. Considering their preferential interaction with biliverdin IXα as endogenous cofactor, they have recently been used for creating optogenetic tools and engineering fluorescent probes. Ideal absorption characteristics for the activation of bacteriophytochrome-based systems in the therapeutic near-infrared window as well the availability of biliverdin in mammalian tissues have resulted in tremendous progress in re-engineering bacteriophytochromes for diverse applications. At the same time, both the structural analysis and the functional characterization of diverse naturally occurring bacteriophytochrome systems have unraveled remarkable differences in signaling mechanisms and have so far only touched the surface of the evolutionary diversity within the family of bacteriophytochromes. This review highlights recent findings and future challenges.

Abstract: Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.

Abstract: Biological networks sense extracellular stimuli and generate appropriate outputs within the cell that determine cellular response. Biological signal generators are becoming an important tool for understanding how information is transmitted in these networks and controlling network behavior. Signal generators produce well-defined, dynamic, intracellular signals of important network components, such as kinase activity or the concentration of a specific transcription factor. Synthetic biology tools coupled with in silico control have enabled the construction of these sophisticated biological signal generators. Here we review recent advances in biological signal generator construction and their use in systems biology studies. Challenges for constructing signal generators for a wider range of biological networks and generalizing their use are discussed.

Abstract: Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the know interaction partners, and could filter out all other proteins.

Abstract: Optogenetic dimerizers are modular domains that can be utilized in a variety of versatile ways to modulate cellular biochemistry. Because of their modularity, many applications using these tools can be easily transferred to new targets without extensive engineering. While a number of photodimerizer systems are currently available, the field remains nascent, with new optimizations for existing systems and new approaches to regulating biological function continuing to be introduced at a steady pace.

Abstract: This chapter describes the use of optogenetic heterodimerization in single cells within whole-vertebrate embryos. This method allows the use of light to reversibly bind together an "anchor" protein and a "bait" protein. Proteins can therefore be directed to specific subcellular compartments, altering biological processes such as cell polarity and signaling. I detail methods for achieving transient expression of fusion proteins encoding the phytochrome heterodimerization system in early zebrafish embryos (Buckley et al., Dev Cell 36(1):117-126, 2016) and describe the imaging parameters used to achieve subcellular light patterning.

Abstract: Gene regulatory networks and the dynamic responses they produce offer a wealth of information about how biological systems process information about their environment. Recently, researchers interested in dissecting these networks have been outsourcing various parts of their experimental workflow to computers. Here we review how, using microfluidic or optogenetic tools coupled with fluorescence imaging, it is now possible to interface cells and computers. These platforms enable scientists to perform informative dynamic stimulations of genetic pathways and monitor their reaction. It is also possible to close the loop and regulate genes in real time, providing an unprecedented view of how signals propagate through the network. Finally, we outline new tools that can be used within the framework of cell-machine interfaces.

Abstract: Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical- and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi- and fully synthetic open- and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals.

Abstract: Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength-specific, and dose- and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots.

Abstract: Optogenetics and photopharmacology are two perspective modern methodologies for control and monitoring of biological processes from an isolated cell to complex cell assemblies and organisms. Both methodologies use optically active components that being introduced into the cells of interest allow for optical control or monitoring of different cellular processes. In optogenetics, genetic materials are introduced into the cells to express light-sensitive proteins or protein constructs. In photopharmacology, photochromic compounds are delivered into a cell directly but not produced inside the cell from a genetic material. The development of both optogenetics and photopharmacology is inseparable from the design of improved tools (protein constructs or organic molecules) optimized for specific applications. Herein, we review the main tools that are used in modern optogenetics and photopharmaclogy and describe the types of cellular processes that can be controlled by these tools. Although a large number of different kinds of optogenetic tools exist, their performance can be evaluated with a limited number of metrics that have to be optimized for specific applications.We classify thesemetrics and describe the ways of their improvement.

Abstract: Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3', 5'-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.