Curated Optogenetic Publication Database

Abstract: Optogenetics, genetically encoded engineering of proteins to respond to light, has enabled precise control of the timing and localization of protein activity in live cells and for specific cell types in animals. Light-sensitive ion channels have become well established tools in neurobiology, and a host of new methods have recently enabled the control of other diverse protein structures as well. This review focuses on approaches to switch proteins between physiologically relevant, naturally occurring conformations using light, accomplished by incorporating light-responsive engineered domains that sterically and allosterically control the active site.

Abstract: Cell morphogenesis is critical for embryonic development, tissue formation, and wound healing. Our ability to manipulate endogenous mechanisms to control cell shape, however, remains limited. Here we combined surface micropatterning of adhesion molecules with optogenetic activation of intracellular signaling pathways to control the nature and morphology of cellular protrusions. We employed geometry-dependent pre-organization of cytoskeletal structures together with acute activation of signaling pathways that control actin assembly to create a tool capable of generating membrane protrusions at defined cellular locations. Further, we find that the size of microfabricated patterns of adhesion molecules influences the molecular mechanism of cell protrusion: larger patterns enable cells to create actin-filled lamellipodia while smaller patterns promote formation of spherical blebs. Optogenetic perturbation of signaling pathways in these cells changes the size of blebs and convert them into lamellipodia. Our results demonstrate how the coordinated manipulation of adhesion geometry and cytoskeletal dynamics can be used to control membrane protrusion and cell morphogenesis.

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: Cell adhesions to the extracellular matrix and to neighboring cells are fundamental to cell behavior and have also been implemented into minimal synthetic cells, which are assembled from molecular building blocks from the bottom-up. Investigating adhesion in cell mimetic models with reduced complexity provides a better understanding of biochemical and biophysical concepts underlying the cell adhesion machinery. In return, implementing cell-matrix and cell-cell adhesions into minimal synthetic cells allows reconstructing cell functions associated with cell adhesions including cell motility, multicellular prototissues, fusion of vesicles, and the self-sorting of different cell types. Cell adhesions have been mimicked using both the native cell receptors and reductionist mimetics providing a variety of specific, reversible, dynamic, and spatiotemporally controlled interactions. This review gives an overview of different minimal adhesion modules integrated into different minimal synthetic cells drawing inspiration from cell and colloidal science.

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: Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction. Consistent with known effects of constraining high concentrations of proteins to a membrane in vitro, there is localized curvature and tension decrease in the plasma membrane. Integrin activity, sensitive to mechanical forces, is activated in this region. Localized mechanical activation of integrin with optogenetics allowed simultaneous imaging of the molecular and cellular response, helping uncover a positive feedback loop comprising SFK- and ERK-dependent RhoA activation, actomyosin contractility, rearward membrane flow, and membrane tension decrease underlying this mode of cell migration.

Abstract: miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments.

Abstract: The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.

Abstract: How a small number of signaling pathways can be re-used in distinct embryonic contexts to control different fates remains unclear. In this issue of Developmental Cell, Johnson and Toettcher (2019) use optogenetic approaches to explore how different dynamic ERK signaling states control specific developmental fates in the Drosophila embryo.

Abstract: Synthetic tools for the control of protein function are valuable for biomedical research to characterize cellular functions of essential proteins or if a rapid switch in protein activity is necessary. The ability to tune protein activity precisely opens another level of investigations that is not available with gene deletion mutants. Control of protein stability is a versatile approach to influence the activity of a target protein by its cellular abundance. Diverse strategies have been developed to achieve efficient proteolysis using external inducers or differentiation-coupled signals. The latter is especially important for the inactivation of a protein during a developmental process. Recently, several approaches to achieve this have been engineered. In this article, we present current synthetic tools for regulation of protein stability that allow fine-tuning of protein abundance, their advantages and disadvantages with an emphasis on methods applicable in the context of cell differentiation and development. We give an outlook toward future developments and discuss main applications of these tools.

Abstract: Modulation of liquid-liquid and liquid-hydrogel phase transitions is central to avoid the cytotoxic aggregation of proteins in eukaryotic cells, but knowledge on its relevance in bacteria is limited. Here the power of optogenetics to engineer proteins as light-responsive switches has been used to control the balance between solubility and aggregation for LOV2-WH1, a chimera between the plant blue light-responsive domain LOV2 and the bacterial prion-like protein RepA-WH1. These proteins were first linked by fusing, as a continuous α-helix, the C-terminal photo-transducer Jα helix in LOV2 with the N-terminal domain-closure α1 helix in RepA-WH1, and then improved for light-responsiveness by including mutations in the Jα moiety. In the darkness and in a crowded solution in vitro, LOV2-WH1 nucleates the irreversible assembly of amyloid fibers into a hydrogel. However, under blue light illumination LOV2-WH1 assembles as soluble oligomers. When expressed in Escherichia coli, LOV2-WH1 forms in the darkness large intracellular amyloid inclusions compatible with bacterial proliferation. Strikingly, under blue light LOV2-WH1 aggregates decrease in size while they become detrimental for bacterial growth. LOV2-WH1 optogenetics governs the assembly of mutually exclusive inert amyloid fibers or cytotoxic oligomers, thus enabling the navigation of the conformational landscape of protein amyloidogenesis to generate potential photo-activated anti-bacterial devices (optobiotics).

Abstract: Abstract not available.

Abstract: The dynamic and spatiotemporal control of integrin‐mediated cell adhesion to RGD motifs in its extracellular matrix (ECM) is important for understating cell biology and biomedical applications because cell adhesion fundamentally regulates cellular behavior. Herein, the first photoswitchable synthetic ECM protein, Photo‐ECM, based on the blue light switchable protein LOV2 is engineered. The Photo‐ECM protein includes a RGD sequence, which is hidden in the folded LOV2 protein structure in the dark and is exposed under blue light so that integrins can bind and cells can adhere. The switchable presentation of the RGD motif allows to reversibly mediate and modulate integrin‐based cell adhesions using noninvasive blue light. With this protein cell adhesions in live cells could be reversed and the dynamics at the cellular level is observed. Hence, the Photo‐ECM opens a new possibility to investigate the spatiotemporal regulation of cell adhesions in cell biology and is the first step toward a genetically encoded and light‐responsive ECM.

Abstract: Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules. VIDEO ABSTRACT.

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: 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: Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.

Abstract: From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.

Abstract: On-demand and long-term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self-replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin-free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light-regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.

Abstract: Nerve growth factor elicits signaling outcomes by interacting with both its high-affinity receptor, TrkA, and its low-affinity receptor, p75NTR. Although these two receptors can regulate distinct cellular outcomes, they both activate the extracellular-signal-regulated kinase pathway upon nerve growth factor stimulation. To delineate TrkA subcircuits in PC12 cell differentiation, we developed an optogenetic system whereby light was used to specifically activate TrkA signaling in the absence of nerve growth factor. By using tyrosine mutants of the optogenetic TrkA in combination with pathway-specific pharmacological inhibition, we find that Y490 and Y785 each contributes to PC12 cell differentiation through the extracellular-signal-regulated kinase pathway in an additive manner. Optogenetic activation of TrkA eliminates the confounding effect of p75NTR and other potential off-target effects of the ligand. This approach can be generalized for the mechanistic study of other receptor-mediated signaling pathways.

Abstract: Developing materials to encapsulate and deliver functional proteins inside the body is a challenging yet rewarding task for therapeutic purposes. High production costs, mostly associated with the purification process, short-term stability in vivo, and controlled and prolonged release are major hurdles for the clinical application of protein-based biopharmaceuticals. In an attempt to overcome these hurdles, herein, the possibility of incorporating bacteria as protein factories into a material and externally controlling protein release using optogenetics is demonstrated. By engineering bacteria to express and secrete a red fluorescent protein in response to low doses of blue light irradiation and embedding them in agarose hydrogels, living materials are fabricated capable of releasing proteins into the surrounding medium when exposed to light. These bacterial hydrogels allow spatially confined protein expression and dosed protein release over several weeks, regulated by the area and extent of light exposure. The possibility of incorporating such complex functions in a material using relatively simple material and genetic engineering strategies highlights the immense potential and versatility offered by living materials for protein-based biopharmaceutical delivery.

Abstract: Synthetic biology has emerged as a multidisciplinary field that provides new tools and approaches to address longstanding problems in biology. It integrates knowledge from biology, engineering, mathematics, and biophysics to build—rather than to simply observe and perturb—biological systems that emulate natural counterparts or display novel properties. The interface between synthetic and developmental biology has greatly benefitted both fields and allowed to address questions that would remain challenging with classical approaches due to the intrinsic complexity and essentiality of developmental processes. This Progress Report provides an overview of how synthetic biology can help to understand a process that is crucial for the development of multicellular organisms: pattern formation. It reviews the major mechanisms of genetically encoded synthetic systems that have been engineered to establish spatial patterns at the population level. Limitations, challenges, applications, and potential opportunities of synthetic pattern formation are also discussed.

Abstract: Cellular activation of RAS GTPases into the GTP-binding "ON" state is a key switch for regulating brain functions. Molecular protein structural elements of rat sarcoma (RAS) and RAS homolog protein enriched in brain (RHEB) GTPases involved in this switch are discussed including their subcellular membrane localization for triggering specific signaling pathways resulting in regulation of synaptic connectivity, axonal growth, differentiation, migration, cytoskeletal dynamics, neural protection, and apoptosis. A beneficial role of neuronal H-RAS activity is suggested from cellular and animal models of neurodegenerative diseases. Recent experiments on optogenetic regulation offer insights into the spatiotemporal aspects controlling RAS/mitogen activated protein kinase (MAPK) or phosphoinositide-3 kinase (PI3K) pathways. As optogenetic manipulation of cellular signaling in deep brain regions critically requires penetration of light through large distances of absorbing tissue, we discuss magnetic guidance of re-growing axons as a complementary approach. In Parkinson's disease, dopaminergic neuronal cell bodies degenerate in the substantia nigra. Current human trials of stem cell-derived dopaminergic neurons must take into account the inability of neuronal axons navigating over a large distance from the grafted site into striatal target regions. Grafting dopaminergic precursor neurons directly into the degenerating substantia nigra is discussed as a novel concept aiming to guide axonal growth by activating GTPase signaling through protein-functionalized intracellular magnetic nanoparticles responding to external magnets.

Abstract: Microplate readers are foundational instruments in ex-perimental biology and bioengineering that enable mul-tiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construc-tion, validation, and benchmarking of an open-source microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs <$3500, a fraction of the cost of commer-cial plate readers, and can detect the fluorescence of common dyes down to ~10 nanomolar concentration. Functional capabilities were demonstrated in context of synthetic biology, optoge¬netics, and photosensory biol-ogy: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing, and by flavin photocycling kinetic measure-ments of a LOV (light-oxygen-voltage) domain photo-receptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and au-tomating it using the custom Python-based API (Appli-cation Program Interface) are provided. This work con-tributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid proto-typing capabilities and low-cost electronics, optoelec-tronics, and microcomputers.

Abstract: Cells sense, process, and respond to extracellular information using signaling networks: collections of proteins that act as precise biochemical sensors. These protein networks are characterized by both complex temporal organization, such as pulses of signaling activity, and by complex spatial organization, where proteins assemble structures at particular locations and times within the cell. Yet despite their ubiquity, studying these spatial and temporal properties has remained challenging because they emerge from the entire protein network rather than a single node, and cannot be easily tuned by drugs or mutations. These challenges are being met by a new generation of optogenetic tools capable of directly controlling the activity of individual signaling nodes over time and the assembly of protein complexes in space. Here, we outline how these recent innovations are being used in conjunction with engineering-influenced experimental design to address longstanding questions in signaling biology.