Showing 1 - 25 of 86 results
Optogenetic tools for manipulation of cyclic nucleotides, functionally coupled to CNG-channels.
The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers that regulate numerous biological processes. Malfunctional cNMP signalling is linked to multiple diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in C. elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarising rhodopsins, yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarisers based on K+ -currents.
Synthetic Biological Approaches for Optogenetics and Tools for Transcriptional Light‐Control in Bacteria.
Light has become established as a tool not only to visualize and investigate but also to steer biological systems. This review starts by discussing the unique features that make light such an effective control input in biology. It then gives an overview of how light‐control came to progress, starting with photoactivatable compounds and leading up to current genetic implementations using optogenetic approaches. The review then zooms in on optogenetics, focusing on photosensitive proteins, which form the basis for optogenetic engineering using synthetic biological approaches. As the regulation of transcription provides a highly versatile means for steering diverse biological functions, the focus of this review then shifts to transcriptional light regulators, which are presented in the biotechnologically highly relevant model organism Escherichia coli.
Real-Time Optogenetics System for Controlling Gene Expression Using a Model-Based Design.
Optimization of engineered biological systems requires precise control over the rates and timing of gene expression. Optogenetics is used to dynamically control gene expression as an alternative to conventional chemical-based methods since it provides a more convenient interface between digital control software and microbial culture. Here, we describe the construction of a real-time optogenetics platform, which performs closed-loop control over the CcaR-CcaS two-plasmid system in Escherichia coli. We showed the first model-based design approach by constructing a nonlinear representation of the CcaR-CcaS system, tuned the model through open-loop experimentation to capture the experimental behavior, and applied the model in silico to inform the necessary changes to build a closed-loop optogenetic control system. Our system periodically induces and represses the CcaR-CcaS system while recording optical density and fluorescence using image processing techniques. We highlight the facile nature of constructing our system and how our model-based design approach will potentially be used to model other systems requiring closed-loop optogenetic control.
Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell–Cell Interactions.
The regulation of cell–cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell–cell interactions with high precision are of great interest to a better understanding of their roles and building tissue‐like structures. Herein, the green light‐responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell–cell interactions form and lead to cell clustering in a concentration‐dependent manner. Upon green light illumination, the CarH based cell–cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell–cell interactions impact cell migration, as observed in a wound‐healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell–cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell–cell interactions in biological processes.
Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.
Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.
Optogenetic control of gut bacterial metabolism to promote longevity.
Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.
The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments.
Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.
The rise and shine of yeast optogenetics.
Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.
Optogenetics in plants.
The last two decades have witnessed the emergence of optogenetics; a field that has given researchers the ability to use light to control biological processes at high spatio-temporal and quantitative resolution, in a reversible manner with minimal side effects. Optogenetics has revolutionised the neurosciences, increased our understanding of cellular signalling and metabolic networks and resulted in variety of applications in biotechnology and biomedicine. However, implementing optogenetics in plants has been less straight forward given their dependency on light for their life cycle. Here, we highlight some of the widely used technologies in microorganisms and animal systems derived from plant photoreceptor proteins and discuss strategies recently implemented to overcome the challenges for using optogenetics in plants.
Optogenetic interrogation and control of cell signaling.
Signaling networks control the flow of information through biological systems and coordinate the chemical processes that constitute cellular life. Optogenetic actuators - genetically encoded proteins that undergo light-induced changes in activity or conformation - are useful tools for probing signaling networks over time and space. They have permitted detailed dissections of cellular proliferation, differentiation, motility, and death, and enabled the assembly of synthetic systems with applications in areas as diverse as photography, chemical synthesis, and medicine. In this review, we provide a brief introduction to optogenetic systems and describe their application to molecular-level analyses of cell signaling. Our discussion highlights important research achievements and speculates on future opportunities to exploit optogenetic systems in the study and assembly of complex biochemical networks.
Optogenetics and biosensors set the stage for metabolic cybergenetics.
Cybergenetic systems use computer interfaces to enable feed-back controls over biological processes in real time. The complex and dynamic nature of cellular metabolism makes cybergenetics attractive for controlling engineered metabolic pathways in microbial fermentations. Cybergenetics would not only create new avenues of research into cellular metabolism, it would also enable unprecedented strategies for pathway optimization and bioreactor operation and automation. Implementation of metabolic cybergenetics, however, will require new capabilities from actuators, biosensors, and control algorithms. The recent application of optogenetics in metabolic engineering, the expanding role of genetically encoded biosensors in strain development, and continued progress in control algorithms for biological processes suggest that this technology will become available in the not so distant future.
Light control of RTK activity: from technology development to translational research.
Inhibition of receptor tyrosine kinases (RTKs) by small molecule inhibitors and monoclonal antibodies is used to treat cancer. Conversely, activation of RTKs with their ligands, including growth factors and insulin, is used to treat diabetes and neurodegeneration. However, conventional therapies that rely on injection of RTK inhibitors or activators do not provide spatiotemporal control over RTK signaling, which results in diminished efficiency and side effects. Recently, a number of optogenetic and optochemical approaches have been developed that allow RTK inhibition or activation in cells and in vivo with light. Light irradiation can control RTK signaling non-invasively, in a dosed manner, with high spatio-temporal precision, and without the side effects of conventional treatments. Here we provide an update on the current state of the art of optogenetic and optochemical RTK technologies and the prospects of their use in translational studies and therapy.
In situ characterisation and manipulation of biological systems with Chi.Bio.
The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.Bio, a parallelised open-source platform that represents a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities, it incorporates tunable light outputs, spectrometry, and advanced automation features. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins in situ. By integrating precise measurement and actuation hardware into a single low-cost platform, Chi.Bio facilitates novel experimental methods for synthetic, systems, and evolutionary biology and broadens access to cutting-edge research capabilities.
Design and Application of Light-Regulated Receptor Tyrosine Kinases.
Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware.
Non-neuromodulatory Optogenetic Tools in Zebrafish.
The zebrafish (Danio rerio) is a popular vertebrate model organism to investigate molecular mechanisms driving development and disease. Due to its transparency at embryonic and larval stages, investigations in the living organism are possible with subcellular resolution using intravital microscopy. The beneficial optical characteristics of zebrafish not only allow for passive observation, but also active manipulation of proteins and cells by light using optogenetic tools. Initially, photosensitive ion channels have been applied for neurobiological studies in zebrafish to dissect complex behaviors on a cellular level. More recently, exciting non-neural optogenetic tools have been established to control gene expression or protein localization and activity, allowing for unprecedented non-invasive and precise manipulation of various aspects of cellular physiology. Zebrafish will likely be a vertebrate model organism at the forefront of in vivo application of non-neural optogenetic tools and pioneering work has already been performed. In this review, we provide an overview of non-neuromodulatory optogenetic tools successfully applied in zebrafish to control gene expression, protein localization, cell signaling, migration and cell ablation.
Flux controlling technology for central carbon metabolism for efficient microbial bio-production.
Syntheses of many commodities that are produced using microorganisms require cofactors such as ATP and NAD(P)H. Thus, optimization of the flux distribution in central carbon metabolism, which plays a key role in cofactor regeneration, is critical for enhancing the production of the target compounds. Since the intracellular and extracellular conditions change over time in the fermentation process, dynamic control of the metabolic system for maintaining the cellular state appropriately is necessary. Here, we review techniques for detecting the intracellular metabolic state with fluorescent sensors and controlling the flux of central carbon metabolism with optogenetic tools, as well as present a prospect of bio-production processes for fine-tuning the flux distribution.
Why is CarH photolytically active in comparison to other B12-dependent enzymes?
The discovery of naturally occurring B12-depedent photoreceptors has allowed for applications of cobalamins (Cbls) in optogenetics and synthetic biology to emerge. However, theoretical investigations of the complex mechanisms of these systems have been lacking. Adenosylcobalamin (AdoCbl)-dependent photoreceptor, CarH, is one example and it relies on daylight to perform its catalytic function. Typically, in enzymes employing AdoCbl as their cofactor, the Co-C5' bond activation and cleavage is triggered by substrate binding. The cleavage of the Co-C5' bond is homolytic resulting in radical pair formation. However, in CarH, this bond is instead activated by light. To explore this peculiarity, the ground and first excited state potential energy surfaces (PESs) were constructed using the quantum mechanics/molecular mechanics (QM/MM) framework and compared with other AdoCbl-dependent enzymes. QM/MM results indicate that CarH is photolytically active as a result of the AdoCbl dual role, acting as a radical generator and as a substrate. Photo-cleavage of the Co-C5' bond and subsequent H-atom abstraction is possible because of the specific orientation of the H-C4' bond with respect to the Co(II) center. Comparison with other AdoCbl-dependent enzymes indicate that the protein environment in the CarH active center alters the photochemistry of AdoCbl by controlling the stereochemistry of the ribose moiety.
Color Sensing and Signal Transmission Diversity of Cyanobacterial Phytochromes and Cyanobacteriochromes.
To perceive fluctuations in light quality, quantity, and timing, higher plants have evolved diverse photoreceptors including UVR8 (a UV-B photoreceptor), cryptochromes, phototropins, and phytochromes (Phys). In contrast to plants, prokaryotic oxygen-evolving photosynthetic organisms, cyanobacteria, rely mostly on bilin-based photoreceptors, namely, cyanobacterial phytochromes (Cphs) and cyanobacteriochromes (CBCRs), which exhibit structural and functional differences compared with plant Phys. CBCRs comprise varying numbers of light sensing domains with diverse color-tuning mechanisms and signal transmission pathways, allowing cyanobacteria to respond to UV-A, visible, and far-red lights. Recent genomic surveys of filamentous cyanobacteria revealed novel CBCRs with broader chromophore-binding specificity and photocycle protochromicity. Furthermore, a novel Cph lineage has been identified that absorbs blue-violet/yellow-orange light. In this minireview, we briefly discuss the diversity in color sensing and signal transmission mechanisms of Cphs and CBCRs, along with their potential utility in the field of optogenetics.
Turning Cell Adhesions ON or OFF with High Spatiotemporal Precision Using the Green Light Responsive Protein CarH.
Spatiotemporal control of integrin-mediated cell adhesions to extracellular matrix regulates cell behavior with has numerous implications for biotechnological applications. In this work, two approaches for regulating cell adhesions in space and time with high precision are reported, both of which utilize green light. In the first design, CarH, which is a tetramer in the dark, is used to mask cRGD adhesion-peptides on a surface. Upon green light illumination, the CarH tetramer dissociates into its monomers, revealing the adhesion peptide so that cells can adhere. In the second design, the RGD motif is incorporated into the CarH protein tetramer such that cells can adhere to surfaces functionalized with this protein. The cell adhesions can be disrupted with green light, due to the disassembly of the CarH-RGD protein. Both designs allow for photoregulation with noninvasive visible light and open new possibilities to investigate the dynamical regulation of cell adhesions in cell biology.
Recent advances in the use of genetically encodable optical tools to elicit and monitor signaling events.
Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
Role of cyclic nucleotides and their downstream signaling cascades in memory function: being at the right time at the right spot.
A plethora of studies indicate the important role of cAMP and cGMP cascades in neuronal plasticity and memory function. As a result, altered cyclic nucleotide signaling has been implicated in the pathophysiology of mnemonic dysfunction encountered in several diseases. In the present review we provide a wide overview of studies regarding the involvement of cyclic nucleotides, as well as their upstream and downstream molecules, in physiological and pathological mnemonic processes. Next, we discuss the regulation of the intracellular concentration of cyclic nucleotides via phosphodiesterases, the enzymes that degrade cAMP and/or cGMP, and via A-kinase-anchoring proteins that refine signal compartmentalization of cAMP signaling. We also provide an overview of the available data pointing to the existence of specific time windows in cyclic nucleotide signaling during neuroplasticity and memory formation and the significance to target these specific time phases for improving memory formation. Finally, we highlight the importance of emerging imaging tools like Förster resonance energy transfer imaging and optogenetics in detecting, measuring and manipulating the action of cyclic nucleotide signaling cascades.
Phytochromes and Cyanobacteriochromes: Photoreceptor Molecules Incorporating a Linear Tetrapyrrole Chromophore.
In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.
Functional Modulation of Receptor Proteins on Cellular Interface with Optogenetic System.
In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.
Multiple-site diversification of regulatory sequences enables inter-species operability of genetic devices.
The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e. ho1 and pcyA genes for synthesis of phycocyanobilin, the ccaS/ccaR system from Synechocystis and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by [i] reassembling them together in a single broad host range, standardized vector and [ii] subjecting the non-coding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species i.e. optimization of relative expression levels and upturning of subcomplex stoichiometry.
Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors.
The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.