Showing 1 - 25 of 64 results
Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.
We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
Dronpa: a light-switchable fluorescent protein for opto-biomechanics.
Since the development of GFP, fluorescent proteins (FP) are indispensable tools in molecular biology. Some FPs change their structure under illumination, which affects their interaction with other biomolecules or proteins. Especially, FPs that are able to form switchable dimers became an important tool in the field of optogenetics. They are widely used for the investigation of signaling pathways, the control of surface recruitment as well as enzyme and gene regulation. However, optogenetics did not yet develop tools for the investigation of biomechanical processes. This could be leveraged if one could find a light-switchable FP dimer, that is able to withstand sufficiently high forces. In this work we measure the rupture force of the switchable interface in pdDronpa1.2 dimers using atomic force microscopy based single molecule force spectroscopy. The most probable dimer rupture force amounts to around 80 pN at a pulling speed of 1600 nm/s. After switching of the dimer using illumination at 488 nm there are hardly any measurable interface interactions, which indicates the successful dissociation of the dimers. Hence this Dronpa dimer could expand the current toolbox in optogenetics with new opto-biomechanical applications like the control of tension in adhesion processes.
A yeast system for discovering optogenetic inhibitors of eukaryotic translation initiation.
The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis non-invasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, LOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photo-activated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate of human eIF4E-depednednt translation initiation in a mechanistically defined manner.
Controlling protein conformation with light.
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.
Photodimerization systems for regulating protein-protein interactions with light.
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.
Perspectives of RAS and RHEB GTPase Signaling Pathways in Regenerating Brain Neurons.
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.
A bright future: optogenetics to dissect the spatiotemporal control of cell behavior.
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.
Programming Bacteria With Light—Sensors and Applications in Synthetic Biology
Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture.
Optogenetic Medicine: Synthetic Therapeutic Solutions Precision-Guided by Light.
Gene- and cell-based therapies are well recognized as central pillars of next-generation medicine, but controllability remains a critical issue for clinical applications. In this context, optogenetics is opening up exciting new opportunities for precision-guided medicine by using illumination with light of appropriate intensity and wavelength as a trigger signal to achieve pinpoint spatiotemporal control of cellular activities, such as transgene expression. In this review, we highlight recent advances in optogenetics, focusing on devices for biomedical applications. We introduce the construction and applications of optogenetic-based biomedical tools to treat neurological diseases, diabetes, heart diseases, and cancer, as well as bioelectronic implants that combine light-interfaced electronic devices and optogenetic systems into portable personalized precision bioelectronic medical tools. Optogenetics-based technology promises the capability to achieve traceless, remotely controlled precision dosing of an enormous range of therapeutic outputs. Finally, we discuss the prospects for optogenetic medicine, as well as some emerging challenges.
Reversible hydrogels with tunable mechanical properties for optically controlling cell migration.
Synthetic hydrogels are widely used as biomimetic in vitro model systems to understand how cells respond to complex microenvironments. The mechanical properties of hydrogels are deterministic for many cellular behaviors, including cell migration, spreading, and differentiation. However, it remains a major challenge to engineer hydrogels that recapture the dynamic mechanical properties of native extracellular matrices. Here, we provide a new hydrogel platform with spatiotemporally tunable mechanical properties to assay and define cellular behaviors under light. The change in the mechanical properties of the hydrogel is effected by a photo-induced switch of the cross-linker fluorescent protein, Dronpa145N, between the tetrameric and monomeric states, which causes minimal changes to the chemical properties of the hydrogel. The mechanical properties can be rapidly and reversibly tuned for multiple cycles using visible light, as confirmed by rheological measurements and atomic force microscopybased nano-indentation. We further demonstrated real-time and reversible modulation of cell migration behaviors on the hydrogels through photo-induced stiffness switching, with minimal invasion to the cultured cells. Hydrogels with a programmable mechanical history and a spatially defined mechanical hierarchy might serve as an ideal model system to better understand complex cellular functions.
Light‐Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology.
The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology‐inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light‐controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non‐neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light‐sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light‐controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.
Discovering selective binders for photoswitchable proteins using phage display.
Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control - precise spatial and temporal resolution - are currently restricted to a few well-defined natural systems. In addition, the affinities and kinetics of native interactions are often sub-optimal and are difficult to engineer in the absence of any structural information. We report a phage display strategy using a small scaffold protein that can be used to discover new binding partners for both light and dark states of a given light-switchable protein. We used our approach to generate binding partners that interact specifically with the light state or the dark state conformation of two light-switchable proteins: PYP, a test case for a protein with no known partners, and AsLOV2 a well-characterized protein. We show that these novel light-switchable protein-protein interactions can function in living cells to control subcellular localization processes.
A compendium of chemical and genetic approaches to light-regulated gene transcription.
On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.
Blue-Light Receptors for Optogenetics.
Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.
Optogenetics: A Primer for Chemists.
The field of optogenetics uses genetically encoded, light-responsive proteins to control physiological processes. This technology has been hailed as the one of the ten big ideas in brain science in the past decade, the breakthrough of the decade, and the method of the year in 2010 and again in 2014. The excitement evidenced by these proclamations is confirmed by a couple of impressive numbers. The term "optogenetics" was coined in 2006. As of December 2017, "optogenetics" is found in the title or abstract of almost 1600 currently funded National Institutes of Health grants. In addition, nearly 600 reviews on optogenetics have appeared since 2006, which averages out to approximately one review per week! However, in spite of these impressive numbers, the potential applications and implications of optogenetics are not even close to being fully realized. This is due, in large part, to the challenges associated with the design of optogenetic analogs of endogenous proteins. This review is written from a chemist's perspective, with a focus on the molecular strategies that have been developed for the construction of optogenetic proteins.
Induction of signal transduction using non-channelrhodopsin-type optogenetic tools.
Signal transductions are the basis for all cellular functions. Previous studies investigating signal transductions mainly relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies do not allow the modulation of protein activity in cells, tissues, and organs in animals with high spatial and temporal precision. Recently, non-channelrhodopsin-type optogenetic tools for regulating signal transduction have emerged. These photoswitches address several disadvantages of previous techniques, and allow us to control a variety of signal transductions such as cell membrane dynamics, calcium signaling, lipid signaling, and apoptosis. In this review, we summarize recent advances in the development of such photoswitches and how these optotools are applied to signaling processes.
Optogenetically controlled protein kinases for regulation of cellular signaling.
Protein kinases are involved in the regulation of many cellular processes including cell differentiation, survival, migration, axon guidance and neuronal plasticity. A growing set of optogenetic tools, termed opto-kinases, allows activation and inhibition of different protein kinases with light. The optogenetic regulation enables fast, reversible and non-invasive manipulation of protein kinase activities, complementing traditional methods, such as treatment with growth factors, protein kinase inhibitors or chemical dimerizers. In this review, we summarize the properties of the existing optogenetic tools for controlling tyrosine kinases and serine-threonine kinases. We discuss how the opto-kinases can be applied for studies of spatial and temporal aspects of protein kinase signaling in cells and organisms. We compare approaches for chemical and optogenetic regulation of protein kinase activity and present guidelines for selection of opto-kinases and equipment to control them with light. We also describe strategies to engineer novel opto-kinases on the basis of various photoreceptors.
Optogenetics in cancer drug discovery.
The discovery and domestication of biomolecules that respond to light has taken a light of its own, providing new molecular tools with incredible spatio-temporal resolution to manipulate cellular behavior. Areas covered: The authors herein analyze the current optogenetic tools in light of their current, and potential, uses in cancer drug discovery, biosafety and cancer biology. Expert opinion: The pipeline from drug discovery to the clinic is plagued with drawbacks, where most drugs fail in either efficacy or safety. These issues require the redesign of the pipeline and the development of more controllable/personalized therapies. Light is, aside from inexpensive, almost harmless if used appropriately, can be directed to single cells or organs with controllable penetration, and comes in a variety of wavelengths. Light-responsive systems can activate, inhibit or compensate cell signaling pathways or specific cellular events, allowing the specific control of the genome and epigenome, and modulate cell fate and transformation. These synthetic molecular tools have the potential to revolutionize drug discovery and cancer research.
Optically controlled reversible protein hydrogels based on photoswitchable fluorescent protein Dronpa.
Exploiting the optically controlled association and dissociation behavior of a photoswitchable fluorescent protein, Dronpa145N, here we demonstrate the engineering of an optically switchable reversible protein hydrogel using Dronpa145N-based protein building blocks. Our results open the possibility to optically tune the mechanical, chemical and structural properties of protein hydrogels.
Emerging approaches for spatiotemporal control of targeted genome with inducible CRISPR-Cas9.
The breakthrough CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nuclease has revolutionized our ability in genome engineering. Although Cas9 is already a powerful tool for simple and efficient target endogenous gene manipulation, further engineering of Cas9 will improve the performance of Cas9, such as gene-editing efficiency and accuracy in vivo, and expand the application possibility of this Cas9 technology. The emerging inducible Cas9 methods, which can control the activity of Cas9 using an external stimulus such as chemicals and light, have the potential to provide spatiotemporal gene manipulation in user-defined cell population at a specific time and improve the accuracy of Cas9-mediated genome editing. In this review, we focus on the recent advance in inducible Cas9 technologies, especially light-inducible Cas9, and related methodologies, and also discuss future directions of this emerging tools.
Optogenetic Tools for Subcellular Applications in Neuroscience.
The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications.
A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription.
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
Applications of optobiology in intact cells and multi-cellular organisms.
Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.
Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.
Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.
Synthetic biological approaches to optogenetically control cell signaling.
Precise spatial and temporal control of cellular processes is in life sciences a highly sought-after capability. In the recent years, this goal has become progressively achievable through the field of optogenetics, which utilizes light as a non-invasive means to control genetically encoded light-responsive proteins. The latest optogenetic systems, such as those for control of subcellular localization or cellular decision-making and tissue morphogenesis provide us with insights to gain a deeper understanding of the cellular inner workings. Besides, they hold a potential for further development into biomedical applications, from in vitro optogenetics-assisted drug candidate screenings to light-controlled gene therapy and tissue engineering.