Qr: switch:"PhyB/PIF"
Showing 76 - 100 of 113 results
76.
Bringing Light to Transcription: The Optogenetics Repertoire.
Abstract:
The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.
77.
Light-Induced Printing of Protein Structures on Membranes in Vitro.
Abstract:
Reconstituting functional modules of biological systems in vitro is an important yet challenging goal of bottom-up synthetic biology, in particular with respect to their precise spatiotemporal regulation. One of the most desirable external control parameters for the engineering of biological systems is visible light, owing to its specificity and ease of defined application in space and time. Here we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on time scales of seconds, and the pattern density can be precisely controlled by protein concentration, laser power, and activation time. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. Thus, light mediated sequential protein assembly in cell-free systems represents a promising approach to hierarchically building up the next level of complexity toward a minimal cell.
78.
Optogenetic regulation of transcription.
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Polesskaya, O
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Baranova, A
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Bui, S
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Kondratev, N
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Kananykhina, E
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Nazarenko, O
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Shapiro, T
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Barg Nardia, F
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Kornienko, V
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Chandhoke, V
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Stadler, I
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Lanzafame, R
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Myakishev‑Rempel, M
Abstract:
Optogenetics has become widely recognized for its success in real-time control of brain neurons by utilizing nonmammalian photosensitive proteins to open or close membrane channels. Here we review a less well known type of optogenetic constructs that employs photosensitive proteins to transduce the signal to regulate gene transcription, and its possible use in medicine. One of the problems with existing gene therapies is that they could remain active indefnitely while not allowing regulated transgene production on demand. Optogenetic regulation of transcription (ORT) could potentially be used to regulate the production of a biological drug in situ, by repeatedly applying light to the tissue, and inducing expression of therapeutic transgenes when needed. Red and near infrared wavelengths, which are capable of penetration into tissues, have potential for therapeutic applications. Existing ORT systems are reviewed herein with these considerations in mind.
79.
CRISPR/dCas9 Switch Systems for Temporal Transcriptional Control.
Abstract:
In a swift revolution, CRISPR/Cas9 has reshaped the means and ease of interrogating biological questions. Particularly, mutants that result in a nuclease-deactivated Cas9 (dCas9) provide scientists with tools to modulate transcription of genomic loci at will by targeting transcriptional effector domains. To interrogate the temporal order of events during transcriptional regulation, rapidly inducible CRISPR/dCas9 systems provide previously unmet molecular tools. In only a few years of time, numerous light and chemical-inducible switches have been applied to CRISPR/dCas9 to generate dCas9 switches. As these inducible switch systems are able to modulate dCas9 directly at the protein level, they rapidly affect dCas9 stability, activity, or target binding and subsequently rapidly influence downstream transcriptional events. Here we review the current state of such biotechnological CRISPR/dCas9 enhancements. Specifically we provide details on their flaws and strengths and on the differences in molecular design between the switch systems. With this we aim to provide a selection guide for researchers with keen interest in rapid temporal control over transcriptional modulation through the CRISPR/dCas9 system.
80.
Optogenetically controlled protein kinases for regulation of cellular signaling.
Abstract:
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.
81.
Optogenetics in cancer drug discovery.
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Kiełbus, M
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Czapiński, J
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Odrzywolski, A
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Stasiak, G
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Szymańska, K
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Kałafut, J
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Kos, M
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Giannopoulos, K
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Stepulak, A
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Rivero-Müller, A
Abstract:
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.
82.
Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics.
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Kyriakakis, P
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Catanho, M
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Hoffner, N
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Thavarajah, W
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Jian-Yu, V
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Chao, SS
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Hsu, A
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Pham, V
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Naghavian, L
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Dozier, LE
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Patrick, G
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Coleman, T
Abstract:
Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics, is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and for the first time, combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.
83.
Optogenetic tools for cell biological applications.
Abstract:
Abstract not available.
84.
Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.
Abstract:
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.
85.
A module for Rac temporal signal integration revealed with optogenetics.
Abstract:
Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades.
86.
At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior.
Abstract:
Cells are bombarded by extrinsic signals that dynamically change in time and space. Such dynamic variations can exert profound effects on behaviors, including cellular signaling, organismal development, stem cell differentiation, normal tissue function, and disease processes such as cancer. Although classical genetic tools are well suited to introduce binary perturbations, new approaches have been necessary to investigate how dynamic signal variation may regulate cell behavior. This fundamental question is increasingly being addressed with optogenetics, a field focused on engineering and harnessing light-sensitive proteins to interface with cellular signaling pathways. Channelrhodopsins initially defined optogenetics; however, through recent use of light-responsive proteins with myriad spectral and functional properties, practical applications of optogenetics currently encompass cell signaling, subcellular localization, and gene regulation. Now, important questions regarding signal integration within branch points of signaling networks, asymmetric cell responses to spatially restricted signals, and effects of signal dosage versus duration can be addressed. This review summarizes emerging technologies and applications within the expanding field of optogenetics.
87.
Optogenetic Modulation of Intracellular Signalling and Transcription: Focus on Neuronal Plasticity.
Abstract:
Several fields in neuroscience have been revolutionized by the advent of optogenetics, a technique that offers the possibility to modulate neuronal physiology in response to light stimulation. This innovative and far-reaching tool provided unprecedented spatial and temporal resolution to explore the activity of neural circuits underlying cognition and behaviour. With an exponential growth in the discovery and synthesis of new photosensitive actuators capable of modulating neuronal networks function, other fields in biology are experiencing a similar re-evolution. Here, we review the various optogenetic toolboxes developed to influence cellular physiology as well as the diverse ways in which these can be engineered to precisely modulate intracellular signalling and transcription. We also explore the processes required to successfully express and stimulate these photo-actuators in vivo before discussing how such tools can enlighten our understanding of neuronal plasticity at the systems level.
88.
Near-Infrared Fluorescent Proteins, Biosensors, and Optogenetic Tools Engineered from Phytochromes.
Abstract:
Phytochrome photoreceptors absorb far-red and near-infrared (NIR) light and regulate light responses in plants, fungi, and bacteria. Their multidomain structure and autocatalytic incorporation of linear tetrapyrrole chromophores make phytochromes attractive molecular templates for the development of light-sensing probes. A subclass of bacterial phytochromes (BphPs) utilizes heme-derived biliverdin tetrapyrrole, which is ubiquitous in mammalian tissues, as a chromophore. Because biliverdin possesses the largest electron-conjugated chromophore system among linear tetrapyrroles, BphPs exhibit the most NIR-shifted spectra that reside within the NIR tissue transparency window. Here we analyze phytochrome structure and photochemistry to describe the molecular mechanisms by which they function. We then present strategies to engineer BphP-based NIR fluorescent proteins and review their properties and applications in modern imaging technologies. We next summarize designs of reporters and biosensors and describe their use in the detection of protein-protein interactions, proteolytic activities, and posttranslational modifications. Finally, we provide an overview of optogenetic tools developed from phytochromes and describe their use in light-controlled cell signaling, gene expression, and protein localization. Our review provides guidelines for the selection of NIR probes and tools for noninvasive imaging, sensing, and light-manipulation applications, specifically focusing on probes developed for use in mammalian cells and in vivo.
89.
The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology.
Abstract:
The zebrafish ( Danio rerio) is a powerful vertebrate model to study cellular and developmental processes in vivo. The optical clarity and their amenability to genetic manipulation make zebrafish a model of choice when it comes to applying optical techniques involving genetically encoded photoresponsive protein technologies. In recent years, a number of fluorescent protein and optogenetic technologies have emerged that allow new ways to visualize, quantify, and perturb developmental dynamics. Here, we explain the principles of these new tools and describe some of their representative applications in zebrafish.
90.
Targeting protein function: the expanding toolkit for conditional disruption.
Abstract:
A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.
91.
Optogenetics in Plants: Red/Far-Red Light Control of Gene Expression.
Abstract:
Optogenetic tools to control gene expression have many advantages over the classical chemically inducible systems, overcoming intrinsic limitations of chemical inducers such as solubility, diffusion, and cell toxicity. They offer an unmatched spatiotemporal resolution and permit quantitative and noninvasive control of the gene expression. Here we describe a protocol of a synthetic light-inducible system for the targeted control of gene expression in plants based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). The synthetic toggle switch system is in the ON state when plant protoplasts are illuminated with red light (660 nm) and can be returned to the OFF state by subsequent illumination with far-red light (760 nm). In this protocol, the implementation of a red light-inducible expression system in plants using Light-Emitting Diode (LED) illumination boxes is described, including the isolation and transient transformation of plant protoplasts from Arabidopsis thaliana and Nicotiana tabacum.
92.
Toward total synthesis of cell function: Reconstituting cell dynamics with synthetic biology.
Abstract:
Biological phenomena, such as cellular differentiation and phagocytosis, are fundamental processes that enable cells to fulfill important physiological roles in multicellular organisms. In the field of synthetic biology, the study of these behaviors relies on the use of a broad range of molecular tools that enable the real-time manipulation and measurement of key components in the underlying signaling pathways. This Review will focus on a subset of synthetic biology tools known as bottom-up techniques, which use technologies such as optogenetics and chemically induced dimerization to reconstitute cellular behavior in cells. These techniques have been crucial not only in revealing causal relationships within signaling networks but also in identifying the minimal signaling components that are necessary for a given cellular function. We discuss studies that used these systems in a broad range of cellular and molecular phenomena, including the time-dependent modulation of protein activity in cellular proliferation and differentiation, the reconstitution of phagocytosis, the reconstitution of chemotaxis, and the regulation of actin reorganization. Finally, we discuss the potential contribution of synthetic biology to medicine.
93.
Natural Resources for Optogenetic Tools.
Abstract:
Photoreceptors are found in all kingdoms of life and mediate crucial responses to environmental challenges. Nature has evolved various types of photoresponsive protein structures with different chromophores and signaling concepts for their given purpose. The abundance of these signaling proteins as found nowadays by (meta-)genomic screens enriched the palette of optogenetic tools significantly. In addition, molecular insights into signal transduction mechanisms and design principles from biophysical studies and from structural and mechanistic comparison of homologous proteins opened seemingly unlimited possibilities for customizing the naturally occurring proteins for a given optogenetic task. Here, a brief overview on the photoreceptor concepts already established as optogenetic tools in natural or engineered form, their photochemistry and their signaling/design principles is given. Finally, so far not regarded photosensitive modules and protein architectures with potential for optogenetic application are described.
94.
Signalling to the nucleus under the control of light and small molecules.
Abstract:
One major regulatory mechanism in cell signalling is the spatio-temporal control of the localization of signalling molecules. We synthetically designed an entire cell signalling pathway, which allows controlling the transport of signalling molecules from the plasma membrane to the nucleus, by using light and small molecules.
95.
Investigating neuronal function with optically controllable proteins.
Abstract:
In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.
96.
Applications of hydrogen deuterium exchange (HDX) for the characterization of conformational dynamics in light-activated photoreceptors.
Abstract:
Rational design of optogenetic tools is inherently linked to the understanding of photoreceptor function. Structural analysis of elements involved in signal integration in individual sensor domains provides an initial idea of their mode of operation, but understanding how local structural rearrangements eventually affect signal transmission to output domains requires inclusion of the effector regions in the characterization. However, the dynamic nature of these assemblies renders their structural analysis challenging and therefore a combination of high- and low-resolution techniques is required to appreciate functional aspects of photoreceptors. This review focuses on the potential of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) for complementing the structural characterization of photoreceptors. In this respect, the ability of HDX-MS to provide information on conformational dynamics and the possibility to address multiple functionally relevant states in solution render this methodology ideally suitable. We highlight recent examples demonstrating the potential of HDX-MS and discuss how these results can help to improve existing optogenetic systems or guide the design of novel optogenetic tools.
97.
Photoreceptor engineering.
Abstract:
Sensory photoreceptors not only control diverse adaptive responses in Nature, but as light-regulated actuators they also provide the foundation for optogenetics, the non-invasive and spatiotemporally precise manipulation of cellular events by light. Novel photoreceptors have been engineered that establish control by light over manifold biological processes previously inaccessible to optogenetic intervention. Recently, photoreceptor engineering has witnessed a rapid development, and light-regulated actuators for the perturbation of a plethora of cellular events are now available. Here, we review fundamental principles of photoreceptors and light-regulated allostery. Photoreceptors dichotomize into associating receptors that alter their oligomeric state as part of light-regulated allostery and non-associating receptors that do not. A survey of engineered photoreceptors pinpoints light-regulated association reactions and order-disorder transitions as particularly powerful and versatile design principles. Photochromic photoreceptors that are bidirectionally toggled by two light colors augur enhanced spatiotemporal resolution and use as photoactivatable fluorophores. By identifying desirable traits in engineered photoreceptors, we provide pointers for the design of future, light-regulated actuators.
98.
Probing Yeast Polarity with Acute, Reversible, Optogenetic Inhibition of Protein Function.
Abstract:
We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inhibition of Bem1 produces profound defects in cell polarization and cell viability that are not observed in bem1Δ. By disrupting Bem1 activity at specific points in the cell cycle, we demonstrate that Bem1 is essential for the establishment of polarity and bud emergence but is dispensable for the growth of an emerged bud. By taking advantage of the reversibility of Bem1 inactivation, we show that pole size scales with cell size, and that this scaling is dependent on the actin cytoskeleton. Our experiments reveal how rapid reversible inactivation of protein function complements traditional genetic approaches. This strategy should be widely applicable to other biological contexts.
99.
Phytochromes: an atomic perspective on photoactivation and signaling.
Abstract:
The superfamily of phytochrome (Phy) photoreceptors regulates a wide array of light responses in plants and microorganisms through their unique ability to reversibly switch between stable dark-adapted and photoactivated end states. Whereas the downstream signaling cascades and biological consequences have been described, the initial events that underpin photochemistry of the coupled bilin chromophore and the ensuing conformational changes needed to propagate the light signal are only now being understood. Especially informative has been the rapidly expanding collection of 3D models developed by x-ray crystallographic, NMR, and single-particle electron microscopic methods from a remarkably diverse array of bacterial Phys. These structures have revealed how the modular architecture of these dimeric photoreceptors engages the buried chromophore through distinctive knot, hairpin, and helical spine features. When collectively viewed, these 3D structures reveal complex structural alterations whereby photoisomerization of the bilin drives nanometer-scale movements within the Phy dimer through bilin sliding, hairpin reconfiguration, and spine deformation that ultimately impinge upon the paired signal output domains. When integrated with the recently described structure of the photosensory module from Arabidopsis thaliana PhyB, new opportunities emerge for the rational redesign of plant Phys with novel photochemistries and signaling properties potentially beneficial to agriculture and their exploitation as optogenetic reagents.
100.
Ultradian oscillations and pulses: coordinating cellular responses and cell fate decisions.
Abstract:
Biological clocks play key roles in organismal development, homeostasis and function. In recent years, much work has focused on circadian clocks, but emerging studies have highlighted the existence of ultradian oscillators - those with a much shorter periodicity than 24 h. Accumulating evidence, together with recently developed optogenetic approaches, suggests that such ultradian oscillators play important roles during cell fate decisions, and analyzing the functional links between ultradian oscillation and cell fate determination will contribute to a deeper understanding of the design principle of developing embryos. In this Review, we discuss the mechanisms of ultradian oscillatory dynamics and introduce examples of ultradian oscillators in various biological contexts. We also discuss how optogenetic technology has been used to elucidate the biological significance of ultradian oscillations.
