Showing 1 - 25 of 62 results
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.
Optogenetic approaches to investigate spatiotemporal signaling during development.
Embryogenesis is coordinated by signaling pathways that pattern the developing organism. Many aspects of this process are not fully understood, including how signaling molecules spread through embryonic tissues, how signaling amplitude and dynamics are decoded, and how multiple signaling pathways cooperate to pattern the body plan. Optogenetic approaches can be used to address these questions by providing precise experimental control over a variety of biological processes. Here, we review how these strategies have provided new insights into developmental signaling and discuss how they could contribute to future investigations.
Structural Basis of Design and Engineering for Advanced Plant Optogenetics.
In optogenetics, light-sensitive proteins are specifically expressed in target cells and light is used to precisely control the activity of these proteins at high spatiotemporal resolution. Optogenetics initially used naturally occurring photoreceptors to control neural circuits, but has expanded to include carefully designed and engineered photoreceptors. Several optogenetic constructs are based on plant photoreceptors, but their application to plant systems has been limited. Here, we present perspectives on the development of plant optogenetics, considering different levels of design complexity. We discuss how general principles of light-driven signal transduction can be coupled with approaches for engineering protein folding to develop novel optogenetic tools. Finally, we explore how the use of computation, networks, circular permutation, and directed evolution could enrich optogenetics.
Single-Molecule Analysis and Engineering of DNA Motors.
Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.
Optogenetics sheds new light on tissue engineering and regenerative medicine.
Optogenetics has demonstrated great potential in the fields of tissue engineering and regenerative medicine, from basic research to clinical applications. Spatiotemporal encoding during individual development has been widely identified and is considered a novel strategy for regeneration. A as a noninvasive method with high spatiotemporal resolution, optogenetics are suitable for this strategy. In this review, we discuss roles of dynamic signal coding in cell physiology and embryonic development. Several optogenetic systems are introduced as ideal optogenetic tools, and their features are compared. In addition, potential applications of optogenetics for tissue engineering are discussed, including light-controlled genetic engineering and regulation of signaling pathways. Furthermore, we present how emerging biomaterials and photoelectric technologies have greatly promoted the clinical application of optogenetics and inspired new concepts for optically controlled therapies. Our summation of currently available data conclusively demonstrates that optogenetic tools are a promising method for elucidating and simulating developmental processes, thus providing vast prospects for tissue engineering and regenerative medicine applications.
Light-induced dimerization approaches to control cellular processes.
Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. The new expansions of the toolbox facilitate control of signal transduction, genome editing, 'painting' patterns of active molecules onto cellular membranes and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches has also seen interesting progress. Here we provide an overview of the optogenetic systems and the emerging chemo-optogenetic systems, and discuss recent applications in tackling complex biological problems.
Regulation of signaling proteins in the brain by light.
In order to study the role of signaling proteins, such as kinases and GTPases, in brain functions it is necessary to control their activity at the appropriate spatiotemporal resolution and to examine the cellular and behavioral effects of such changes in activity. Reduced spatiotemporal resolution in the regulation of these proteins activity will impede the ability to understand the proteins normal functions as longer modification of their activity in non-normal locations could lead to effects different from their natural functions. To control intracellular signaling proteins at the highest temporal resolution recent innovative optogenetic approaches were developed to allow the control of photoactivable signaling proteins activity by light. These photoactivatable proteins can be activated in selected cell population in brain and in specific subcellular compartments. Minimal-invasive tools are being developed to photoactivate these proteins for study and therapy. Together these techniques afford an unprecedented spatiotemporal control of signaling proteins activity to unveil the function of brain proteins with high accuracy in behaving animals. As dysfunctional signaling proteins are involved in brain diseases, the optogenetic technique has also the potential to be used as a tool to treat brain diseases.
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.
Synthetic switches and regulatory circuits in plants.
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.
Perspective Tools for Optogenetics and Photopharmacology: From Design to Implementation.
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.
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.
Bringing Light to Transcription: The Optogenetics Repertoire.
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.
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.
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.
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.
New approaches for solving old problems in neuronal protein trafficking.
Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision.
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.
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.
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.
Recent advances in the development of light-inducible transgene expression systems have overcome many inherent drawbacks of conventional chemically regulated systems. The latest generation of those light-regulated systems that are specifically responsive to different wavelengths allows spatiotemporal control of gene expression in a so far unprecedented manner.In this chapter, we first describe the available light-inducible gene expression systems compatible with mammalian cells and explain their underlying mechanisms. Afterward, we give a detailed protocol for the implementation of a UVB light-inducible expression system in mammalian 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.
At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior.
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.
Optogenetic Modulation of Intracellular Signalling and Transcription: Focus on Neuronal Plasticity.
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.