Curated Optogenetic Publication Database

Abstract: Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.

Abstract: The ability to stimulate mammalian cells with light, brought along by optogenetic control, has significantly broadened our understanding of electrically excitable tissues. Backed by advanced (bio)materials, it has recently paved the way towards novel biosensing concepts supporting bio-analytics applications transversal to the main biomedical stream. The advancements concerning enabling biomaterials and related novel biosensing concepts involving optogenetics are reviewed with particular focus on the use of engineered cells for cell-based sensing platforms and the available toolbox (from mere actuators and reporters to novel multifunctional opto-chemogenetic tools) for optogenetic-enabled real-time cellular diagnostics and biosensor development. The key advantages of these modified cell-based biosensors concern both significantly faster (minutes instead of hours) and higher sensitivity detection of low concentrations of bioactive/toxic analytes (below the threshold concentrations in classical cellular sensors) as well as improved standardization as warranted by unified analytic platforms. These novel multimodal functional electro-optical label-free assays are reviewed among the key elements for optogenetic-based biosensing standardization. This focused review is a potential guide for materials researchers interested in biosensing based on light-responsive biomaterials and related analytic tools.

Abstract: Many organs and tissues have an intrinsic ability to regenerate from a dedicated, tissue-specific stem cell pool. As organisms age, the process of self-regulation or homeostasis begins to slow down with fewer stem cells available for tissue repair. Tissues become more fragile and organs less efficient. This slowdown of homeostatic processes leads to the development of cellular and neurodegenerative diseases. In this review, we highlight the recent use and future potential of optogenetic approaches to study homeostasis. Optogenetics uses photosensitive molecules and genetic engineering to modulate cellular activity in vivo, allowing precise experiments with spatiotemporal control. We look at applications of this technology for understanding the mechanisms governing homeostasis and degeneration as applied to widely used model organisms, such as Drosophila melanogaster, where other common tools are less effective or unavailable.

Abstract: Even though transcriptional repressors are studied with ever-increasing molecular resolution, the temporal aspects of gene repression remain poorly understood. Here, we address the dynamics of transcriptional repression by Capicua (Cic), which is essential for normal development and is commonly mutated in human cancers and neurodegenerative diseases.1,2 We report the speed limit for Cic-dependent gene repression based on live imaging and optogenetic perturbations in the early Drosophila embryo, where Cic was originally discovered.3 Our measurements of Cic concentration and intranuclear mobility, along with real-time monitoring of the activity of Cic target genes, reveal remarkably fast transcriptional repression within minutes of removing an optogenetic de-repressive signal. In parallel, quantitative analyses of transcriptional bursting of Cic target genes support a repression mechanism providing a fast-acting brake on burst generation. This work sets quantitative constraints on potential mechanisms for gene regulation by Cic.

Abstract: Materials in nature have fascinating properties that serve as a continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization, or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here, we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.

Abstract: Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

Abstract: Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Abstract: 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.

Abstract: 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.

Abstract: Dissection of cell signaling requires tools that can mimic spatiotemporal dynamics of individual pathways in living cells. Optogenetic methods enable manipulation of signaling processes with precise timing and local control. In this review, we describe recent optogenetic approaches for regulation of cell signaling, highlight their advantages and limitations, and discuss examples of their application.

Abstract: 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.

Abstract: 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.

Abstract: Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).

Abstract: Since the neurobiological inception of optogenetics, light-controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light-sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle-specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Physiology of Model Organisms Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Cellular Models.

Abstract: Since the breakthrough discoveries that CRISPR-Cas9 nucleases can be easily programmed and employed to induce targeted double-strand breaks in mammalian cells, the gene editing field has grown exponentially. Today, CRISPR technologies based on engineered class II CRISPR effectors facilitate targeted modification of genes and RNA transcripts. Moreover, catalytically impaired CRISPR-Cas variants can be employed as programmable DNA binding domains and used to recruit effector proteins, such as transcriptional regulators, epigenetic modifiers or base-modifying enzymes, to selected genomic loci. The juxtaposition of CRISPR and optogenetics enables spatiotemporally confined and highly dynamic genome perturbations in living cells and animals and holds unprecedented potential for biology and biomedicine.Here, we provide an overview of the state-of-the-art methods for light-control of CRISPR effectors. We will detail the plethora of exciting applications enabled by these systems, including spatially confined genome editing, timed activation of endogenous genes, as well as remote control of chromatin-chromatin interactions. Finally, we will discuss limitations of current optogenetic CRISPR tools and point out routes for future innovation in this emerging field.

Abstract: G protein-coupled receptors (GPCRs) form the largest class of membrane receptors in the mammalian genome with nearly 800 human genes encoding for unique subtypes. Accordingly, GPCR signaling is implicated in nearly all physiological processes. However, GPCRs have been difficult to study due in part to the complexity of their function which can lead to a plethora of converging or diverging downstream effects over different time and length scales. Classic techniques such as pharmacological control, genetic knockout and biochemical assays often lack the precision required to probe the functions of specific GPCR subtypes. Here we describe the rapidly growing set of optogenetic tools, ranging from methods for optical control of the receptor itself to optical sensing and manipulation of downstream effectors. These tools permit the quantitative measurements of GPCRs and their downstream signaling with high specificity and spatiotemporal precision.

Abstract: 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.

Abstract: Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.

Abstract: Nanoscale membrane curvature is now understood to play an active role in essential cellular processes such as endocytosis, exocytosis and actin dynamics. Previous studies have shown that membrane curvature can directly affect protein function and intracellular signaling. However, few methods are able to precisely manipulate membrane curvature in live cells. Here, we report the development of a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. For this purpose, we make use of BAR domain proteins, a family of well-studied membrane-remodeling and membrane-sculpting proteins. Specifically, we engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of positive and negative membrane curvature, respectively. Using opto-FBAR, blue light activation results in the formation of tubular membrane invaginations (positive curvature), controllable down to the subcellular level. Using opto-IBAR, blue light illumination results in the formation of membrane protrusions or filopodia (negative curvature). These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells.

Abstract: 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.

Abstract: Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer. Nonnative cues for synthetically regulating kinases are thus much sought after, both for dissecting cell signaling pathways and for pharmaceutical development. In recent years advances in protein engineering and sequence analysis have led to new approaches for manipulating kinase activity, localization, and in some instances specificity. These tools have revealed fundamental principles of intracellular signaling and suggest paths forward for the design of therapeutic allosteric kinase regulators.

Abstract: 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.

Abstract: Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

Abstract: 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.

Abstract: 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.