Showing 1 - 25 of 107 results
Lights up on organelles: Optogenetic tools to control subcellular structure and organization.
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.
Optogenetic Rescue of a Patterning Mutant.
Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90 min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.
Optogenetic control of gene expression in plants in the presence of ambient white light.
Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.
The Association Kinetics Encode the Light Dependence of Arabidopsis Phytochrome B Interactions.
Plant phytochromes enable vital adaptations to red and far-red light. At the molecular level, these responses are mediated by light-regulated interactions between phytochromes and partner proteins, foremost the phytochrome-interacting factors (PIF). Although known for decades, quantitative analyses of these interactions have long been sparse. To address this deficit, we here studied by an integrated fluorescence-spectroscopic approach the equilibrium and kinetics of Arabidopsis thaliana phytochrome B (AtPhyB) binding to a tetramerized PIF6 variant. Several readouts consistently showed the stringently light-regulated interaction to be little affected by PIF tetramerization. Analysis of the binding kinetics allowed the determination of bimolecular association and unimolecular dissociation rate constants as a function of light. Unexpectedly, the stronger affinity of AtPhyB under red light relative to far-red light is entirely due to accelerated association rather than decelerated dissociation. The association reaction under red light is highly efficient and only threefold slower than the diffusion limit. The present findings pertain equally to the analysis of signal transduction in plants and to the biotechnological application of phytochromes.
Non-neuromodulatory Optogenetic Tools in Zebrafish.
The zebrafish (Danio rerio) is a popular vertebrate model organism to investigate molecular mechanisms driving development and disease. Due to its transparency at embryonic and larval stages, investigations in the living organism are possible with subcellular resolution using intravital microscopy. The beneficial optical characteristics of zebrafish not only allow for passive observation, but also active manipulation of proteins and cells by light using optogenetic tools. Initially, photosensitive ion channels have been applied for neurobiological studies in zebrafish to dissect complex behaviors on a cellular level. More recently, exciting non-neural optogenetic tools have been established to control gene expression or protein localization and activity, allowing for unprecedented non-invasive and precise manipulation of various aspects of cellular physiology. Zebrafish will likely be a vertebrate model organism at the forefront of in vivo application of non-neural optogenetic tools and pioneering work has already been performed. In this review, we provide an overview of non-neuromodulatory optogenetic tools successfully applied in zebrafish to control gene expression, protein localization, cell signaling, migration and cell ablation.
Using optogenetics to tackle systems-level questions of multicellular morphogenesis.
Morphogenesis of multicellular systems is governed by precise spatiotemporal regulation of biochemical reactions and mechanical forces which together with environmental conditions determine the development of complex organisms. Current efforts in the field aim at decoding the system-level principles underlying the regulation of developmental processes. Toward this goal, optogenetics, the science of regulation of protein function with light, is emerging as a powerful new tool to quantitatively perturb protein function in vivo with unprecedented precision in space and time. In this review, we provide an overview of how optogenetics is helping to address system-level questions of multicellular morphogenesis and discuss future directions.
Lights, cytoskeleton, action: Optogenetic control of cell dynamics.
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.
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.
Intracellular signaling dynamics and their role in coordinating tissue repair.
Tissue repair is a complex process that requires effective communication and coordination between cells across multiple tissues and organ systems. Two of the initial intracellular signals that encode injury signals and initiate tissue repair responses are calcium and extracellular signal-regulated kinase (ERK). However, calcium and ERK signaling control a variety of cellular behaviors important for injury repair including cellular motility, contractility, and proliferation, as well as the activity of several different transcription factors, making it challenging to relate specific injury signals to their respective repair programs. This knowledge gap ultimately hinders the development of new wound healing therapies that could take advantage of native cellular signaling programs to more effectively repair tissue damage. The objective of this review is to highlight the roles of calcium and ERK signaling dynamics as mechanisms that link specific injury signals to specific cellular repair programs during epithelial and stromal injury repair. We detail how the signaling networks controlling calcium and ERK can now also be dissected using classical signal processing techniques with the advent of new biosensors and optogenetic signal controllers. Finally, we advocate the importance of recognizing calcium and ERK dynamics as key links between injury detection and injury repair programs that both organize and execute a coordinated tissue repair response between cells across different tissues and organs. This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Biological Mechanisms > Cell Signaling Laboratory Methods and Technologies > Imaging Models of Systems Properties and Processes > Organ, Tissue, and Physiological Models.
New Pioneers of Optogenetics in Neuroscience.
Optogenetics have recently increased in popularity as tools to study behavior in response to the brain and how these trends relate back to a neuronal circuit. Additionally, the high demand for human cerebral tissue in research has led to the generation of a new model to investigate human brain development and disease. Human Pluripotent Stem Cells (hPSCs) have been previously used to recapitulate the development of several tissues such as intestine, stomach and liver and to model disease in a human context, recently new improvements have been made in the field of hPSC-derived brain organoids to better understand overall brain development but more specifically, to mimic inter-neuronal communication. This review aims to highlight the recent advances in these two separate approaches of brain research and to emphasize the need for overlap. These two novel approaches would combine the study of behavior along with the specific circuits required to produce the signals causing such behavior. This review is focused on the current state of the field, as well as the development of novel optogenetic technologies and their potential for current scientific study and potential therapeutic use.
Light-mediated control of Gene expression in mammalian cells.
Taking advantage of the recent development of genetically-defined photo-activatable actuator molecules, cellular functions, including gene expression, can be controlled by exposure to light. Such optogenetic strategies enable precise temporal and spatial manipulation of targeted single cells or groups of cells at a level hitherto impossible. In this review, we introduce light-controllable gene expression systems exploiting blue or red/far-red wavelengths and discuss their inherent properties potentially affecting induced downstream gene expression patterns. We also discuss recent advances in optical devices that will extend the application of optical gene expression control technologies into many different areas of biology and medicine.
Strategies for Engineering and Rewiring Kinase Regulation.
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.
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.
Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors.
Phytochrome photoreceptors mediate adaptive responses of plants to red and far-red light. These responses generally entail light-regulated association between phytochromes and other proteins, among them the phytochrome-interacting factors (PIF). The interaction with Arabidopsis thaliana phytochrome B (AtPhyB) localizes to the bipartite APB motif of the A. thaliana PIFs (AtPIF). To address a dearth of quantitative interaction data, we construct and analyze numerous AtPIF3/6 variants. Red-light-activated binding is predominantly mediated by the APB N-terminus, whereas the C-terminus modulates binding and underlies the differential affinity of AtPIF3 and AtPIF6. We identify AtPIF variants of reduced size, monomeric or homodimeric state, and with AtPhyB affinities between 10 and 700 nM. Optogenetically deployed in mammalian cells, the AtPIF variants drive light-regulated gene expression and membrane recruitment, in certain cases reducing basal activity and enhancing regulatory response. Moreover, our results provide hitherto unavailable quantitative insight into the AtPhyB:AtPIF interaction underpinning vital light-dependent responses in plants.
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.
CreLite: An Optogenetically Controlled Cre/loxP System Using Red Light.
Precise manipulation of gene expression with temporal and spatial control is essential for functional studies and the determination of cell lineage relationships in complex biological systems.The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre or CreER (tamoxifen-inducible). Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing the inactive halves with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. In addition, PhyB-PIF6 binding requires phycocyanobilin (PCB), providing an additional layer of control. Upon exposure to red light (660 nm) illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of PCB to restore Cre activity. Red-light exposure of transgenic zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter (ubi:zebrabow) injected with CreLite mRNAs and PCB, resulted in Cre activity as measured by the generation of multi-spectral cell labeling in various tissues, including heart, skeletal muscle and epithelium. We show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different stages of development. CreLite provides a novel optogenetic tool for precise temporal and spatial control of gene expression in zebrafish embryos that may also be useful in cell culture, ex vivo organ culture, and other animal models for developmental biology studies.
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.
Principles and applications of optogenetics in developmental biology.
The development of multicellular organisms is controlled by highly dynamic molecular and cellular processes organized in spatially restricted patterns. Recent advances in optogenetics are allowing protein function to be controlled with the precision of a pulse of laser light in vivo, providing a powerful new tool to perturb developmental processes at a wide range of spatiotemporal scales. In this Primer, we describe the most commonly used optogenetic tools, their application in developmental biology and in the nascent field of synthetic morphogenesis.
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.
Red/Far-Red Light Switchable Cargo Attachment and Release in Bacteria-Driven Microswimmers.
In bacteria-driven microswimmers, i.e., bacteriabots, artificial cargos are attached to flagellated chemotactic bacteria for active delivery with potential applications in biomedical technology. Controlling when and where bacteria bind and release their cargo is a critical step for bacteriabot fabrication and efficient cargo delivery/deposition at the target site. Toward this goal, photoregulating the cargo integration and release in bacteriabots using red and far-red light, which are noninvasive stimuli with good tissue penetration and provide high spatiotemporal control, is proposed. In the bacteriabot design, the surfaces of E. coli and microsized model cargo particles with the proteins PhyB and PIF6, which bind to each other under red light and dissociate from each other under far-red light are functionalized. Consequently, the engineered bacteria adhere and transport the model cargo under red light and release it on-demand upon far-red light illumination due to the photoswitchable PhyB-PIF6 protein interaction. Overall, the proof-of-concept for red/far-red light switchable bacteriabots, which opens new possibilities in the photoregulation in biohybrid systems for bioengineering, targeted drug delivery, and lab-on-a-chip devices, is demonstrated.
Production of Phytochromes by High-Cell-Density E. coli Fermentation.
Phytochromes are important photoreceptors of plants, bacteria, and fungi responsive to light in the red and far-red spectrum. For increasing applications in basic research, synthetic biology, and materials sciences, it is required to recombinantly produce and purify phytochromes in high amounts. An ideal host organism for this purpose is E. coli due to its widespread use, fast growth, and ability for high-cell-density fermentation. Here, we describe the development of a generic platform for the production of phytochromes in E. coli that is compatible with high-cell-density fermentation. We exemplify our approach by the production of the photosensory domains of phytochrome B (PhyB) from A. thaliana and of the cyanobacterial phytochrome 1 (Cph1) from Synechocystis PCC 6803 in the multigram scale per 10 L fermentation run.
High-throughput multicolor optogenetics in microwell plates.
Optogenetic probes can be powerful tools for dissecting complexity in cell biology, but there is a lack of instrumentation to exploit their potential for automated, high-information-content experiments. This protocol describes the construction and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments that allows simultaneous manipulation of common red- and blue-light-sensitive optogenetic probes. The optoPlate-96 enables illumination of individual wells in 96-well microwell plates or in groups of wells in 384-well plates. Its design ensures that there will be no cross-illumination between microwells in 96-well plates, and an active cooling system minimizes sample heating during light-intensive experiments. This protocol details the steps to assemble, test, and use the optoPlate-96. The device can be fully assembled without specialized equipment beyond a 3D printer and a laser cutter, starting from open-source design files and commercially available components. We then describe how to perform a typical optogenetics experiment using the optoPlate-96 to stimulate adherent mammalian cells. Although optoPlate-96 experiments are compatible with any plate-based readout, we describe analysis using quantitative single-cell immunofluorescence. This workflow thus allows complex optogenetics experiments (independent control of stimulation colors, intensity, dynamics, and time points) with high-dimensional outputs at single-cell resolution. Starting from 3D-printed and laser-cut components, assembly and testing of the optoPlate-96 can be accomplished in 3-4 h, at a cost of ~$600. A full optoPlate-96 experiment with immunofluorescence analysis can be performed within ~24 h, but this estimate is variable depending on the cell type and experimental parameters.
Optogenomic Interfaces: Bridging Biological Networks With the Electronic Digital World.
The development of optical nano-bio interfaces is a fundamental step toward connecting biological networks and traditional electronic computing systems. Compared to conventional chemical and electrical nano-bio interfaces, the use of light as a mediator enables new type of interfaces with unprecedented spatial and temporal resolutions. In this paper, the state of the art and future research directions in optogenomic interfaces are discussed. Optogenomic interfaces are light-mediated nano-bio interfaces that allow the control of the genome, i.e., the genes and their interactions in the cell nucleus (and, thus, of all the cell functionalities) with (sub) cellular resolution and high temporal accuracy. Given its fundamental role in the process of cell development, the study is focused on the interactions with the fibroblast growth factor receptor 1 (FGFR1) gene and the integrative nuclear FGFR1 signaling (INFS) module in stem cells and in neuronal cells, whose control opens the door to transformative applications, including reconstructive medicine and cancer therapy. Three stages of optogenomic interfaces are described, ranging from already experimentally validated interfaces activating broad cellular responses and expressing individual genes to more advanced interfaces able to regulate and correct DNA topology, chromatin structure, and cellular development.
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.
Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor.
The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.