Showing 1 - 25 of 120 results
Optogenetic Protein Cleavage in Zebrafish Embryos.
A wide array of optogenetic tools is available that allow for precise spatiotemporal control over many cellular processes. These tools have been especially popular among zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and apoptosis.
Optophysiology: Illuminating cell physiology with optogenetics.
Optogenetics combines light and genetics to enable precise control of living cells, tissues, and organisms with tailored functions. Optogenetics has the advantages of noninvasiveness, rapid responsiveness, tunable reversibility, and superior spatiotemporal resolution. Following the initial discovery of microbial opsins as light-actuated ion channels, a plethora of naturally occurring or engineered photoreceptors or photosensitive domains that respond to light at varying wavelengths has ushered in the next chapter of optogenetics. Through protein engineering and synthetic biology approaches, genetically encoded photoswitches can be modularly engineered into protein scaffolds or host cells to control a myriad of biological processes, as well as to enable behavioral control and disease intervention in vivo. Here, we summarize these optogenetic tools on the basis of their fundamental photochemical properties to better inform the chemical basis and design principles. We also highlight exemplary applications of opsin-free optogenetics in dissecting cellular physiology (designated "optophysiology") and describe the current progress, as well as future trends, in wireless optogenetics, which enables remote interrogation of physiological processes with minimal invasiveness. This review is anticipated to spark novel thoughts on engineering next-generation optogenetic tools and devices that promise to accelerate both basic and translational studies.
Towards translational optogenetics.
Optogenetics is widely used to interrogate the neural circuits underlying disease and has most recently been harnessed for therapeutic applications. The optogenetic toolkit consists of light-responsive proteins that modulate specific cellular functions, vectors for the delivery of the transgenes that encode the light-responsive proteins to targeted cellular populations, and devices for the delivery of light of suitable wavelengths at effective fluence rates. A refined toolkit with a focus towards translational uses would include efficient and safer viral and non-viral gene-delivery vectors, increasingly red-shifted photoresponsive proteins, nanomaterials that efficiently transduce near-infrared light deep into tissue, and wireless implantable light-delivery devices that allow for spatiotemporally precise interventions at clinically relevant tissue depths. In this Review, we examine the current optogenetics toolkit and the most notable preclinical and translational uses of optogenetics, and discuss future methodological and translational developments and bottlenecks.
Optogenetic approaches in biotechnology and biomaterials.
Advances in genetic engineering, combined with the development of optical technologies, have allowed optogenetics to broaden its area of possible applications in recent years. However, the application of optogenetic tools in industry, including biotechnology and the production of biomaterials, is still limited, because each practical task requires the engineering of a specific optogenetic system. In this review, we discuss recent advances in the use of optogenetic tools in the production of biofuels and valuable chemicals, the synthesis of biomedical and polymer materials, and plant agrobiology. We also offer a comprehensive analysis of the properties and industrial applicability of light-controlled and other smart biomaterials. These data allow us to outline the prospects for the future use of optogenetics in bioindustry.
Gasdermin D pores are dynamically regulated by local phosphoinositide circuitry.
Gasdermin D forms large, ~21 nm diameter pores in the plasma membrane to drive the cell death program pyroptosis. These pores are thought to be permanently open, and the resultant osmotic imbalance is thought to be highly damaging. Yet some cells mitigate and survive pore formation, suggesting an undiscovered layer of regulation over the function of these pores. However, no methods exist to directly reveal these mechanistic details. Here, we combine optogenetic tools, live cell fluorescence biosensing, and electrophysiology to demonstrate that gasdermin pores display phosphoinositide-dependent dynamics. We quantify repeated and fast opening-closing of these pores on the tens of seconds timescale, visualize the dynamic pore geometry, and identify the signaling that controls dynamic pore activity. The identification of this circuit allows pharmacological tuning of pyroptosis and control of inflammatory cytokine release by living cells.
Directed evolution approaches for optogenetic tool development.
Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field.
Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering.
The robust modularity of biological components that are assembled into complex functional systems is central to synthetic biology. Here we apply modular “plug and play” design principles to a microscale solid phase protein display system that enables protein purification and functional assays for biotherapeutics. Specifically, we capture protein molecules from cell lysates on polyacrylamide hydrogel display beads (‘PHD beads’), made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA). Anchors form covalent bonds with fusion proteins bearing cognate tag recognition (SNAP and Halo-tags) in specific, orthogonal and stable fashion. Given that these anchors are copolymerised throughout the 3D structure of the beads, proteins are also distributed across the entire bead sphere, allowing attachment of ∼109 protein molecules per bead (Ø 20 μm). This mode of attachment reaches a higher density than possible on widely used surface-modified beads, and additionally mitigates surface effects that often complicate studies with proteins on beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher and SnpTag). Proteins can be displayed in their monomeric forms, but also reformatted as a multivalent display (using secondary capture modules that create branches) to test the contributions of avidity and multivalency towards protein function. Finally, controlled release of modules by irradiation of light is achieved by incorporating the photocleavable protein PhoCl: irradiation severs the displayed protein from the solid support, so that functional assays can be carried out in solution. As a demonstration of the utility of valency engineering, an antibody drug screen is performed, in which an anti-TRAIL-R1 scFv protein is released into solution as monomers-hexamers, showing a ∼50-fold enhanced potency in the pentavalent format. The ease of protein purification on solid support, quantitative control over presentation and release of proteins and choice of valency make this experimental format a versatile, modular platform for large scale functional analysis of proteins, in bioassays of protein-protein interactions, enzymatic catalysis and bacteriolysis.
Modularly Built Synthetic Membraneless Organelles Enabling Targeted Protein Sequestration and Release.
Abstract not available.
Structure-based design of a photoswitchable affibody scaffold.
Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.
Engineering Photoresponsive Ligand Tethers for Mechanical Regulation of Stem Cells.
Regulating stem cell functions by precisely controlling the nanoscale presentation of bioactive ligands has a substantial impact on tissue engineering and regenerative medicine but remains a major challenge. Here it is shown that bioactive ligands can become mechanically "invisible" by increasing their tether lengths to the substrate beyond a critical length, providing a way to regulate mechanotransduction without changing the biochemical conditions. Building on this finding, light switchable tethers are rationally designed, whose lengths can be modulated reversibly by switching a light-responsive protein, pdDronpa, in between monomer and dimer states. This allows the regulation of the adhesion, spreading, and differentiation of stem cells by light on substrates of well-defined biochemical and physical properties. Spatiotemporal regulation of differential cell fates on the same substrate is further demonstrated, which may represent an important step toward constructing complex organoids or mini tissues by spatially defining the mechanical cues of the cellular microenvironment with light.
Applications of Upconversion Nanoparticles in Cellular Optogenetics.
Upconversion-mediated optogenetics is an emerging powerful technique to remotely control and manipulate the deep-tissue protein functions and signaling pathway activation. This technique uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and through near-infrared light to indirectly activate the traditional optogenetic proteins. With the merits of high spatiotemporal resolution and minimal invasiveness, this technique enables cell-type specific manipulation of cellular activities in deep tissues as well as in living animals. In this review, we introduce the latest development of optogenetic modules and UCNPs, with emphasis on the integration of UCNPs with cellular optogenetics and their biomedical applications on the control of neural/brain activity, cancer therapy and cardiac optogenetics in vivo. Furthermore, we analyze the current developed strategies to optimize and advance the upconversion-mediated optogenetics and discuss the remaining challenges of its further applications in biomedical study and clinical translational research. STATEMENT OF SIGNIFICANCE: Optogenetics harnesses photoactivatable proteins to optically stimulate and control intracellular activities. UCNPs-mediated NIR-activatable optogenetics uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and utilizes near-infrared (NIR) light to indirectly activate the traditional optogenetic proteins. The integration of UCNPs with cellular optogenetics has showed great promise in biomedical applications in regulating neural/brain activity, cancer therapy and cardiac optogenetics in vivo. The evolution and optimization of functional UCNPs and the discovery and engineering of novel optogenetic modules would both contribute to the advance of such unique hybrid technology, which may lead to discoveries in biomedical research and provide new treatments for human diseases.
A guide to the optogenetic regulation of endogenous molecules.
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.
A unique photochromic UV-A sensor protein, Rc-PYP, interacting with the PYP-binding protein.
Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.
Designer membraneless organelles sequester native factors for control of cell behavior.
Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.
Advanced Optogenetic-Based Biosensing and Related Biomaterials.
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.
Clinical applicability of optogenetic gene regulation.
The field of optogenetics is rapidly growing in relevance and number of developed tools. Amongst other things, the optogenetic repertoire includes light-responsive ion channels and methods for gene regulation. This review will be confined to the optogenetic control of gene expression in mammalian cells as suitable models for clinical applications. Here optogenetic gene regulation might offer an excellent method for spatially and timely regulated gene and protein expression in cell therapeutic approaches. Well-known systems for gene regulation, such as the LOV-, CRY2/CIB-, PhyB/PIF-systems, as well as other, in mammalian cells not yet fully established systems will be described. Advantages and disadvantages with regard to clinical applications are outlined in detail. Among the many unanswered questions concerning the application of optogenetics, we discuss items such as the use of exogenous chromophores and their effects on the biology of the cells and methods for a gentle, but effective gene transfection method for optogenetic tools for in vivo applications. This article is protected by copyright. All rights reserved.
Optogenetic approaches for understanding homeostatic and degenerative processes in Drosophila.
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.
Capicua is a fast-acting transcriptional brake.
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.
Synthetic biology as driver for the biologization of materials sciences.
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.
Optogenetic Approaches for the Spatiotemporal Control of Signal Transduction Pathways.
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.
Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude.
How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.
Signaling, Deconstructed: Using Optogenetics to Dissect and Direct Information Flow in Biological Systems.
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.
Synthetic Biological Approaches for Optogenetics and Tools for Transcriptional Light‐Control in Bacteria.
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.
Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches.
To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre‐defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.
Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.
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.