Showing 26 - 50 of 81 results
Physical Plasma Membrane Perturbation Using Subcellular Optogenetics Drives Integrin-Activated Cell Migration.
Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction. Consistent with known effects of constraining high concentrations of proteins to a membrane in vitro, there is localized curvature and tension decrease in the plasma membrane. Integrin activity, sensitive to mechanical forces, is activated in this region. Localized mechanical activation of integrin with optogenetics allowed simultaneous imaging of the molecular and cellular response, helping uncover a positive feedback loop comprising SFK- and ERK-dependent RhoA activation, actomyosin contractility, rearward membrane flow, and membrane tension decrease underlying this mode of cell migration.
Synthetic Control of Protein Degradation during Cell Proliferation and Developmental Processes.
Synthetic tools for the control of protein function are valuable for biomedical research to characterize cellular functions of essential proteins or if a rapid switch in protein activity is necessary. The ability to tune protein activity precisely opens another level of investigations that is not available with gene deletion mutants. Control of protein stability is a versatile approach to influence the activity of a target protein by its cellular abundance. Diverse strategies have been developed to achieve efficient proteolysis using external inducers or differentiation-coupled signals. The latter is especially important for the inactivation of a protein during a developmental process. Recently, several approaches to achieve this have been engineered. In this article, we present current synthetic tools for regulation of protein stability that allow fine-tuning of protein abundance, their advantages and disadvantages with an emphasis on methods applicable in the context of cell differentiation and development. We give an outlook toward future developments and discuss main applications of these tools.
Engineering Improved Photoswitches for the Control of Nucleocytoplasmic Distribution.
Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control sub-cellular localization and activity. We previously engineered two optogenetic systems, the Light Activated Nuclear Shuttle (LANS) and the Light-Inducible Nuclear eXporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favourably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic 'tool kit' for the research community.
Programming the Dynamic Control of Bacterial Gene Expression with a Chimeric Ligand- and Light-Based Promoter System.
To program cells in a dynamic manner, synthetic biologists require precise control over the threshold levels and timing of gene expression. However, in practice, modulating gene expression is widely carried out using prototypical ligand-inducible promoters, which have limited tunability and spatiotemporal resolution. Here, we built two dual-input hybrid promoters, each retaining the function of the ligand-inducible promoter while being enhanced with a blue-light-switchable tuning knob. Using the new promoters, we show that both ligand and light inputs can be synchronously modulated to achieve desired amplitude or independently regulated to generate desired frequency at a specific amplitude. We exploit the versatile programmability and orthogonality of the two promoters to build the first reprogrammable logic gene circuit capable of reconfiguring into logic OR and N-IMPLY logic on the fly in both space and time without the need to modify the circuit. Overall, we demonstrate concentration- and time-based combinatorial regulation in live bacterial cells with potential applications in biotechnology and synthetic biology.
Discovering selective binders for photoswitchable proteins using phage display.
Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control - precise spatial and temporal resolution - are currently restricted to a few well-defined natural systems. In addition, the affinities and kinetics of native interactions are often sub-optimal and are difficult to engineer in the absence of any structural information. We report a phage display strategy using a small scaffold protein that can be used to discover new binding partners for both light and dark states of a given light-switchable protein. We used our approach to generate binding partners that interact specifically with the light state or the dark state conformation of two light-switchable proteins: PYP, a test case for a protein with no known partners, and AsLOV2 a well-characterized protein. We show that these novel light-switchable protein-protein interactions can function in living cells to control subcellular localization processes.
A Single-Component Optogenetic System Allows Stringent Switch of Gene Expression in Yeast Cells.
Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔN Sic1 genes, respectively. Furthermore, we engineered a bidirectional expression module that allows the simultaneous control of the expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative, and spatially specific degradation of ssrA-tagged protein was observed. We suggest that this single-component optogenetic system will be immensely helpful in understanding cellular gene regulatory networks and in the design of robust genetic circuits for synthetic biology.
Optical activation of TrkA signaling.
Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 (CRY2) and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports the survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.
Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light Switchable Proteins.
Towards the bottom-up assembly of synthetic cells from molecular building blocks it is an ongoing challenge to assemble micrometer sized compartments that host different processes into precise multicompartmental assemblies, also called prototissues. The difficulty lies in controlling interactions between different compartments dynamically both in space and time, as these interactions determine how they organize with respect to each other and how they work together. In this study, we have been able to control the self-assembly and social self-sorting of four different types of colloids, which we use as a model for synthetic cells, into two separate families with visible light. For this purpose we used two photoswitchable protein pairs (iLID/Nano and nHagHigh/pMagHigh) that both reversibly heterodimerize upon blue light exposure and dissociate from each other in the dark. These photoswitchable proteins provide non-invasive, dynamic and reversible remote control under biocompatible conditions over the self-assembly process with unprecedented spatial and temporal precision. In addition, each protein pair brings together specifically two different types of colloids. The orthogonality of the two protein pairs enables social self-sorting of a four component mixture into two distinct families of colloidal aggregates with controlled arrangements. These results will ultimately pave the way for the bottom-up assembly of multicompartment synthetic prototissues of a higher complexity, enabling us to control precisely and dynamically the organization of different compartments in space and time.
OptoBase: A web platform for molecular optogenetics.
OptoBase is an online platform for molecular optogenetics. At its core is a hand-annotated and ontology-supported database that aims to cover all existing optogenetic switches and publications, which is further complemented with a collection of convenient optogenetics-related web tools. OptoBase is meant for both expert optogeneticists, to easily keep track of the field, as well as for all researchers who find optogenetics inviting as a powerful tool to address their biological questions of interest. It is available at https://www.optobase.org. This work also presents OptoBase-based analysis of the trends in molecular optogenetics.
LOV Domains in the Design of Photoresponsive Enzymes.
In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occuring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest. Immediate advantages of these new photoresponsive proteins such as genetic encoding, no requirement of chemical modification, and reversibility are paid by difficulties in predicting the envisaged activity or type and site of domain fusion. In this article, we summarize recent advances and give a survey on currently available design concepts for engineering photoswitchable proteins.
Optogenetic inhibition of Gαq protein signaling reduces calcium oscillation stochasticity.
As fast terminators of G-protein coupled receptor (GPCR) signaling, regulators of G-protein signaling (RGS) serve critical roles in fine-tuning second messenger levels and, consequently, cellular responses to external stimuli. Here, we report the creation of an optogenetic RGS2 (opto-RGS2) that suppresses agonist-evoked calcium oscillations by the inactivation of Gαq protein. In this system, cryptochrome-mediated hetero-dimerization of the catalytic RGS2-box with its N-terminal amphipathic helix reconstitutes a functional membrane-localized complex that can dynamically suppress store-operated release of calcium. Engineered opto-RGS2 cell lines were used to establish the role of RGS2 as a key inhibitory feedback regulator of the stochasticity of the Gαq-mediated calcium spike timing. RGS2 reduced the stochasticity of carbachol-stimulated calcium oscillations, and the feedback inhibition was coupled to the global calcium elevation by calmodulin/RGS2 interactions. The identification of a critical negative feedback circuit exemplifies the utility of optogenetic approaches for interrogating RGS/GPCR biology and calcium encoding principles through temporally precise molecular gain-of-function.
Bioprinting Living Biofilms through Optogenetic Manipulation.
In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living bacteria to autonomically form patterned biofilms following prescribed illumination. The results showed that through optogenetic manipulation, patterned bacterial communities with high spatial resolution (approximately 10 μm) could be constructed in 6 h. Thus, optogenetic manipulation greatly increases the range of available bioprinting techniques.
A green light-responsive system for the control of transgene expression in mammalian and plant cells.
The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH6 and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene expression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.
Cell-free optogenetic gene expression system.
Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, its vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modelling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of target gene to generate oscillatory waveform. Deterministic model developed was able to recapitulate the system behaviour and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.
Rewiring Calcium Signaling for Precise Transcriptional Reprogramming.
Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.
Generation of Optogenetically Modified Adenovirus Vector for Spatiotemporally Controllable Gene Therapy.
Gene therapy is expected to be utilized for the treatment of various diseases. However, the spatiotemporal resolution of current gene therapy technology is not high enough. In this study, we generated a new technology for spatiotemporally controllable gene therapy. We introduced optogenetic and CRISPR/Cas9 techniques into a recombinant adenovirus (Ad) vector, which is widely used in clinical trials and exhibits high gene transfer efficiency, to generate an illumination-dependent spatiotemporally controllable gene regulation system (designated the Opt/Cas-Ad system). We generated an Opt/Cas-Ad system that could regulate a potential tumor suppressor gene, and we examined the effectiveness of this system in cancer treatment using a xenograft tumor model. With the Opt/Cas-Ad system, highly selective tumor treatment could be performed by illuminating the tumor. In addition, Opt/Cas-Ad system-mediated tumor treatment could be stopped simply by turning off the light. We believe that our Opt/Cas-Ad system can enhance both the safety and effectiveness of gene therapy.
Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics.
Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics, is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and for the first time, combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.
Spatiotemporal Control of TGF-β Signaling with Light.
Cells employ signaling pathways to make decisions in response to changes in their immediate environment. Transforming growth factor beta (TGF-β) is an important growth factor that regulates many cellular functions in development and disease. Although the molecular mechanisms of TGF-β signaling have been well studied, our understanding of this pathway is limited by the lack of tools that allow the control of TGF-β signaling with high spatiotemporal resolution. Here, we developed an optogenetic system (optoTGFBRs) that enables the precise control of TGF-β signaling in time and space. Using the optoTGFBRs system, we show that TGF-β signaling can be selectively and sequentially activated in single cells through the modulation of the pattern of light stimulations. By simultaneously monitoring the subcellular localization of TGF-β receptor and Smad2 proteins, we characterized the dynamics of TGF-β signaling in response to different patterns of blue light stimulations. The spatial and temporal precision of light control will make the optoTGFBRs system as a powerful tool for quantitative analyses of TGF-β signaling at the single cell level.
Optogenetic Control of Endoplasmic Reticulum-Mitochondria Tethering.
The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.
Engineering an E. coli Near-Infrared Light Sensor.
Optogenetics is a technology wherein researchers combine light and genetically engineered photoreceptors to control biological processes with unrivaled precision. Near-infrared (NIR) wavelengths (>700 nm) are desirable optogenetic inputs due to their low phototoxicity and spectral isolation from most photoproteins. The bacteriophytochrome photoreceptor 1 (BphP1), found in several purple photosynthetic bacteria, senses NIR light and activates transcription of photosystem promoters by binding to and inhibiting the transcriptional repressor PpsR2. Here, we examine the response of a library of output promoters to increasing levels of Rhodopseudomonas palustris PpsR2 expression, and we identify that of Bradyrhizobium sp. BTAi1 crtE as the most strongly repressed in Escherichia coli. Next, we optimize Rps. palustris bphP1 and ppsR2 expression in a strain engineered to produce the required chromophore biliverdin IXα in order to demonstrate NIR-activated transcription. Unlike a previously engineered bacterial NIR photoreceptor, our system does not require production of a second messenger, and it exhibits rapid response dynamics. It is also the most red-shifted bacterial optogenetic tool yet reported by approximately 50 nm. Accordingly, our BphP1-PpsR2 system has numerous applications in bacterial optogenetics.
Optogenetics Manipulation Enables Prevention of Biofilm Formation of Engineered Pseudomonas aeruginosa on Surfaces.
Synthetic biologists have attempted to solve real-world problems, such as those of bacterial biofilms, that are involved in the pathogenesis of many clinical infections and difficult to eliminate. To address this, we employed a blue light responding system and integrated it into the chromosomes of Pseudomonas aeruginosa. With making rational adaptions and improvements of the light-activated system, we provided a robust and convenient means to spatiotemporally control gene expression and manipulate biological processes with minimal perturbation in P. aeruginosa. It increased the light-induced gene expression up to 20-fold. Moreover, we deliberately introduced a functional protein gene PA2133 containing an EAL domain to degrade c-di-GMP into the modified system, and showed that the optimally engineered optogenetic tool inhibited the formation of P. aeruginosa biofilms through the induction of blue light, resulting in much sparser and thinner biofilms. Our approach establishes a methodology for leveraging the tools of synthetic biology to guide biofilm formation and engineer biofilm patterns with unprecedented spatiotemporal resolution. Furthermore, the findings suggest that the synthetic optogenetic system may provide a promising strategy that could be applied to control and fight biofilms.
Near-Infrared Light Triggered Upconversion Optogenetic Nanosystem for Cancer Therapy.
In vivo the application of optogenetic manipulation in deep tissue is seriously obstructed by the limited penetration depth of visible light that is continually applied to activate a photoactuator. Herein, we designed a versatile upconversion optogenetic nanosystem based on a blue-light-mediated heterodimerization module and rare-earth upconversion nanoparticles (UCNs). The UCNs worked as a nanotransducer to convert external deep-tissue-penetrating near-infrared (NIR) light to local blue light to noninvasively activate photoreceptors for optogenetic manipulation in vivo. In this, we demonstrated that deeply penetrating NIR light could be used to control the apoptotic signaling pathway of cancer cells in both mammalian cells and mice by UCNs. We believe that this interesting NIR-light-responsive upconversion optogenetic nanotechnology has significant application potentials for both basic research and clinical applications in vivo.
Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control.
Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.
A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription.
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
Blue Light Switchable Bacterial Adhesion as a Key Step toward the Design of Biofilms.
The control of where and when bacteria adhere to a substrate is a key step toward controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E. coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light. These adhesions are reversible in the dark and can be repeatedly turned on and off. Further, the number of bacteria that can adhere to the substrate as well as the attachment and detachment dynamics are adjustable by using different point mutants of pMag and altering light intensity. Overall, the blue light switchable bacteria adhesions offer reversible, tunable and bioorthogonal control with exceptional spatial and temporal resolution. This enables us to pattern bacteria on substrates with great flexibility.