Showing 201 - 225 of 678 results
Shaping bacterial population behavior through computer-interfaced control of individual cells.
Bacteria in groups vary individually, and interact with other bacteria and the environment to produce population-level patterns of gene expression. Investigating such behavior in detail requires measuring and controlling populations at the single-cell level alongside precisely specified interactions and environmental characteristics. Here we present an automated, programmable platform that combines image-based gene expression and growth measurements with on-line optogenetic expression control for hundreds of individual Escherichia coli cells over days, in a dynamically adjustable environment. This integrated platform broadly enables experiments that bridge individual and population behaviors. We demonstrate: (i) population structuring by independent closed-loop control of gene expression in many individual cells, (ii) cell-cell variation control during antibiotic perturbation, (iii) hybrid bio-digital circuits in single cells, and freely specifiable digital communication between individual bacteria. These examples showcase the potential for real-time integration of theoretical models with measurement and control of many individual cells to investigate and engineer microbial population behavior.
Gradients of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling.
Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.
Shedding light on the role of cAMP in mammalian sperm physiology.
Mammalian fertilization relies on sperm finding the egg and penetrating the egg vestments. All steps in a sperm's lifetime crucially rely on changes in the second messenger cAMP (cyclic adenosine monophosphate). In recent years, it has become clear that signal transduction in sperm is not a continuum, but rather organized in subcellular domains, e.g. the sperm head and the sperm flagellum, with the latter being further separated into the midpiece, principal piece, and endpiece. To understand the underlying signaling pathways controlling sperm function in more detail, experimental approaches are needed that allow to study sperm signaling with spatial and temporal precision. Here, we will give a comprehensive overview on cAMP signaling in mammalian sperm, describing the molecular players involved in these pathways and the sperm functions that are controlled by cAMP. Furthermore, we will highlight recent advances in analyzing and manipulating sperm signaling with spatio-temporal precision using light.
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.
Real-time observation of light-controlled transcription in living cells.
Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.
Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC.
RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCCRho) and without (GCRho) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GCRho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GCRho monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.
Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell.
β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance.
Optogenetic regulation of artificial microRNA improves H2 production in green alga Chlamydomonas reinhardtii.
Chlamydomonas reinhardtii is an ideal model organism not only for the study of basic metabolic processes in both plants and animals but also the production of biofuels including hydrogen. Transgenic analysis of C. reinhardtii is now well established and very convenient, but inducible exogenous gene expression systems remain under-studied. The most commonly used heat shock-inducible system has serious effects on algal cell growth and is difficult and costly to control in large-scale culture. Previous studies of hydrogen photoproduction in Chlamydomonas also use this heat-inducible system to activate target gene transcription and hydrogen synthesis.
Propagating Wave of ERK Activation Orients Collective Cell Migration.
The biophysical framework of collective cell migration has been extensively investigated in recent years; however, it remains elusive how chemical inputs from neighboring cells are integrated to coordinate the collective movement. Here, we provide evidence that propagation waves of extracellular signal-related kinase (ERK) mitogen-activated protein kinase activation determine the direction of the collective cell migration. A wound-healing assay of Mardin-Darby canine kidney (MDCK) epithelial cells revealed two distinct types of ERK activation wave, a "tidal wave" from the wound, and a self-organized "spontaneous wave" in regions distant from the wound. In both cases, MDCK cells collectively migrated against the direction of the ERK activation wave. The inhibition of ERK activation propagation suppressed collective cell migration. An ERK activation wave spatiotemporally controlled actomyosin contraction and cell density. Furthermore, an optogenetic ERK activation wave reproduced the collective cell migration. These data provide new mechanistic insight into how cells sense the direction of collective cell migration.
Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions.
Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.
Optogenetic Tools for Subcellular Applications in Neuroscience.
The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications.
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.
Design and Profiling of a Subcellular Targeted Optogenetic cAMP-Dependent Protein Kinase.
Although the cAMP-dependent protein kinase (PKA) is ubiquitously expressed, it is sequestered at specific subcellular locations throughout the cell, thereby resulting in compartmentalized cellular signaling that triggers site-specific behavioral phenotypes. We developed a three-step engineering strategy to construct an optogenetic PKA (optoPKA) and demonstrated that, upon illumination, optoPKA migrates to specified intracellular sites. Furthermore, we designed intracellular spatially segregated reporters of PKA activity and confirmed that optoPKA phosphorylates these reporters in a light-dependent fashion. Finally, proteomics experiments reveal that light activation of optoPKA results in the phosphorylation of known endogenous PKA substrates as well as potential novel substrates.
Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling.
Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.
Optogenetic control of focal adhesion kinase signaling.
Focal adhesion kinase (FAK) integrates signaling from integrins, growth factor receptors and mechanical stress to control cell adhesion, motility, survival and proliferation. Here, we developed a single-component, photo-activatable FAK, termed optoFAK, by using blue light-induced oligomerization of cryptochrome 2 (CRY2) to activate FAK-CRY2 fusion proteins. OptoFAK functions uncoupled from physiological stimuli and activates downstream signaling rapidly and reversibly upon blue light exposure. OptoFAK stimulates SRC creating a positive feedback loop on FAK activation, facilitating phosphorylation of paxillin and p130Cas in adherent cells. In detached cells or in mechanically stressed adherent cells, optoFAK is autophosphorylated upon exposure to blue light, however, downstream signaling is hampered indicating that the accessibility to these substrates is disturbed. OptoFAK may prove to be a useful tool to study the biological function of FAK in growth factor and integrin signaling, tension-mediated focal adhesion maturation or anoikis and could additionally serve as test system for kinase inhibitors.
Re-engineering the two-component systems as light-regulated in Escherichia coli.
Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make use of various sensor-response circuits, called two-component systems (TCSs). A TCS comprises a histidine protein kinase (HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two components are coupled via a phosphorylation control mechanism. In a recent study, we adopted an optogenetics approach to re-engineer the sensor HKs in Escherichia coli as a light-sensing fusion protein. We constructed a light-controllable HK by replacing the original signal-specific sensing domain of HK with the light-sensing domain of Cph1 from Cyanobacteria Synechocystis, so that HK can be investigated by red light. Here, we extended the study to other 16 HK-RR TCSs and constructed a library of light-responsible HK-Cph1 chimeras. By taking the NarX-NarL system as an example, we demonstrated the light responsiveness of the constructed chimera and investigated the frequency response of the NarXNarL system. The constructed library serves as a toolkit for future TCS study using optogenetics approach.
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.
Optimized light-inducible transcription in mammalian cells using Flavin Kelch-repeat F-box1/GIGANTEA and CRY2/CIB1.
Light-inducible systems allow spatiotemporal control of a variety of biological activities. Here, we report newly optimized optogenetic tools to induce transcription with light in mammalian cells, using the Arabidopsis photoreceptor Flavin Kelch-repeat F-box 1 (FKF1) and its binding partner GIGANTEA (GI) as well as CRY2/CIB1. By combining the mutagenesis of FKF1 with the optimization of a split FKF1/GI dimerized Gal4-VP16 transcriptional system, we identified constructs enabling significantly improved light-triggered transcriptional induction. In addition, we have improved the CRY2/CIB1-based light-inducible transcription with split construct optimization. The improvements regarding the FKF1/GI- and CRY2/CIB1-based systems will be widely applicable for the light-dependent control of transcription in mammalian cells.
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.
Modulation of cyclic nucleotide-mediated cellular signaling and gene expression using photoactivated adenylyl cyclase as an optogenetic tool.
Cyclic nucleotide signaling pathway plays a significant role in various biological processes such as cell growth, transcription, inflammation, in microbial pathogenesis, etc. Modulation of cyclic nucleotide levels by optogenetic tools has overcome certain limitations of studying transduction cascade by pharmacological agents and has allowed several ways to modulate biological processes in a spatiotemporal manner. Here, we have shown the optogenetic modulation of the cyclooxygenase 2 (Cox-2) gene expression and their downstream effector molecule (PGE2) in HEK-293T cells and the development process of Dictyostelium discoideum via modulating the cyclic nucleotide (cAMP) signaling pathway utilizing photoactivated adenylyl cyclases (PACs) as an optogenetic tool. Light-induced activation of PACs in HEK-293T cells increases the cAMP level that leads to activation of cAMP response element-binding protein (CREB) transcription factor and further upregulates downstream Cox-2 gene expression and their downstream effector molecule prostaglandin E2. In D. discoideum, the light-regulated increase in cAMP level affects the starvation-induced developmental process. These PACs could modulate the cAMP levels in a light-dependent manner and have a potential to control gene expression and their downstream effector molecules with varying magnitude. It would enable one to utilize PAC as a tool to decipher cyclic nucleotide mediated signaling pathway regulations and their mechanism.
Understanding CRY2 interactions for optical control of intracellular signaling.
Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2-CRY2 homo-oligomerization and CRY2-CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2-CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2-CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2-CIB1 and CRY2-CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2-CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems.Cryptochrome 2 (CRY2) can form light-regulated CRY2-CRY2 homo-oligomers or CRY2-CIB1 hetero-dimers, but modulating these interactions is difficult owing to the lack of interaction mechanism. Here the authors identify the interactions facilitating homo-oligomers and introduce mutations to create low and high oligomerization versions.
Engineering a light-activated caspase-3 for precise ablation of neurons in vivo.
The circuitry of the brain is characterized by cell heterogeneity, sprawling cellular anatomy, and astonishingly complex patterns of connectivity. Determining how complex neural circuits control behavior is a major challenge that is often approached using surgical, chemical, or transgenic approaches to ablate neurons. However, all these approaches suffer from a lack of precise spatial and temporal control. This drawback would be overcome if cellular ablation could be controlled with light. Cells are naturally and cleanly ablated through apoptosis due to the terminal activation of caspases. Here, we describe the engineering of a light-activated human caspase-3 (Caspase-LOV) by exploiting its natural spring-loaded activation mechanism through rational insertion of the light-sensitive LOV2 domain that expands upon illumination. We apply the light-activated caspase (Caspase-LOV) to study neurodegeneration in larval and adult Drosophila Using the tissue-specific expression system (UAS)-GAL4, we express Caspase-LOV specifically in three neuronal cell types: retinal, sensory, and motor neurons. Illumination of whole flies or specific tissues containing Caspase-LOV-induced cell death and allowed us to follow the time course and sequence of neurodegenerative events. For example, we find that global synchronous activation of caspase-3 drives degeneration with a different time-course and extent in sensory versus motor neurons. We believe the Caspase-LOV tool we engineered will have many other uses for neurobiologists and others for specific temporal and spatial ablation of cells in complex organisms.
CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation.
Our improved CRISPR-Cas9-based photoactivatable transcription systems, CPTS2.0 and Split-CPTS2.0, enable high blue-light-inducible activation of endogenous target genes in various human cell lines. We achieved reversible activation of target genes with CPTS2.0 and induced neuronal differentiation in induced pluripotent stem cells (iPSCs) by upregulating NEUROD1 with Split-CPTS2.0.
Using Light-Activated Enzymes for Modulating Intracellular c-di-GMP Levels in Bacteria.
Signaling pathways involving second messenger c-di-GMP regulate various aspects of bacterial physiology and behavior. We describe the use of a red light-activated diguanylate cyclase (c-di-GMP synthase) and a blue light-activated c-di-GMP phosphodiesterase (hydrolase) for manipulating intracellular c-di-GMP levels in bacterial cells. We illustrate the application of these enzymes in regulating several c-di-GMP-dependent phenotypes, i.e., motility and biofilm phenotypes in E. coli and chemotactic behavior in the alphaproteobacterium Azospirillum brasilense. We expect these light-activated enzymes to be also useful in regulating c-di-GMP-dependent processes occurring at the fast timescale, for spatial control of bacterial populations, as well as for analyzing c-di-GMP-dependent phenomena at the single-cell level.