Showing 226 - 250 of 647 results
Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development.
Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.
Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres.
Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.
Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation.
Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because the activated Rac1 reduced the phosphorylation levels of myosin light chain, failure of the cup formation were probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with FRET imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup.
At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior.
Cells are bombarded by extrinsic signals that dynamically change in time and space. Such dynamic variations can exert profound effects on behaviors, including cellular signaling, organismal development, stem cell differentiation, normal tissue function, and disease processes such as cancer. Although classical genetic tools are well suited to introduce binary perturbations, new approaches have been necessary to investigate how dynamic signal variation may regulate cell behavior. This fundamental question is increasingly being addressed with optogenetics, a field focused on engineering and harnessing light-sensitive proteins to interface with cellular signaling pathways. Channelrhodopsins initially defined optogenetics; however, through recent use of light-responsive proteins with myriad spectral and functional properties, practical applications of optogenetics currently encompass cell signaling, subcellular localization, and gene regulation. Now, important questions regarding signal integration within branch points of signaling networks, asymmetric cell responses to spatially restricted signals, and effects of signal dosage versus duration can be addressed. This review summarizes emerging technologies and applications within the expanding field of optogenetics.
Hydrogen Bonding Environment of the N3-H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins.
The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light-induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3-H group of flavin mononucleotide (FMN) of the LOV2 domain from Adiantum neochrome (neo) 1 was investigated by low-temperature Fourier transform infrared spectroscopy. Using specifically (15)N-labeled FMN, [1,3-(15)N2]FMN, the N3-H stretch was identified at 2831 cm(-1) for the unphotolyzed state at 150 K, indicating that the N3-H group forms a fairly strong hydrogen bond. The N3-H stretch showed temperature dependence, with a shift to lower frequencies at ≤200 K and to higher frequencies at ≥250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from Arabidopsis phot1 and phot2. By contrast, the N3-H stretch of the Q1029L mutant of neo1-LOV2 and neo1-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the β-sheet region and/or the adjacent Jα helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neo1-LOV1 domain. The environment around the N3-H group was also investigated.
Mini Photobioreactors for in Vivo Real-Time Characterization and Evolutionary Tuning of Bacterial Optogenetic Circuit.
The current standard protocols for characterizing the optogenetic circuit of bacterial cells using flow cytometry in light tubes and light exposure of culture plates are tedious, labor-intensive, and cumbersome. In this work, we engineer a bioreactor with working volume of ∼10 mL for in vivo real-time optogenetic characterization of E. coli with a CcaS-CcaR light-sensing system. In the bioreactor, optical density measurements, reporter protein fluorescence detection, and light input stimuli are provided by four light-emitting diode sources and two photodetectors. Once calibrated, the device can cultivate microbial cells and record their growth and gene expression without human intervention. We measure gene expression during cell growth with different organic substrates (glucose, succinate, acetate, pyruvate) as carbon sources in minimal medium and demonstrate evolutionary tuning of the optogenetic circuit by serial dilution passages.
Optical control of membrane tethering and interorganellar communication at nanoscales.
Endoplasmic reticulum (ER) forms an extensive intracellular membranous network in eukaryotes that dynamically connects and communicates with diverse subcellular compartments such as plasma membrane (PM) through membrane contact sites (MCSs), with the inter-membrane gaps separated by a distance of 10-40 nm. Phosphoinositides (PI) constitute an important class of cell membrane phospholipids shared by many MCSs to regulate a myriad of cellular events, including membrane trafficking, calcium homeostasis and lipid metabolism. By installing photosensitivity into a series of engineered PI-binding domains with minimal sizes, we have created an optogenetic toolkit (designated as 'OptoPB') to enable rapid and reversible control of protein translocation and inter-membrane tethering at MCSs. These genetically-encoded, single-component tools can be used as scaffolds for grafting lipid-binding domains to dissect molecular determinants that govern protein-lipid interactions in living cells. Furthermore, we have demonstrated the use of OptoPB as a versatile fusion tag to photomanipulate protein translocation toward PM for reprogramming of PI metabolism. When tethered to the ER membrane with the insertion of flexible spacers, OptoPB can be applied to reversibly photo-tune the gap distances at nanometer scales between the two organellar membranes at MCSs, and to gauge the distance requirement for the free diffusion of protein complexes into MCSs. Our modular optical tools will find broad applications in non-invasive and remote control of protein subcellular localization and interorganellar contact sites that are critical for cell signaling.
Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond.
Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, in particular tools for all-optical electrophysiology, now provide a means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline - from target identification and validation, to target-based and phenotypic screens, to clinical trials.
B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release.
Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.
Engineering RGB color vision into Escherichia coli.
Optogenetic tools use colored light to rapidly control gene expression in space and time. We designed a genetically encoded system that gives Escherichia coli the ability to distinguish between red, green, and blue (RGB) light and respond by changing gene expression. We use this system to produce 'color photographs' on bacterial culture plates by controlling pigment production and to redirect metabolic flux by expressing CRISPRi guide RNAs.
Engineering genetically-encoded tools for optogenetic control of protein activity.
Optogenetic tools offer fast and reversible control of protein activity with subcellular spatial precision. In the past few years, remarkable progress has been made in engineering photoactivatable systems regulating the activity of cellular proteins. In this review, we discuss general strategies in designing and optimizing such optogenetic tools and highlight recent advances in the field, with specific focus on applications regulating protein catalytic activity.
Illuminating developmental biology through photochemistry.
Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.
Optogenetics: Switching with red and blue.
Abstract not available.
A simple optogenetic MAPK inhibitor design reveals resonance between transcription-regulating circuitry and temporally-encoded inputs.
Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.
Time-Resolved X-Ray Solution Scattering Reveals the Structural Photoactivation of a Light-Oxygen-Voltage Photoreceptor.
Light-oxygen-voltage (LOV) receptors are sensory proteins controlling a wide range of organismal adaptations in multiple kingdoms of life. Because of their modular nature, LOV domains are also attractive for use as optogenetic actuators. A flavin chromophore absorbs blue light, forms a bond with a proximal cysteine residue, and induces changes in the surroundings. There is a gap of knowledge on how this initial signal is relayed further through the sensor to the effector module. To characterize these conformational changes, we apply time-resolved X-ray scattering to the homodimeric LOV domain from Bacillus subtilis YtvA. We observe a global structural change in the LOV dimer synchronous with the formation of the chromophore photoproduct state. Using molecular modeling, this change is identified as splaying apart and relative rotation of the two monomers, which leads to an increased separation at the anchoring site of the effector modules.
Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii.
RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one with red-shifted (D380E, λmax = 533 nm) absorption maxima relative to the wild-type protein (λmax = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.
Optogenetic Modulation of Intracellular Signalling and Transcription: Focus on Neuronal Plasticity.
Several fields in neuroscience have been revolutionized by the advent of optogenetics, a technique that offers the possibility to modulate neuronal physiology in response to light stimulation. This innovative and far-reaching tool provided unprecedented spatial and temporal resolution to explore the activity of neural circuits underlying cognition and behaviour. With an exponential growth in the discovery and synthesis of new photosensitive actuators capable of modulating neuronal networks function, other fields in biology are experiencing a similar re-evolution. Here, we review the various optogenetic toolboxes developed to influence cellular physiology as well as the diverse ways in which these can be engineered to precisely modulate intracellular signalling and transcription. We also explore the processes required to successfully express and stimulate these photo-actuators in vivo before discussing how such tools can enlighten our understanding of neuronal plasticity at the systems level.
Distinctive Properties of Dark Reversion Kinetics between Two Red/Green-Type Cyanobacteriochromes and their Application in the Photoregulation of cAMP Synthesis.
Cyanobacteriochromes (CBCRs) are photoreceptors that bind to a linear tetrapyrrole within a conserved cGMP-phosphodiesterase/adenylate cyclase/FhlA (GAF) domain and exhibit reversible photoconversion. Red/green-type CBCR GAF domains that photoconvert between red- (Pr) and green-absorbing (Pg) forms occur widely in various cyanobacteria. A putative phototaxis regulator, AnPixJ, contains multiple red/green-type CBCR GAF domains. We previously reported that AnPixJ's second domain (AnPixJg2) but not its fourth domain (AnPixJg4) shows red/green reversible photoconversion. Herein, we found that AnPixJg4 showed Pr-to-Pg photoconversion and rapid Pg-to-Pr dark reversion, whereas AnPixJg2 showed a barely detectable dark reversion. Site-directed mutagenesis revealed the involvement of six residues in Pg stability. Replacement at the Leu294/Ile660 positions of AnPixJg2/AnPixJg4 showed the highest influence on dark reversion kinetics. AnPixJg2_DR6, wherein the six residues of AnPixJg2 were entirely replaced with those of AnPixJg4, showed a 300-fold faster dark reversion than that of the wild type. We constructed chimeric proteins by fusing the GAF domains with adenylate cyclase catalytic regions, such as AnPixJg2-AC, AnPixJg4-AC and AnPixJg2_DR6-AC. We detected successful enzymatic activation under red light for both AnPixJg2-AC and AnPixJg2_DR6-AC, and repression under green light for AnPixJg2-AC and under dark incubation for AnPixJg2_DR6-AC. These results provide platforms to develop cAMP synthetic optogenetic tools.
Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice.
With the increasingly dominant role of smartphones in our lives, mobile health care systems integrating advanced point-of-care technologies to manage chronic diseases are gaining attention. Using a multidisciplinary design principle coupling electrical engineering, software development, and synthetic biology, we have engineered a technological infrastructure enabling the smartphone-assisted semiautomatic treatment of diabetes in mice. A custom-designed home server SmartController was programmed to process wireless signals, enabling a smartphone to regulate hormone production by optically engineered cells implanted in diabetic mice via a far-red light (FRL)-responsive optogenetic interface. To develop this wireless controller network, we designed and implanted hydrogel capsules carrying both engineered cells and wirelessly powered FRL LEDs (light-emitting diodes). In vivo production of a short variant of human glucagon-like peptide 1 (shGLP-1) or mouse insulin by the engineered cells in the hydrogel could be remotely controlled by smartphone programs or a custom-engineered Bluetooth-active glucometer in a semiautomatic, glucose-dependent manner. By combining electronic device-generated digital signals with optogenetically engineered cells, this study provides a step toward translating cell-based therapies into the clinic.
A photoconversion model for full spectral programming and multiplexing of optogenetic systems.
Optogenetics combines externally applied light signals and genetically engineered photoreceptors to control cellular processes with unmatched precision. Here, we develop a mathematical model of wavelength- and intensity-dependent photoconversion, signaling, and output gene expression for our two previously engineered light-sensing Escherichia coli two-component systems. To parameterize the model, we develop a simple set of spectral and dynamical calibration experiments using our recent open-source "Light Plate Apparatus" device. In principle, the parameterized model should predict the gene expression response to any time-varying signal from any mixture of light sources with known spectra. We validate this capability experimentally using a suite of challenging light sources and signals very different from those used during the parameterization process. Furthermore, we use the model to compensate for significant spectral cross-reactivity inherent to the two sensors in order to develop a new method for programming two simultaneous and independent gene expression signals within the same cell. Our optogenetic multiplexing method will enable powerful new interrogations of how metabolic, signaling, and decision-making pathways integrate multiple input signals.
Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2.
Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators. We used this knowledge to develop two different approaches to regulate cellular protein levels with light: a system using CRY2 and CIB1 to induce protein expression with light through stimulation of transcription, and a system using CRY2 and a LOV-fused degron to simultaneously block transcription and deplete protein levels with light. These tools will allow precise, bi-directional control of gene expression in a variety of cells and model systems.
Cell-matrix adhesion and cell-cell adhesion differentially control basal myosin oscillation and Drosophila egg chamber elongation.
Pulsatile actomyosin contractility, important in tissue morphogenesis, has been studied mainly in apical but less in basal domains. Basal myosin oscillation underlying egg chamber elongation is regulated by both cell-matrix and cell-cell adhesions. However, the mechanism by which these two adhesions govern basal myosin oscillation and tissue elongation is unknown. Here we demonstrate that cell-matrix adhesion positively regulates basal junctional Rho1 activity and medio-basal ROCK and myosin activities, thus strongly controlling tissue elongation. Differently, cell-cell adhesion governs basal myosin oscillation through controlling medio-basal distributions of both ROCK and myosin signals, which are related to the spatial limitations of cell-matrix adhesion and stress fibres. Contrary to cell-matrix adhesion, cell-cell adhesion weakly affects tissue elongation. In vivo optogenetic protein inhibition spatiotemporally confirms the different effects of these two adhesions on basal myosin oscillation. This study highlights the activity and distribution controls of basal myosin contractility mediated by cell-matrix and cell-cell adhesions, respectively, during tissue morphogenesis.
Near-Infrared Fluorescent Proteins, Biosensors, and Optogenetic Tools Engineered from Phytochromes.
Phytochrome photoreceptors absorb far-red and near-infrared (NIR) light and regulate light responses in plants, fungi, and bacteria. Their multidomain structure and autocatalytic incorporation of linear tetrapyrrole chromophores make phytochromes attractive molecular templates for the development of light-sensing probes. A subclass of bacterial phytochromes (BphPs) utilizes heme-derived biliverdin tetrapyrrole, which is ubiquitous in mammalian tissues, as a chromophore. Because biliverdin possesses the largest electron-conjugated chromophore system among linear tetrapyrroles, BphPs exhibit the most NIR-shifted spectra that reside within the NIR tissue transparency window. Here we analyze phytochrome structure and photochemistry to describe the molecular mechanisms by which they function. We then present strategies to engineer BphP-based NIR fluorescent proteins and review their properties and applications in modern imaging technologies. We next summarize designs of reporters and biosensors and describe their use in the detection of protein-protein interactions, proteolytic activities, and posttranslational modifications. Finally, we provide an overview of optogenetic tools developed from phytochromes and describe their use in light-controlled cell signaling, gene expression, and protein localization. Our review provides guidelines for the selection of NIR probes and tools for noninvasive imaging, sensing, and light-manipulation applications, specifically focusing on probes developed for use in mammalian cells and in vivo.
Light-induced protein degradation in human-derived cells.
Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies.
The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology.
The zebrafish ( Danio rerio) is a powerful vertebrate model to study cellular and developmental processes in vivo. The optical clarity and their amenability to genetic manipulation make zebrafish a model of choice when it comes to applying optical techniques involving genetically encoded photoresponsive protein technologies. In recent years, a number of fluorescent protein and optogenetic technologies have emerged that allow new ways to visualize, quantify, and perturb developmental dynamics. Here, we explain the principles of these new tools and describe some of their representative applications in zebrafish.