Showing 951 - 975 of 1060 results
951.
Benchmarking of optical dimerizer systems.
Abstract:
Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein-protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast.
952.
The optogenetic promise for oncology: Episode I.
Abstract:
As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial-mesenchymal transition, and angiogenic sprouting-cell behaviors central to cancer progression.
953.
Orthogonal optogenetic triple-gene control in Mammalian cells.
Abstract:
Optogenetic gene switches allow gene expression control at an unprecedented spatiotemporal resolution. Recently, light-responsive transgene expression systems that are activated by UV-B, blue, or red light have been developed. These systems perform well on their own, but their integration into genetic networks has been hampered by the overlapping absorbance spectra of the photoreceptors. We identified a lack of orthogonality between UV-B and blue light-controlled gene expression as the bottleneck and employed a model-based approach that identified the need for a blue light-responsive gene switch that is insensitive to low-intensity light. Based on this prediction, we developed a blue light-responsive and rapidly reversible expression system. Finally, we employed this expression system to demonstrate orthogonality between UV-B, blue, and red/far-red light-responsive gene switches in a single mammalian cell culture. We expect this approach to enable the spatiotemporal control of gene networks and to expand the applications of optogenetics in synthetic biology.
954.
Ultradian oscillations and pulses: coordinating cellular responses and cell fate decisions.
Abstract:
Biological clocks play key roles in organismal development, homeostasis and function. In recent years, much work has focused on circadian clocks, but emerging studies have highlighted the existence of ultradian oscillators - those with a much shorter periodicity than 24 h. Accumulating evidence, together with recently developed optogenetic approaches, suggests that such ultradian oscillators play important roles during cell fate decisions, and analyzing the functional links between ultradian oscillation and cell fate determination will contribute to a deeper understanding of the design principle of developing embryos. In this Review, we discuss the mechanisms of ultradian oscillatory dynamics and introduce examples of ultradian oscillators in various biological contexts. We also discuss how optogenetic technology has been used to elucidate the biological significance of ultradian oscillations.
955.
Photochemistry of flavoprotein light sensors.
Abstract:
Three major classes of flavin photosensors, light oxygen voltage (LOV) domains, blue light sensor using FAD (BLUF) proteins and cryptochromes (CRYs), regulate diverse biological activities in response to blue light. Recent studies of structure, spectroscopy and chemical mechanism have provided unprecedented insight into how each family operates at the molecular level. In general, the photoexcitation of the flavin cofactor leads to changes in redox and protonation states that ultimately remodel protein conformation and molecular interactions. For LOV domains, issues remain regarding early photochemical events, but common themes in conformational propagation have emerged across a diverse family of proteins. For BLUF proteins, photoinduced electron transfer reactions critical to light conversion are defined, but the subsequent rearrangement of hydrogen bonding networks key for signaling remains highly controversial. For CRYs, the relevant photocycles are actively debated, but mechanistic and functional studies are converging. Despite these challenges, our current understanding has enabled the engineering of flavoprotein photosensors for control of signaling processes within cells.
956.
Optogenetic approaches to cell migration and beyond.
Abstract:
Optogenetics, the use of genetically encoded tools to control protein function with light, can generate localized changes in signaling within living cells and animals. For years it has been focused on channel proteins for neurobiology, but has recently expanded to cover many different types of proteins, using a broad array of different protein engineering approaches. These methods have largely been directed at proteins involved in motility, cytoskeletal regulation and gene expression. This review provides a survey of non-channel proteins that have been engineered for optogenetics. Existing molecules are used to illustrate the advantages and disadvantages of the many imaginative new approaches that the reader can use to create light-controlled proteins.
957.
Optogenetic control of signaling in mammalian cells.
Abstract:
Molecular signals are sensed by their respective receptors and information is transmitted and processed by a sophisticated intracellular network controlling various biological functions. Optogenetic tools allow the targeting of specific signaling nodes for a precise spatiotemporal control of downstream effects. These tools are based on photoreceptors such as phytochrome B (PhyB), cryptochrome 2, or light-oxygen-voltage-sensing domains that reversibly bind to specific interaction partners in a light-dependent manner. Fusions of a protein of interest to the photoreceptor or their interaction partners may enable the control of the protein function by light-mediated dimerization, a change of subcellular localization, or due to photocaging/-uncaging of effectors. In this review, we summarize the photoreceptors and the light-based mechanisms utilized for the modulation of signaling events in mammalian cells focusing on non-neuronal applications. We discuss in detail optogenetic tools and approaches applied to control signaling events mediated by second messengers, Rho GTPases and growth factor-triggered signaling cascades namely the RAS/RAF and phosphatidylinositol-3-kinase pathways. Applying the latest generation of optogenetic tools allows to control cell fate decisions such as proliferation and differentiation or to deliver therapeutic substances in a spatiotemporally controlled manner.
958.
Structure and Function of the ZTL/FKF1/LKP2 Group Proteins in Arabidopsis.
Abstract:
The ZTL/FKF1/LKP2 group proteins are LOV-domain-based blue-light photoreceptors that control protein degradation by ubiquitination. These proteins were identified relatively recently and are known to be involved in the regulation of the circadian clock and photoperiodic flowering in Arabidopsis. In this review, we focus on two topics. First, we summarize the molecular mechanisms by which ZTL and FKF1 regulate these biological phenomena based on genetic and biochemical analyses. Next, we discuss the chemical properties of the ZTL family LOV domains obtained from structural, biophysical, and photochemical characterizations of the LOV domains. These two different levels of approach unveiled the molecular mechanisms by which plants utilize ZTL and FKF1 proteins to monitor light for daily and seasonal adaptation.
959.
A cyanobacterial light activated adenylyl cyclase partially restores development of a Dictyostelium discoideum, adenylyl cyclase a null mutant.
Abstract:
A light-regulated adenylyl cyclase, mPAC, was previously identified from the cyanobacterium Microcoleus chthonoplastes PCC7420. MPAC consists of a flavin-based blue light-sensing LOV domain and a catalytic domain. In this work, we expressed mPAC in an adenylate cyclase A null mutant (aca-) of the eukaryote Dictyostelium discoideum and tested to what extent light activation of mPAC could restore the cAMP-dependent developmental programme of this organism. Amoebas of Dictyostelium, a well-established model organism, generate and respond to cAMP pulses, which cause them to aggregate and construct fruiting bodies. mPAC was expressed under control of a constitutive actin-15 promoter in D. discoideum and displayed low basal adenylyl cyclase activity in darkness that was about five-fold stimulated by blue light. mPAC expression in aca- cells marginally restored aggregation and fruiting body formation in darkness. However, more and larger fruiting bodies were formed when mPAC expressing cells were incubated in light. Extending former applications of light-regulated AC, these results demonstrate that mPAC can be used to manipulate multicellular development in eukaryotes in a light dependent manner.
960.
Remote control of myosin and kinesin motors using light-activated gearshifting.
Abstract:
Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.
961.
Aureochrome 1 illuminated: structural changes of a transcription factor probed by molecular spectroscopy.
Abstract:
Aureochrome 1 from Vaucheria frigida is a recently identified blue-light receptor that acts as a transcription factor. The protein comprises a photosensitive light-, oxygen- and voltage-sensitive (LOV) domain and a basic zipper (bZIP) domain that binds DNA rendering aureochrome 1 a prospective optogenetic tool. Here, we studied the photoreaction of full-length aureochrome 1 by molecular spectroscopy. The kinetics of the decay of the red-shifted triplet state and the blue-shifted signaling state were determined by time-resolved UV/Vis spectroscopy. It is shown that the presence of the bZIP domain further prolongs the lifetime of the LOV390 signaling state in comparison to the isolated LOV domain whereas bound DNA does not influence the photocycle kinetics. The light-dark Fourier transform infrared (FTIR) difference spectrum shows the characteristic features of the flavin mononucleotide chromophore except that the S-H stretching vibration of cysteine 254, which is involved in the formation of the thio-adduct state, is significantly shifted to lower frequencies compared to other LOV domains. The presence of the target DNA influences the light-induced FTIR difference spectrum of aureochrome 1. Vibrational bands that can be assigned to arginine and lysine side chains as well to the phosphate backbone, indicate crucial changes in interactions between transcription factor and DNA.
962.
Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.
Abstract:
The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.
963.
Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.
Abstract:
Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.
964.
How to control proteins with light in living systems.
Abstract:
The possibility offered by photocontrolling the activity of biomolecules in vivo while recording physiological parameters is opening up new opportunities for the study of physiological processes at the single-cell level in a living organism. For the last decade, such tools have been mainly used in neuroscience, and their application in freely moving animals has revolutionized this field. New photochemical approaches enable the control of various cellular processes by manipulating a wide range of protein functions in a noninvasive way and with unprecedented spatiotemporal resolution. We are at a pivotal moment where biologists can adapt these cutting-edge technologies to their system of study. This user-oriented review presents the state of the art and highlights technical issues to be resolved in the near future for wide and easy use of these powerful approaches.
965.
Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides.
Abstract:
Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently inhibited kinases in the light but were sterically blocked from kinase interaction in the dark. Photoactivatable inhibitors for cyclic-AMP dependent kinase (PKA) and myosin light chain kinase (MLCK) are described, together with studies that shed light on proper positioning of the peptides in the LOV domain. These inhibitors altered endogenous signaling in living cells and produced light-dependent changes in cell morphodynamics.
966.
Light-mediated control of gene expression in filamentous fungus Trichoderma reesei.
Abstract:
We developed a light-mediated system based on synthetic light-switchable transactivators. The transactivators bind promoter upon blue-light exposure and rapidly initiate transcription of target transgenes in filamentous fungus Trichoderma reesei. Light is inexpensive to apply, easily delivered, and instantly removed, and thus has significant advantages over chemical inducers.
967.
Rac1-dependent lamellipodial motility in prostate cancer PC-3 cells revealed by optogenetic control of Rac1 activity.
Abstract:
The lamellipodium, an essential structure for cell migration, plays an important role in the invasion and metastasis of cancer cells. Although Rac1 recognized as a key player in the formation of lamellipodia, the molecular mechanisms underlying lamellipodial motility are not fully understood. Optogenetic technology enabled us to spatiotemporally control the activity of photoactivatable Rac1 (PA-Rac1) in living cells. Using this system, we revealed the role of phosphatidylinositol 3-kinase (PI3K) in Rac1-dependent lamellipodial motility in PC-3 prostate cancer cells. Through local blue laser irradiation of PA-Rac1-expressing cells, lamellipodial motility was reversibly induced. First, outward extension of a lamellipodium parallel to the substratum was observed. The extended lamellipodium then showed ruffling activity at the periphery. Notably, PI(3,4,5)P3 and WAVE2 were localized in the extending lamellipodium in a PI3K-dependent manner. We confirmed that the inhibition of PI3K activity greatly suppressed lamellipodial extension, while the ruffling activity was less affected. These results suggest that Rac1-induced lamellipodial motility consists of two distinct activities, PI3K-dependent outward extension and PI3K-independent ruffling.
968.
Optical control of protein function through unnatural amino acid mutagenesis and other optogenetic approaches.
Abstract:
Biological processes are naturally regulated with high spatial and temporal resolution at the molecular, cellular, and systems level. To control and study processes with the same resolution, light-sensitive groups and domains have been employed to optically activate and deactivate protein function. Optical control is a noninvasive technique in which the amplitude, wavelength, spatial location, and timing of the light illumination can be easily controlled. This review focuses on applications of genetically encoded unnatural amino acids containing light-removable protecting groups to optically trigger protein function, while also discussing select optogenetic approaches using natural light-sensitive domains to engineer optical control of biological processes.
969.
Blue light-induced dimerization of monomeric aureochrome-1 enhances its affinity for the target sequence.
Abstract:
Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in stramenopile alga, Vaucheria frigida. AUREO1 contains a basic leucine zipper (bZIP) domain in the central region and a light-oxygen-voltage sensing (LOV) domain at the C terminus, and has been suggested to function as a light-regulated transcription factor. We have previously reported that preparations of recombinant AUREO1 contained the complete coding sequence (full-length, FL) and N-terminal truncated protein (ZL) containing bZIP and LOV domains, and suggested that wild-type ZL (ZLwt2) was in a dimer form with intermolecular disulfide linkages at Cys(162) and Cys(182) (Hisatomi, O., Takeuchi, K., Zikihara, K., Ookubo, Y., Nakatani, Y., Takahashi, F., Tokutomi, S., and Kataoka, H. (2013) Plant Cell Physiol. 54, 93-106). In the present study, we report the photoreactions, oligomeric structures, and DNA binding of monomeric cysteine to serine-mutated ZL (ZLC2S), DTT-treated ZL (DTT-ZL), and FL (DTT-FL). Recombinant AUREO1 showed similar spectral properties and dark regeneration kinetics to those of dimeric ZLwt2. Dynamic light scattering and size exclusion chromatography revealed that ZLC2S and DTT-ZL were monomeric in the dark state. Dissociation of intermolecular disulfide bonds of ZLwt2 was in equilibrium with a midpoint oxidation-redox potential of approximately -245 ± 15 mV. BL induced the dimerization of monomeric ZL, which subsequently increased its affinity for the target sequence. Also, DTT-FL was monomeric in the dark state and underwent BL-induced dimerization, which led to formation of the FL2·DNA complex. Taken together, our results suggest that monomeric AUREO1 is present in vivo, with dimerization playing a key role in its role as a BL-regulated transcription factor.
970.
Optical control of the Ca2+ concentration in a live specimen with a genetically encoded Ca2+-releasing molecular tool.
Abstract:
Calcium ion (Ca2+) is an important second messenger implicated in the control of many different cellular processes in living organisms. Ca2+ is typically studied by direct visualization using chemically or genetically encoded indicators. A complementary, and perhaps more useful, approach involves direct manipulation of Ca2+ concentration; tools for this exist but are rather poorly developed compared to the indicators at least. Here, we report a photoactivatable Ca2+-releasing protein, photoactivatable Ca2+ releaser (PACR), made by the insertion of a photosensitive protein domain (LOV2) into a Ca2+ binding protein (calmodulin fused with the M13 peptide). As the PACR is genetically encoded, and unlike conventional optical control tools (e.g., channel rhodopsin) not membrane bound, we are able to restrict expression within the cell, to allow subcellular perturbation of Ca2+ levels. In whole animals, we are able to control the behavior of Caenorhabditis elegans with light by expressing the PACR only in the touch neuron.
971.
Light-inducible gene regulation with engineered zinc finger proteins.
Abstract:
The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.
972.
Biophysical, mutational, and functional investigation of the chromophore-binding pocket of light-oxygen-voltage photoreceptors.
Abstract:
As light-regulated actuators, sensory photoreceptors underpin optogenetics and numerous applications in synthetic biology. Protein engineering has been applied to fine-tune the properties of photoreceptors and to generate novel actuators. For the blue-light-sensitive light-oxygen-voltage (LOV) photoreceptors, mutations near the flavin chromophore modulate response kinetics and the effective light responsiveness. To probe for potential, inadvertent effects on receptor activity, we introduced these mutations into the engineered LOV photoreceptor YF1 and determined their impact on light regulation. While several mutations severely impaired the dynamic range of the receptor (e.g., I39V, R63K, and N94A), residue substitutions in a second group were benign with little effect on regulation (e.g., V28T, N37C, and L82I). Electron paramagnetic resonance and absorption spectroscopy identified correlated effects for certain of the latter mutations on chromophore environment and response kinetics in YF1 and the LOV2 domain from Avena sativa phototropin 1. Carefully chosen mutations provide a powerful means to adjust the light-response function of photoreceptors as demanded for diverse applications.
973.
Optogenetic control of ROS production.
Abstract:
Reactive Oxygen Species (ROS) are known to cause oxidative damage to DNA, proteins and lipids. In addition, recent evidence suggests that ROS can also initiate signaling cascades that respond to stress and modify specific redox-sensitive moieties as a regulatory mechanism. This suggests that ROS are physiologically-relevant signaling molecules. However, these sensor/effector molecules are not uniformly distributed throughout the cell. Moreover, localized ROS damage may elicit site-specific compensatory measures. Thus, the impact of ROS can be likened to that of calcium, a ubiquitous second messenger, leading to the prediction that their effects are exquisitely dependent upon their location, quantity and even the timing of generation. Despite this prediction, ROS signaling is most commonly intuited through the global administration of chemicals that produce ROS or by ROS quenching through global application of antioxidants. Optogenetics, which uses light to control the activity of genetically-encoded effector proteins, provides a means of circumventing this limitation. Photo-inducible genetically-encoded ROS-generating proteins (RGPs) were originally employed for their phototoxic effects and cell ablation. However, reducing irradiance and/or fluence can achieve sub-lethal levels of ROS that may mediate subtle signaling effects. Hence, transgenic expression of RGPs as fusions to native proteins gives researchers a new tool to exert spatial and temporal control over ROS production. This review will focus on the new frontier defined by the experimental use of RGPs to study ROS signaling.
974.
Factors that control the chemistry of the LOV domain photocycle.
Abstract:
Algae, plants, bacteria and fungi contain Light-Oxygen-Voltage (LOV) domains that function as blue light sensors to control cellular responses to light. All LOV domains contain a bound flavin chromophore that is reduced upon photon absorption and forms a reversible, metastable covalent bond with a nearby cysteine residue. In Avena sativa LOV2 (AsLOV2), the photocycle is accompanied by an allosteric conformational change that activates the attached phototropin kinase in the full-length protein. Both the conformational change and formation of the cysteinyl-flavin adduct are stabilized by the reduction of the N5 atom in the flavin's isoalloxazine ring. In this study, we perform a mutational analysis to investigate the requirements for LOV2 to photocycle. We mutated all the residues that interact with the chromophore isoalloxazine ring to inert functional groups but none could fully inhibit the photocycle except those to the active-site cysteine. However, electronegative side chains in the vicinity of the chromophore accelerate the N5 deprotonation and the return to the dark state. Mutations to the N414 and Q513 residues identify a potential water gate and H₂O coordination sites. These residues affect the electronic nature of the chromophore and photocycle time by helping catalyze the N5 reduction leading to the completion of the photocycle. In addition, we demonstrate that dehydration leads to drastically slower photocycle times. Finally, to investigate the requirements of an active-site cysteine for photocycling, we moved the nearby cysteine to alternative locations and found that some variants can still photocycle. We propose a new model of the LOV domain photocycle that involves all of these components.
975.
Genetically engineered photoinducible homodimerization system with improved dimer-forming efficiency.
Abstract:
Vivid (VVD) is a photoreceptor derived from Neurospora Crassa that rapidly forms a homodimer in response to blue light. Although VVD has several advantages over other photoreceptors as photoinducible homodimerization system, VVD has a critical limitation in its low dimer-forming efficiency. To overcome this limitation of wild-type VVD, here we conduct site-directed saturation mutagenesis in the homodimer interface of VVD. We have found that the Ile52Cys mutation of VVD (VVD-52C) substantially improves its homodimer-forming efficiency up to 180%. We have demonstrated the utility of VVD-52C for making a light-inducible gene expression system more robust. In addition, using VVD-52C, we have developed photoactivatable caspase-9, which enables optical control of apoptosis of mammalian cells. The present genetically engineered photoinducible homodimerization system can provide a powerful tool to optically control a broad range of molecular processes in the cell.