Qr: switch:"CRY2/CIB1"
Showing 401 - 425 of 517 results
401.
Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2.
Abstract:
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.
402.
Cell-matrix adhesion and cell-cell adhesion differentially control basal myosin oscillation and Drosophila egg chamber elongation.
Abstract:
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.
403.
The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology.
Abstract:
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.
404.
Temporally precise labeling and control of neuromodulatory circuits in the mammalian brain.
Abstract:
Few tools exist to visualize and manipulate neurons that are targets of neuromodulators. We present iTango, a light- and ligand-gated gene expression system based on a light-inducible split tobacco etch virus protease. Cells expressing the iTango system exhibit increased expression of a marker gene in the presence of dopamine and blue-light exposure, both in vitro and in vivo. We demonstrated the iTango system in a behaviorally relevant context, by inducing expression of optogenetic tools in neurons under dopaminergic control during a behavior of interest. We thereby gained optogenetic control of these behaviorally relevant neurons. We applied the iTango system to decipher the roles of two classes of dopaminergic neurons in the mouse nucleus accumbens in a sensitized locomotor response to cocaine. Thus, the iTango platform allows for control of neuromodulatory circuits in a genetically and functionally defined manner with spatial and temporal precision.
405.
Light-Regulated Protein Kinases Based on the CRY2-CIB1 System.
Abstract:
Optogenetic approaches enable the control of biological processes in a time- and space-resolved manner. These light-based methods are noninvasive and by using light as sole activator minimize side effects in contrast to chemical inducers. Here, we provide a protocol for the targeted control of the activity of protein kinases in mammalian cells based on the photoreceptor cryptochrome 2 (CRY2) of Arabidopsis thaliana and its interaction partner CIB1. Blue light (450 nm)-induced binding of CRY2 to CIB1 allows the recruitment of a chimeric cytosolic protein kinase AKT1 to the plasma membrane accompanied with stimulation of its kinase activity. This protocol comprises the transient and stable implementation of the light-regulated system into mammalian cells and its stimulation by blue light-emitting diodes (450 nm) irradiation as well as analysis of the light-activated AKT1.
406.
Optogenetic switches for light-controlled gene expression in yeast.
Abstract:
Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.
407.
Optogenetic control of cellular forces and mechanotransduction.
Abstract:
Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space.
408.
Epigenetic Editing of Ascl1 Gene in Neural Stem Cells by Optogenetics.
Abstract:
Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.
409.
Optogenetic Control of Synaptic Composition and Function.
Abstract:
The molecular composition of the postsynaptic membrane is sculpted by synaptic activity. During synaptic plasticity at excitatory synapses, numerous structural, signaling, and receptor molecules concentrate at the postsynaptic density (PSD) to regulate synaptic strength. We developed an approach that uses light to tune the abundance of specific molecules in the PSD. We used this approach to investigate the relationship between the number of AMPA-type glutamate receptors in the PSD and synaptic strength. Surprisingly, adding more AMPA receptors to excitatory contacts had little effect on synaptic strength. Instead, we observed increased excitatory input through the apparent addition of new functional sites. Our data support a model where adding AMPA receptors is sufficient to activate synapses that had few receptors to begin with, but that additional remodeling events are required to strengthen established synapses. More broadly, this approach introduces the precise spatiotemporal control of optogenetics to the molecular control of synaptic function.
410.
Drive the Car(go)s-New Modalities to Control Cargo Trafficking in Live Cells.
Abstract:
Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.
411.
Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.
Abstract:
Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.
412.
The STIM-Orai Pathway: Light-Operated Ca2+ Entry Through Engineered CRAC Channels.
Abstract:
Ca2+ signals regulate a plethora of cellular functions that include muscle contraction, heart beating, hormone secretion, lymphocyte activation, gene expression, and metabolism. To study the impact of Ca2+ signals on biological processes, pharmacological tools and caged compounds have been commonly applied to induce fluctuations of intracellular Ca2+ concentrations. These conventional approaches, nonetheless, lack rapid reversibility and high spatiotemporal resolution. To overcome these disadvantages, we and others have devised a series of photoactivatable genetically encoded Ca2+ actuators (GECAs) by installing light sensitivities into a bona fide highly selective Ca2+ channel, the Ca2+ release-activated Ca2+ (CRAC) channel. Store-operated CRAC channel serves as a major route for Ca2+ entry in many cell types. These GECAs enable remote and precise manipulation of Ca2+ signaling in both excitable and non-excitable cells. When combined with nanotechnology, it becomes feasible to wirelessly photo-modulate Ca2+-dependent activities in vivo. In this chapter, we briefly review most recent advances in engineering CRAC channels to achieve optical control over Ca2+ signaling, outline their design principles and kinetic features, and present exemplary applications of GECAs engineered from CRAC channels.
413.
A Photoactivatable Innate Immune Receptor for Optogenetic Inflammation.
Abstract:
Although spatial and temporal elements of immune activation mediate the intensity of the immune response, few tools exist to directly examine these effects. To elucidate the spatiotemporal aspects of innate immune responses, we designed an optogenetic pattern recognition receptor that activates in response to blue light. We demonstrate direct receptor activation, leading to spatial and temporal control of downstream signaling pathways in a variety of relevant cell types. We combined our platform with Bi-molecular Fluorescence Complementation (BiFC), resulting in selective fluorescent labeling of cells in which receptor activation has occurred.
414.
Plasma Membrane Association but Not Midzone Recruitment of RhoGEF ECT2 Is Essential for Cytokinesis.
Abstract:
Cytokinesis, the final step of cell division, begins with the formation of a cleavage furrow. How the mitotic spindle specifies the furrow at the equator in animal cells remains unknown. Current models propose that the concentration of the RhoGEF ECT2 at the spindle midzone and the equatorial plasma membrane directs furrow formation. Using chemical genetic and optogenetic tools, we demonstrate that the association of ECT2 with the plasma membrane during anaphase is required and sufficient for cytokinesis. Local membrane targeting of ECT2 leads to unilateral furrowing, highlighting the importance of local ECT2 activity. ECT2 mutations that prevent centralspindlin binding compromise concentration of ECT2 at the midzone and equatorial membrane but sustain cytokinesis. While the association of ECT2 with the plasma membrane is essential for cytokinesis, our data suggest that ECT2 recruitment to the spindle midzone is insufficient to account for equatorial furrowing and may act redundantly with yet-uncharacterized signals.
415.
Strategies for the photo-control of endogenous protein activity.
Abstract:
Photo-controlled or 'optogenetic' effectors interfacing with endogenous protein machinery allow the roles of endogenous proteins to be probed. There are two main approaches being used to develop optogenetic effectors: (i) caging strategies using photo-controlled conformational changes, and (ii) protein relocalization strategies using photo-controlled protein-protein interactions. Numerous specific examples of these approaches have been reported and efforts to develop general methods for photo-control of endogenous proteins are a current focus. The development of improved screening and selection methods for photo-switchable proteins would advance the field.
416.
Optogenetic inhibition of apical constriction during Drosophila embryonic development.
Abstract:
Morphogenesis of multicellular organisms is driven by changes in cell behavior, which happen at precise locations and defined developmental stages. Therefore, the studying of morphogenetic events would greatly benefit from tools that allow the perturbation of cell activity with spatial and temporal precision. We recently developed an optogenetic approach to modulate cell contractility with cellular precision and on fast (seconds) timescales during Drosophila embryogenesis. We present here a protocol to handle genetically engineered photosensitive Drosophila embryos and achieve light-mediated inhibition of apical constriction during tissue invagination. The possibility to modulate the levels of optogenetic activation at different laser powers makes this method suited also for studying how mechanical stresses are sensed and interpreted in vivo. Given the conserved function of cell contractility during animal development, the application of this method to other morphogenetic processes will facilitate our understanding of tissue mechanics and cell-cell interaction during morphogenesis.
417.
Optogenetics - Bringing light into the darkness of mammalian signal transduction.
Abstract:
Cells receive many different environmental clues to which they must adapt accordingly. Therefore, a complex signal transduction network has evolved. Cellular signal transduction is a highly dynamic process, in which the specific outcome is a result of the exact spatial and temporal resolution of single sub-events. While conventional techniques, like chemical inducer systems, have led to a sound understanding of the architecture of signal transduction pathways, the spatiotemporal aspects were often impossible to resolve. Optogenetics, based on genetically encoded light-responsive proteins, has the potential to revolutionize manipulation of signal transduction processes. Light can be easily applied with highest precision and minimal invasiveness. This review focuses on examples of optogenetic systems which were generated and applied to manipulate non-neuronal mammalian signaling processes at various stages of signal transduction, from cell membrane through cytoplasm to nucleus. Further, the future of optogenetic signaling will be discussed.
418.
An open-hardware platform for optogenetics and photobiology.
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Gerhardt, KP
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Olson, EJ
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Castillo-Hair, SM
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Hartsough, LA
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Landry, BP
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Ekness, F
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Yokoo, R
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Gomez, EJ
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Ramakrishnan, P
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Suh, J
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Savage, DF
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Tabor, JJ
Abstract:
In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.
419.
A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.
Abstract:
Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m(-2)) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.
420.
Optogenetic Control of Protein Function: From Intracellular Processes to Tissue Morphogenesis.
Abstract:
Optogenetics is an emerging and powerful technique that allows the control of protein activity with light. The possibility of inhibiting or stimulating protein activity with the spatial and temporal precision of a pulse of laser light is opening new frontiers for the investigation of developmental pathways and cell biological bases underlying organismal development. With this powerful technique in hand, it will be possible to address old and novel questions about how cells, tissues, and organisms form. In this review, we focus on the applications of existing optogenetic tools for addressing issues in animal morphogenesis.
421.
Reversible optogenetic control of kinase activity during differentiation and embryonic development.
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Krishnamurthy, VV
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Khamo, JS
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Mei, W
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Turgeon, AJ
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Ashraf, HM
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Mondal, P
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Patel, DB
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Risner, N
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Cho, EE
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Yang, J
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Zhang, K
Abstract:
A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.
422.
Targeting protein function: the expanding toolkit for conditional disruption.
Abstract:
A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.
423.
Following Optogenetic Dimerizers and Quantitative Prospects.
Abstract:
Optogenetics describes the use of genetically encoded photosensitive proteins to direct intended biological processes with light in recombinant and native systems. While most of these light-responsive proteins were originally discovered in photosynthetic organisms, the past few decades have been punctuated by experiments that not only commandeer but also engineer and enhance these natural tools to explore a wide variety of physiological questions. In addition, the ability to tune dynamic range and kinetic rates of optogenetic actuators is a challenging question that is heavily explored with computational methods devised to facilitate optimization of these systems. Here, we explain the basic mechanisms of a few popular photodimerizing optogenetic systems, discuss applications, compare optogenetic tools against more traditional chemical methods, and propose a simple quantitative understanding of how actuators exert their influence on targeted processes.
424.
Optogenetic regulation of site-specific subtelomeric DNA methylation.
Abstract:
Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest.
425.
Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.
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Yim, N
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Ryu, SW
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Choi, K
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Lee, KR
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Lee, S
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Choi, H
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Kim, J
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Shaker, MR
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Sun, W
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Park, JH
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Kim, D
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Heo, WD
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Choi, C
Abstract:
Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.
