Showing 101 - 125 of 146 results
Labelling and optical erasure of synaptic memory traces in the motor cortex.
Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), that can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning task induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble.
Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.
Light generation of intracellular Ca(2+) signals by a genetically encoded protein BACCS.
Ca(2+) signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca(2+) channel switch (BACCS), as an optogenetic tool for generating Ca(2+) signals. BACCS opens Ca(2+)-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca(2+) concentration more rapidly, such that Ca(2+) elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca(2+). Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca(2+) signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.
Photoactivatable CRISPR-Cas9 for optogenetic genome editing.
We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.
Regulation of endogenous transmembrane receptors through optogenetic Cry2 clustering.
Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artefacts. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signalling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.
Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins.
Optogenetic methods take advantage of photoswitches to control the activity of cellular proteins. Here, we completed a multi-directional engineering of the fungal photoreceptor Vivid to develop pairs of distinct photoswitches named Magnets. These new photoswitches were engineered to recognize each other based on the electrostatic interactions, thus preventing homodimerization and enhancing light-induced heterodimerization. Furthermore, we tuned the switch-off kinetics by four orders of magnitude and developed several variants, including those with substantially faster kinetics than any of the other conventional dimerization-based blue spectrum photoswitches. We demonstrate the utility of Magnets as powerful tools that can optogenetically manipulate molecular processes in biological systems.
A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.
Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.
Optogenetic control of organelle transport and positioning.
Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.
Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant.
Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.
An optimized optogenetic clustering tool for probing protein interaction and function.
The Arabidopsis photoreceptor cryptochrome 2 (CRY2) was previously used as an optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we report the development of a new CRY2-derived optogenetic module, 'CRY2olig', which induces rapid, robust, and reversible protein oligomerization in response to light. Using this module, we developed a novel protein interaction assay, Light-Induced Co-clustering, that can be used to interrogate protein interaction dynamics in live cells. In addition to use probing protein interactions, CRY2olig can also be used to induce and reversibly control diverse cellular processes with spatial and temporal resolution. Here we demonstrate disrupting clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with light. These new CRY2-based approaches expand the growing arsenal of optogenetic strategies to probe cellular function.
Photochemistry of flavoprotein light sensors.
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.
Remote control of myosin and kinesin motors using light-activated gearshifting.
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.
Illuminating cell signalling with optogenetic tools.
The light-based control of ion channels has been transformative for the neurosciences, but the optogenetic toolkit does not stop there. An expanding number of proteins and cellular functions have been shown to be controlled by light, and the practical considerations in deciding between reversible optogenetic systems (such as systems that use light-oxygen-voltage domains, phytochrome proteins, cryptochrome proteins and the fluorescent protein Dronpa) are well defined. The field is moving beyond proof of concept to answering real biological questions, such as how cell signalling is regulated in space and time, that were difficult or impossible to address with previous tools.
Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.
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.
Optogenetic characterization methods overcome key challenges in synthetic and systems biology.
Systems biologists aim to understand how organism-level processes, such as differentiation and multicellular development, are encoded in DNA. Conversely, synthetic biologists aim to program systems-level biological processes, such as engineered tissue growth, by writing artificial DNA sequences. To achieve their goals, these groups have adapted a hierarchical electrical engineering framework that can be applied in the forward direction to design complex biological systems or in the reverse direction to analyze evolved networks. Despite much progress, this framework has been limited by an inability to directly and dynamically characterize biological components in the varied contexts of living cells. Recently, two optogenetic methods for programming custom gene expression and protein localization signals have been developed and used to reveal fundamentally new information about biological components that respond to those signals. This basic dynamic characterization approach will be a major enabling technology in synthetic and systems biology.
How to control proteins with light in living systems.
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.
Light-inducible receptor tyrosine kinases that regulate neurotrophin signalling.
Receptor tyrosine kinases (RTKs) are a family of cell-surface receptors that have a key role in regulating critical cellular processes. Here, to understand and precisely control RTK signalling, we report the development of a genetically encoded, photoactivatable Trk (tropomyosin-related kinase) family of RTKs using a light-responsive module based on Arabidopsis thaliana cryptochrome 2. Blue-light stimulation (488 nm) of mammalian cells harbouring these receptors robustly upregulates canonical Trk signalling. A single light stimulus triggers transient signalling activation, which is reversibly tuned by repetitive delivery of blue-light pulses. In addition, the light-provoked process is induced in a spatially restricted and cell-specific manner. A prolonged patterned illumination causes sustained activation of extracellular signal-regulated kinase and promotes neurite outgrowth in a neuronal cell line, and induces filopodia formation in rat hippocampal neurons. These light-controllable receptors are expected to create experimental opportunities to spatiotemporally manipulate many biological processes both in vitro and in vivo.
Reversible protein inactivation by optogenetic trapping in cells.
We present a versatile platform to inactivate proteins in living cells using light, light-activated reversible inhibition by assembled trap (LARIAT), which sequesters target proteins into complexes formed by multimeric proteins and a blue light-mediated heterodimerization module. Using LARIAT, we inhibited diverse proteins that modulate cytoskeleton, lipid signaling and cell cycle with high spatiotemporal resolution. Use of single-domain antibodies extends the method to target proteins containing specific epitopes, including GFP.
Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals.
Gene circuits are dynamical systems that regulate cellular behaviors, often using protein signals as inputs and outputs. Here we have developed an optogenetic 'function generator' method for programming tailor-made gene expression signals in live bacterial cells. We designed precomputed light sequences based on experimentally calibrated mathematical models of light-switchable two-component systems and used them to drive intracellular protein levels to match user-defined reference time courses. We used this approach to generate accelerated and linearized dynamics, sinusoidal oscillations with desired amplitudes and periods, and a complex waveform, all with unprecedented accuracy and precision. We also combined the function generator with a dual fluorescent protein reporter system, analogous to a dual-channel oscilloscope, to reveal that a synthetic repressible promoter linearly transforms repressor signals with an approximate 7-min delay. Our approach will enable a new generation of dynamical analyses of synthetic and natural gene circuits, providing an essential step toward the predictive design and rigorous understanding of biological systems.
Control of gene expression using a red- and far-red light-responsive bi-stable toggle switch.
Light-triggered gene expression systems offer an unprecedented spatiotemporal resolution that cannot be achieved with classical chemically inducible genetic tools. Here we describe a protocol for red light-responsive gene expression in mammalian cells. This system can be toggled between stable ON and OFF states by short pulses of red and far-red light, respectively. In the protocol, CHO-K1 cells are transfected to allow red light-inducible expression of the secreted alkaline phosphatase (SEAP) reporter, and gene expression is tuned by illumination with light of increasing wavelengths. As a starting point for elaborate red light-responsive gene expression, we outline the reversible activation of gene expression and describe how a spatial pattern can be 'printed' on a monolayer of cells by using a photomask. The core protocol requires only 4 d from seeding of the cells to reporter quantification, and other than light-emitting diode (LED) illumination boxes no elaborate equipment is required.
An optogenetic gene expression system with rapid activation and deactivation kinetics.
Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.
A fully genetically encoded protein architecture for optical control of peptide ligand concentration.
Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K(+) channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.
Optical control of mammalian endogenous transcription and epigenetic states.
The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.
Techniques: Optogenetics takes more control.
Abstract not available.
Optogenetic protein clustering and signaling activation in mammalian cells.
We report an optogenetic method based on Arabidopsis thaliana cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the β-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.