Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 25 of 41 results

Membrane dynamics induced by a PIP3 optogenetic tool.

blue CRY2/CIB1 Cos-7 HEK293 NIH/3T3 Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Anal Sci, 2 Nov 2018 DOI: 10.2116/analsci.18sdp06 Link to full text
Abstract: Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a crucial lipid that regulates PIP3 dynamics. Investigations of how PIP3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information of membrane dynamics upon PIP3 production. In the present study, we applied an optogenetic approach using the CRY2–CIBN system. Using this system, we revealed that local PIP3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP3-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells.

blue AsLOV2 EL222 CHO-K1 Cos-7 HEK293 HEK293T HeLa isolated MEFs NIH/3T3 Cell death
Sci Rep, 9 Oct 2018 DOI: 10.1038/s41598-018-32929-7 Link to full text
Abstract: Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch (‘Blue-OFF’), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.

A green light-responsive system for the control of transgene expression in mammalian and plant cells.

green TtCBD A. thaliana leaf protoplasts Cos-7 HEK293 HeLa NIH/3T3
ACS Synth Biol, 10 Apr 2018 DOI: 10.1021/acssynbio.7b00450 Link to full text
Abstract: The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH6 and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene expression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.

Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells.

blue near-infrared AsLOV2 BphP1/Q-PAS1 Cos-7 HEK293 HeLa Neuro-2a rat cortical neurons SH-SY5Y U-2 OS Multichromatic
Chembiochem, 21 Feb 2018 DOI: 10.1002/cbic.201700642 Link to full text
Abstract: Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner QPAS1 is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested functionality of two BphP1-QPAS1-based optogenetic tools, such as an NIR and blue light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA), in several cell types including cortical neurons. We found that the performance of these optogenetic tools often rely on physiological properties of a specific cell type, such as nuclear transport, which may limit applicability of blue light-sensitive component of iRIS. In contrast, the NIR-light-sensing part of iRIS performed well in all tested cell types. The TA system showed the best performance in HeLa, U-2 OS and HEK-293 cells. Small size of the QPAS1 component allows designing AAV viral particles, which were applied to deliver the TA system to neurons.

Light-activated protein interaction with high spatial subcellular confinement.

blue CRY2/CIB1 iLID Magnets Cos-7 HeLa human primary dermal fibroblasts primary mouse cortical neurons primary mouse hippocampal neurons Benchmarking
Proc Natl Acad Sci USA, 20 Feb 2018 DOI: 10.1073/pnas.1713845115 Link to full text
Abstract: Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications.

Optogenetic activation of EphB2 receptor in dendrites induced actin polymerization by activating Arg kinase.

blue CRY2olig 3T3MEF Cos-7 HEK293 rat hippocampal neurons Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Biol Open, 20 Nov 2017 DOI: 10.1242/bio.029900 Link to full text
Abstract: Erythropoietin-producing hepatocellular (Eph) receptors regulate a wide array of developmental processes by responding to cell-cell contacts. EphB2 is well-expressed in brain and known to be important for dendritic spine development, as well as for the maintenance of the synapses, although the mechanisms of these functions have not been fully understood. Here we studied EphB2's functions in hippocampal neurons with an optogenetic approach, which allows us to specify spatial regions of signal activation and monitor in real-time the consequences of signal activation. We designed and constructed OptoEphB2, a genetically encoded photoactivatable EphB2. Photoactivation of OptoEphB2 in fibroblast cells induced receptor phosphorylation and resulted in cell rounding - a well-known cellular response to EphB2 activation. In contrast, local activation of OptoEphb2 in dendrites of hippocampal neurons induces rapid actin polymerization, resulting dynamic dendritic filopodial growth. Inhibition of Rac1 and CDC42 did not abolish OptoEphB2-induced actin polymerization. Instead, we identified Abelson Tyrosine-Protein Kinase 2 (Abl2/Arg) as a necessary effector in OptoEphB2-induced filopodia growth in dendrites. These findings provided new mechanistic insight into EphB2's role in neural development and demonstrated the advantage of OptoEphB as a new tool for studying EphB signaling.

Gradients of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling.

blue CRY2/CIB1 Cos-7
Cell Rep, 14 Nov 2017 DOI: 10.1016/j.celrep.2017.10.069 Link to full text
Abstract: Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.

Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions.

blue iLID Cos-7 Organelle manipulation
Nat Mater, 6 Nov 2017 DOI: 10.1038/nmat5006 Link to full text
Abstract: Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.

Understanding CRY2 interactions for optical control of intracellular signaling.

blue CRY2/CIB1 CRY2/CRY2 CRY2olig Cos-7 HEK293T Signaling cascade control
Nat Commun, 15 Sep 2017 DOI: 10.1038/s41467-017-00648-8 Link to full text
Abstract: Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2-CRY2 homo-oligomerization and CRY2-CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2-CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2-CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2-CIB1 and CRY2-CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2-CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems.Cryptochrome 2 (CRY2) can form light-regulated CRY2-CRY2 homo-oligomers or CRY2-CIB1 hetero-dimers, but modulating these interactions is difficult owing to the lack of interaction mechanism. Here the authors identify the interactions facilitating homo-oligomers and introduce mutations to create low and high oligomerization versions.

Optogenetic activation of Plexin-B1 reveals contact repulsion between osteoclasts and osteoblasts.

blue CRY2/CIB1 CRY2/CRY2 Cos-7 MC3T3-E1 primary mouse calvarial osteoblasts Control of cytoskeleton / cell motility / cell shape
Nat Commun, 21 Jun 2017 DOI: 10.1038/ncomms15831 Link to full text
Abstract: During bone remodelling, osteoclasts induce chemotaxis of osteoblasts and yet maintain spatial segregation. We show that osteoclasts express the repulsive guidance factor Semaphorin 4D and induce contact inhibition of locomotion (CIL) in osteoblasts through its receptor Plexin-B1. To examine causality and elucidate how localized Plexin-B1 stimulation may spatiotemporally coordinate its downstream targets in guiding cell migration, we develop an optogenetic tool for Plexin-B1 designated optoPlexin. Precise optoPlexin activation at the leading edge of migrating osteoblasts readily induces local retraction and, unexpectedly, distal protrusions to steer cells away. These morphological changes are accompanied by reorganization of Myosin II, PIP3, adhesion and active Cdc42. We attribute the resultant repolarization to RhoA/ROCK-mediated redistribution of β-Pix, which activates Cdc42 and promotes protrusion. Thus, our data demonstrate a causal role of Plexin-B1 for CIL in osteoblasts and reveals a previously unknown effect of Semaphorin signalling on spatial distribution of an activator of cell migration.

Optical control of membrane tethering and interorganellar communication at nanoscales.

blue AsLOV2 Cos-7 HeLa in vitro Organelle manipulation
Chem Sci, 31 May 2017 DOI: 10.1039/c7sc01115f Link to full text
Abstract: 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.

A simple optogenetic MAPK inhibitor design reveals resonance between transcription-regulating circuitry and temporally-encoded inputs.

blue AsLOV2 Cos-7 HEK293T in vitro rat cerebellar granule neurons Signaling cascade control
Nat Commun, 12 May 2017 DOI: 10.1038/ncomms15017 Link to full text
Abstract: 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.

A Phytochrome-Derived Photoswitch for Intracellular Transport.

blue red PhyB/PIF6 TULIP Cos-7 U-2 OS Organelle manipulation Multichromatic
ACS Synth Biol, 30 Mar 2017 DOI: 10.1021/acssynbio.6b00333 Link to full text
Abstract: Cells depend on the proper positioning of their organelles, suggesting that active manipulation of organelle positions can be used to explore spatial cell biology and to restore cellular defects caused by organelle misplacement. Recently, blue-light dependent recruitment of specific motors to selected organelles has been shown to alter organelle motility and positioning, but these approaches lack rapid and active reversibility. The light-dependent interaction of phytochrome B with its interacting factors has been shown to function as a photoswitch, dimerizing under red light and dissociating under far-red light. Here we engineer phytochrome domains into photoswitches for intracellular transport that enable the reversible interaction between organelles and motor proteins. Using patterned illumination and live-cell imaging, we demonstrate that this system provides unprecedented spatiotemporal control. We also demonstrate that it can be used in combination with a blue-light dependent system to independently control the positioning of two different organelles. Precise optogenetic control of organelle motility and positioning will provide a better understanding of and control over the spatial biology of cells.

Optogenetic control of the Dab1 signaling pathway.

blue CRY2olig Cos-7 HEK293 NIH/3T3 primary mouse cortical neurons Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Sci Rep, 8 Mar 2017 DOI: 10.1038/srep43760 Link to full text
Abstract: The Reelin-Dab1 signaling pathway regulates development of the mammalian brain, including neuron migrations in various brain regions, as well as learning and memory in adults. Extracellular Reelin binds to cell surface receptors and activates phosphorylation of the intracellular Dab1 protein. Dab1 is required for most effects of Reelin, but Dab1-independent pathways may contribute. Here we developed a single-component, photoactivatable Dab1 (opto-Dab1) by using the blue light-sensitive dimerization/oligomerization property of A. thaliana Cryptochrome 2 (Cry2). Opto-Dab1 can activate downstream signals rapidly, locally, and reversibly upon blue light illumination. The high spatiotemporal resolution of the opto-Dab1 probe also allows us to control membrane protrusion, retraction and ruffling by local illumination in both COS7 cells and in primary neurons. This shows that Dab1 activation is sufficient to orient cell movement in the absence of other signals. Opto-Dab1 may be useful to study the biological functions of the Reelin-Dab1 signaling pathway both in vitro and in vivo.

Assembly Domain-Based Optogenetic System for the Efficient Control of Cellular Signaling.

blue Magnets Cos-7 HEK293T Control of cytoskeleton / cell motility / cell shape
ACS Synth Biol, 3 Mar 2017 DOI: 10.1021/acssynbio.7b00022 Link to full text
Abstract: We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein-protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca(2+)/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation.

LOVTRAP: A Versatile Method to Control Protein Function with Light.

blue LOVTRAP Cos-7 HEK293 HeLa
Curr Protoc Cell Biol, 1 Dec 2016 DOI: 10.1002/cpcb.12 Link to full text
Abstract: We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered. Light-induced protein release occurs in less than a second, and the half-life of return can be adjusted using LOV point mutations (∼2 to 500 sec). © 2016 by John Wiley & Sons, Inc.

Optical manipulation of the alpha subunits of heterotrimeric G proteins using photoswitchable dimerization systems.

blue red Magnets PhyB/PIF6 Cos-7 HEK293 HeLa Immediate control of second messengers
Sci Rep, 21 Oct 2016 DOI: 10.1038/srep35777 Link to full text
Abstract: Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca(2+) and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.

A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.

blue CRY2/CIB1 Magnets CHO-K1 Cos-7 HEK293 HeLa mouse in vivo NIH/3T3
Nat Chem Biol, 10 Oct 2016 DOI: 10.1038/nchembio.2205 Link to full text
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.

Light-induced nuclear export reveals rapid dynamics of epigenetic modifications.

blue AsLOV2 iLID C. elegans in vivo Cos-7 HeLa mouse IA32 fibroblasts S. cerevisiae Epigenetic modification
Nat Chem Biol, 18 Apr 2016 DOI: 10.1038/nchembio.2068 Link to full text
Abstract: We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.

Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking.

blue CRY2/CIB1 Cos-7 HeLa NIH/3T3 PC-12 rat hippocampal neurons Control of cytoskeleton / cell motility / cell shape Control of vesicular transport
Nat Chem Biol, 11 Apr 2016 DOI: 10.1038/nchembio.2064 Link to full text
Abstract: Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems.

Optical Control of Peroxisomal Trafficking.

blue AsLOV2 Cos-7 HEK293T HeLa S. cerevisiae
ACS Synth Biol, 2 Nov 2015 DOI: 10.1021/acssynbio.5b00144 Link to full text
Abstract: The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times.

Optogenetic control of endogenous Ca(2+) channels in vivo.

blue AsLOV2 CRY2/CRY2 Cos-7 HEK293 HeLa hESCs HUVEC mouse astrocytes mouse hippocampal slices mouse in vivo NIH/3T3 primary mouse hippocampal neurons zebrafish in vivo Immediate control of second messengers
Nat Biotechnol, 14 Sep 2015 DOI: 10.1038/nbt.3350 Link to full text
Abstract: Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.

Optogenetic apoptosis: light-triggered cell death.

blue CRY2/CIB1 Cos-7 HeLa MTLn3 Cell death
Angew Chem Int Ed Engl, 25 Aug 2015 DOI: 10.1002/anie.201506346 Link to full text
Abstract: An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

A light-switchable bidirectional expression module allowing simultaneous regulation of multiple genes.

blue VVD Cos-7 HEK293 mouse in vivo NCI-H1299 U-87 MG Transgene expression
Biochem Biophys Res Commun, 21 Aug 2015 DOI: 10.1016/j.bbrc.2015.08.085 Link to full text
Abstract: Several light-regulated genetic circuits have been applied to spatiotemporally control transgene expression in mammalian cells. However, simultaneous regulation of multiple genes using one genetic device by light has not yet been reported. In this study, we engineered a bidirectional expression module based on LightOn system. Our data showed that both reporter genes could be regulated at defined and quantitative levels. Simultaneous regulation of four genes was further achieved in cultured cells and mice. Additionally, we successfully utilized the bidirectional expression module to monitor the expression of a suicide gene, showing potential for photodynamic gene therapy. Collectively, we provide a robust and useful tool to simultaneously control multiple genes expression by light, which will be widely used in biomedical research and biotechnology.

Control of Protein Activity and Cell Fate Specification via Light-Mediated Nuclear Translocation.

blue AsLOV2 C. elegans in vivo Cos-7 HEK293 HeLa S. cerevisiae Developmental processes
PLoS ONE, 17 Jun 2015 DOI: 10.1371/journal.pone.0128443 Link to full text
Abstract: Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.
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