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 548 results
1.

OpEn-Tag: a customizable optogenetic toolbox to dissect subcellular signaling.

blue CRY2/CIB1 HeLa Signaling cascade control
ACS Synth Biol, 11 Jun 2019 DOI: 10.1021/acssynbio.9b00059 Link to full text
Abstract: Subcellular localization of signal molecules is a hallmark in organizing the signal network. OpEn-Tag is a modular optogenetic endomembrane targeting toolbox that allows alteration of the localization and therefore the activity of signaling processes with the spatio-temporal resolution of optogenetics. OpEn-Tag is a two-component system employing 1) a variety of targeting peptides, fused to and thereby dictating the localization of mCherry-labeled cryptochrome 2 binding protein CIBN towards distinct endomembranes and 2) the cytosolic, fluorescence-labeled blue light photoreceptor cryptochrome 2 as customizable building block that can be fused to proteins of interest. The combination of OpEn-Tag with growth factor stimulation or the use of two membrane anchor sequences allows investigation of multilayered signal transduction processes as demonstrated here for the protein kinase AKT.
2.

Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.

blue Fluorescent proteins Background
Biochemistry, 30 May 2019 DOI: 10.1021/acs.biochem.9b00279 Link to full text
Abstract: We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
3.

Photocontrollable mononegaviruses.

blue Magnets BHK-21 mouse in vivo Vero/hSLAM Nucleic acid editing
Proc Natl Acad Sci USA, 28 May 2019 DOI: 10.1073/pnas.1906531116 Link to full text
Abstract: Mononegaviruses are promising tools as oncolytic vectors and transgene delivery vectors for gene therapy and regenerative medicine. By using the Magnet proteins, which reversibly heterodimerize upon blue light illumination, photocontrollable mononegaviruses (measles and rabies viruses) were generated. The Magnet proteins were inserted into the flexible domain of viral polymerase, and viruses showed strong replication and oncolytic activities only when the viral polymerases were activated by blue light illumination.
4.

A molecular toolbox for interrogation of membrane contact sites.

blue LOV domains Review
J Physiol (Lond), 23 May 2019 DOI: 10.1113/jp277761 Link to full text
Abstract: Membrane contact sites (MCSs) are specialized subcellular compartments formed by closely apposed membranes from two organelles. The intermembrane gap is separated by a distance ranging from 10 nm to 35 nm. MCSs are typically maintained through dynamic protein-protein and protein-lipid interactions. These inter-membrane contact sites constitute important intracellular signaling hotspots to mediate a plethora of cellular processes, including calcium homeostasis, lipid metabolism, membrane biogenesis and organelle remodeling. In recent years, a series of genetically-encoded probes and chemogenetic or optogenetic actuators have been invented to aid the visualization and interrogation of MCSs in both fixed and living cells. These molecular tools have greatly accelerated the pace of mechanistic dissection of membrane contact sites at the molecular level. In this review, we present an overview on the latest progress in this endeavor, and provide a general guide to the selection of methods and molecular tools for probing interorganellar membrane contact sites. This article is protected by copyright. All rights reserved.
5.

Independent Blue and Red Light Triggered Narcissistic Self-Sorting Self-Assembly of Colloidal Particles.

blue red Cph1 VVD in vitro Multichromatic
Small, 21 May 2019 DOI: 10.1002/smll.201901801 Link to full text
Abstract: The ability of living systems to self-sort different cells into separate assemblies and the ability to independently regulate different structures are one ingredient that gives rise to their spatiotemporal complexity. Here, this self-sorting behavior is replicated in a synthetic system with two types of colloidal particles; where each particle type independently self-assembles either under blue or red light into distinct clusters, known as narcissistic self-sorting. For this purpose, each particle type is functionalized either with the light-switchable protein VVDHigh or Cph1, which homodimerize under blue and red light, respectively. The response to different wavelengths of light and the high specificity of the protein interactions allows for the independent self-assembly of each particle type with blue or red light and narcissistic self-sorting. Moreover, as both of the photoswitchable protein interactions are reversible in the dark; also, the self-sorting is reversible and dynamic. Overall, the independent blue and red light controlled self-sorting in a synthetic system opens new possibilities to assemble adaptable, smart, and advanced materials similar to the complexity observed in tissues.
6.

Light-based control of metabolic flux through assembly of synthetic organelles.

blue CRY2/CRY2 CRY2olig PixD/PixE S. cerevisiae Organelle manipulation
Nat Chem Biol, 13 May 2019 DOI: 10.1038/s41589-019-0284-8 Link to full text
Abstract: To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
7.

Engineering proteins for allosteric control by light or ligands.

blue AsLOV2 HEK293T HeLa
Nat Protoc, 10 May 2019 DOI: 10.1038/s41596-019-0165-3 Link to full text
Abstract: Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.
8.

NF-κB signaling dynamics is controlled by a dose-sensing autoregulatory loop.

blue CRY2olig NIH/3T3 Signaling cascade control
Sci Signal, 30 Apr 2019 DOI: 10.1126/scisignal.aau3568 Link to full text
Abstract: Over the last decade, multiple studies have shown that signaling proteins activated in different temporal patterns, such as oscillatory, transient, and sustained, can result in distinct gene expression patterns or cell fates. However, the molecular events that ensure appropriate stimulus- and dose-dependent dynamics are not often understood and are difficult to investigate. Here, we used single-cell analysis to dissect the mechanisms underlying the stimulus- and dose-encoding patterns in the innate immune signaling network. We found that Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling dynamics relied on a dose-dependent, autoinhibitory loop that rendered cells refractory to further stimulation. Using inducible gene expression and optogenetics to perturb the network at different levels, we identified IL-1R-associated kinase 1 (IRAK1) as the dose-sensing node responsible for limiting signal flow during the innate immune response. Although the kinase activity of IRAK1 was not required for signal propagation, it played a critical role in inhibiting the nucleocytoplasmic oscillations of the transcription factor NF-κB. Thus, protein activities that may be "dispensable" from a topological perspective can nevertheless be essential in shaping the dynamic response to the external environment.
9.

RhoA mediates epithelial cell shape changes via mechanosensitive endocytosis.

blue TULIP Caco-2 Signaling cascade control Control of cytoskeleton / cell motility / cell shape
bioRxiv, 10 Apr 2019 DOI: 10.1101/605485 Link to full text
Abstract: Morphogenetic movements require tight spatiotemporal control over cell-cell junction lengths. Contractile forces, acting at adherens junctions, alter cell-cell contact lengths in a cyclic fashion as a mechanical ratchet. Pulsatile RhoA activity is thought to drive ratcheting through acute periods of junction contraction followed by stabilization. Currently, we lack a mechanistic under-standing of if and how RhoA activity governs junction length and subsequent cell shape within epi-thelia. In this study we use optogenetics to exogenously control RhoA activity in model Caco-2 epi-thelium. We find that at short timescales, RhoA activation drives reversible junction contraction. Sus-tained RhoA activity drives irreversible junction shortening but the amount of shortening saturates for a single pulse. To capture these data, we develop a vertex model modified to include strain-dependent junction length and tension remodeling. We find that, to account for experimental data, tension remodeling requires a strain-dependent threshold. Our model predicts that temporal structur-ing of RhoA activity allows for subsequent tension remodeling events to overcome the limited short-ening within a single pulse and this is confirmed by our experimental data. We find that RhoA-mediated junction remodeling requires activities of formin and dynamin, indicating the closely inter-connected activities of contractility, E-cadherin clustering, and endocytosis. Junction length is there-fore regulated by the coordinated action of RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling. Altogether these data provide insights into the underlying molecular and biophysical mechanisms of RhoA-mediated regulation of epithelial cell shape.
10.

Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos.

blue CRY2/CIB1 D. melanogaster in vivo Control of cytoskeleton / cell motility / cell shape Organelle manipulation
Cell, 9 Apr 2019 DOI: 10.1016/j.cell.2019.03.007 Link to full text
Abstract: The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.
11.

Optogenetic downregulation of protein levels with an ultrasensitive switch.

blue AsLOV2 AtLOV2 iLID LOVTRAP S. cerevisiae Cell cycle control Transgene expression
ACS Synth Biol, 8 Apr 2019 DOI: 10.1021/acssynbio.8b00471 Link to full text
Abstract: Optogenetic control of protein activity is a versatile technique to gain control over cellular processes, e.g. for biomedical and biotechnological applications. Among other techniques, the regulation of protein abundance by controlling either transcription or protein stability found common use as this controls the activity of any type of target protein. Here, we report modules of an improved variant of the photosensitive degron module and a light-sensitive transcription factor, which we compared to doxycycline-dependent transcriptional control. Given their modularity the combined control of synthesis and stability of a given target protein resulted in the synergistic down regulation of its abundance by light. This combined module exhibits very high switching ratios, profound downregulation of protein abundance at low light-fluxes as well as fast protein depletion kinetics. Overall, this synergistic optogenetic multistep control (SOMCo) module is easy to implement and results in a regulation of protein abundance superior to each individual component.
12.

Noise-Reducing Negative-Feedback Optogenetic Circuits in Mammalian Cells.

blue VVD Flp-In-T-REx293 Signaling cascade control
bioRxiv, 7 Apr 2019 DOI: 10.1101/601005 Link to full text
Abstract: Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noisein cells. Optogenetic systems have recently been employed for controlling gene expression levelsin mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback. Here, we engineer optogenetic negative-feedback gene circuits inmammalian cellsto achievenoise-reduction forprecisegene expression control. We builda toolset of these noise-reducingLight-Inducible Tuner (LITer)gene circuits usingtheTetR repressor fused witha Tet-Inhibitory peptide (TIP)oradegradation tagthrough the light-sensitive LOV2 protein domain. These LITers provide nearly a range of 4-fold gene expression control and up to five-fold noise reductionfrom existing optogenetic systems. Moreover, we usethe LITergene circuit architecture to controlgene expression of the cancer oncogene KRAS(G12V)and studyits downstream effectsthrough phospho-ERK levels and cellular proliferation.Overall, these novel LITer optogenetic platformsshould enable precisespatiotemporal perturbations for studyingmulticellular phenotypes in developmental biology, oncology,and other biomedical fields of research.
13.

Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling.

blue LOVTRAP Jurkat Signaling cascade control
Elife, 5 Apr 2019 DOI: 10.7554/elife.42498 Link to full text
Abstract: T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the mechanical stability of the receptor-ligand interaction. Here we develop an optogenetic approach to specifically tune the binding half-life of a chimeric antigen receptor without changing other binding parameters and provide direct evidence of kinetic proofreading in T cell signaling. This half-life discrimination is executed in the proximal signaling pathway, downstream of ZAP70 recruitment and upstream of diacylglycerol accumulation. Our methods represent a general tool for temporal and spatial control of T cell signaling and extend the reach of optogenetics to probe pathways where the individual molecular kinetics, rather than the ensemble average, gates downstream signaling.
14.

Optical induction of autophagy via Transcription factor EB (TFEB) reduces pathological tau in neurons.

blue EL222 HEK293T human IPSCs Neuro-2a Transgene expression
bioRxiv, 5 Apr 2019 DOI: 10.1101/600908 Link to full text
Abstract: Aggregation and accumulation of microtubule associated protein tau in neurons is major neuropathological hallmark of Alzheimer’s disease (AD) and related tauopathies. Attempts have been made to promote clearance of pathological tau (p-Tau) from neurons via autophagy. Over expression of transcription factor EB (TFEB), has shown to clear pTau from neurons via autophagy. However, sustained TFEB activation and/or autophagy can create burden on cellular bioenergetics and can be deleterious. Thus, we engineered a minimally invasive optical system that could transiently alter autophagic flux. We optimized and testedan optogenetic gene expression system derived from apreviouslyengineered bacterial transcription factor, EL222. For the first time, our group utilized this system not only to spatial-temporally control nuclear TFEB expression, we also show light-induced TFEB has the capacity to reduce p-Tau burden in AD patient-derived human iPSC-neurons. Together, these results suggest that optically-regulatable gene expression of TFEB unlocks opto-therapeutics to treat AD and other dementias.
15.

Reversible induction of mitophagy by an optogenetic bimodular system.

blue iLID ETNA HEK293T HeLa human T cells zebrafish in vivo Organelle manipulation
Nat Commun, 4 Apr 2019 DOI: 10.1038/s41467-019-09487-1 Link to full text
Abstract: Autophagy-mediated degradation of mitochondria (mitophagy) is a key process in cellular quality control. Although mitophagy impairment is involved in several patho-physiological conditions, valuable methods to induce mitophagy with low toxicity in vivo are still lacking. Herein, we describe a new optogenetic tool to stimulate mitophagy, based on light-dependent recruitment of pro-autophagy protein AMBRA1 to mitochondrial surface. Upon illumination, AMBRA1-RFP-sspB is efficiently relocated from the cytosol to mitochondria, where it reversibly mediates mito-aggresome formation and reduction of mitochondrial mass. Finally, as a proof of concept of the biomedical relevance of this method, we induced mitophagy in an in vitro model of neurotoxicity, fully preventing cell death, as well as in human T lymphocytes and in zebrafish in vivo. Given the unique features of this tool, we think it may turn out to be very useful for a wide range of both therapeutic and research applications.
16.

Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions.

blue LOVTRAP HEK293T
Elife, 3 Apr 2019 DOI: 10.7554/elife.43826 Link to full text
Abstract: Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested 'AND' gate design of SPARK2-in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest-yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.
17.

Use of Exogenous and Endogenous Photomediators as Efficient ROS Modulation Tools: Results and Perspectives for Therapeutic Purposes.

blue LOV domains Review
Oxid Med Cell Longev, 31 Mar 2019 DOI: 10.1155/2019/2867516 Link to full text
Abstract: Reactive Oxygen Species (ROS) play an essential dual role in living systems. Healthy levels of ROS modulate several signaling pathways, but at the same time, when they exceed normal physiological amounts, they work in the opposite direction, playing pivotal functions in the pathophysiology of multiple severe medical conditions (i.e., cancer, diabetes, neurodegenerative and cardiovascular diseases, and aging). Therefore, the research for methods to detect their levels via light-sensitive fluorescent probes has been extensively studied over the years. However, this is not the only link between light and ROS. In fact, the modulation of ROS mediated by light has been exploited already for a long time. In this review, we report the state of the art, as well as recent developments, in the field of photostimulation of oxidative stress, from photobiomodulation (PBM) mediated by naturally expressed light-sensitive proteins to the most recent optogenetic approaches, and finally, we describe the main methods of exogenous stimulation, in particular highlighting the new insights based on optically driven ROS modulation mediated by polymeric materials.
18.

Cortical mitochondria regulate insulin secretion by local Ca2+ buffering.

blue CRY2/CIB1 MIN6 Control of cytoskeleton / cell motility / cell shape Organelle manipulation
J Cell Sci, 29 Mar 2019 DOI: 10.1242/jcs.228544 Link to full text
Abstract: Mitochondria play an essential role in regulating insulin secretion from beta cells by providing ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine if the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria 2-fold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely important for the generation of Ca2+ microdomains required for efficient insulin granule release.
19.

Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth.

blue AsLOV2 A. thaliana in vivo
Science, 29 Mar 2019 DOI: 10.1126/science.aaw0046 Link to full text
Abstract: Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K+ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K+ conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation.
20.

Direct observation and analysis of the dynamics of the photoresponsive transcription factor GAL4.

blue VVD in vitro
Angew Chem Int Ed Engl, 25 Mar 2019 DOI: 10.1002/anie.201900610 Link to full text
Abstract: We report direct visualization of the dynamic behavior of transcription factor GAL4 with photo-switching function (GAL4-VVD) in the DNA origami structure. Using high-speed atomic force microscopy (HS-AFM), we observed photo-induced complex formation of GAL4-VVD and substrate DNAs. Dynamic behaviors of GAL4-VVD such as binding, sliding, stalling, and dissociation with two substrate DNA strands, containing specific GAL4 binding sites, were observed. We also observed inter-strand hopping on two double-stranded (ds) DNAs. On a long substrate DNA strand that contained five binding sites, a series of GAL4-VVD/DNA interactions including binding, sliding, stalling, and dissociation could be identified while interacting with the surface. We also found the clear difference in the movement of GAL4-VVD between sliding and stalling in the AFM images. Detailed analysis revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping for rapidly searching specific binding sites, and then stalled to the specific sites for the stable complex formation. The results suggest the existence of the different conformational mode of the protein for sliding and stalling. This single-molecule imaging system at the nanoscale resolution provides the insight of the searching mechanism of the DNA binding proteins.
21.

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions.

blue BcLOV4 in vitro
Meth Enzymol, 23 Mar 2019 DOI: 10.1016/bs.mie.2019.02.015 Link to full text
Abstract: The ability to rapidly screen interactions between proteins and membrane-like interfaces would aid in establishing the structure-function of protein-lipid interactions, provide a platform for engineering lipid-interacting protein tools, and potentially inform the signaling mechanisms and dynamics of membrane-associated proteins. Here, we describe the preparation and application of water-in-oil (w/o) emulsions with lipid-stabilized droplet interfaces that emulate the plasma membrane inner leaflet with tunable composition. Fluorescently labeled proteins are easily visualized in these synthetic cell-like droplets on an automated inverted fluorescence microscope, thus allowing for both rapid screening of relative binding and spatiotemporally resolved analyses of for example, protein-interface association and dissociation dynamics and competitive interactions, using commonplace instrumentation. We provide protocols for droplet formation, automated imaging assays and analysis, and the production of the positive control protein BcLOV4, a natural photoreceptor with a directly light-regulated interaction with anionic membrane phospholipids that is useful for optogenetic membrane recruitment.
22.

A yeast system for discovering optogenetic inhibitors of eukaryotic translation initiation.

blue cyan AsLOV2 Dronpa145K/N PYP RsLOV S. cerevisiae
ACS Synth Biol, 22 Mar 2019 DOI: 10.1021/acssynbio.8b00386 Link to full text
Abstract: The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis non-invasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, LOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photo-activated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate of human eIF4E-depednednt translation initiation in a mechanistically defined manner.
23.

Advances in optogenetic regulation of gene expression in mammalian cells using cryptochrome 2 (CRY2).

blue CRY2/CIB1 HEK293T
Methods, 21 Mar 2019 DOI: 10.1016/j.ymeth.2019.03.011 Link to full text
Abstract: Synthetic regulation of gene expression provides a powerful approach to reprogram molecular and cellular processes and test the function of specific genes and gene products. In the last decade, optogenetic systems that allow light-dependent gene regulation have become valuable tools, providing tight spatiotemporal control of protein levels. Here we discuss and build on recent optogenetic approaches for regulating gene expression in mammalian cells using cryptochrome 2 (CRY2), a photoreceptor protein from Arabidopsis. We provide detailed protocols for using light to manipulate activity of a CRY2-based engineered photoactivatable Cre DNA recombinase, and to induce or disrupt transcription factor function. In addition, we provide instructions and software for building an inexpensive Rasberry-Pi-based programable LED device for optogenetic experiments, delivering pulsed light with customized control of illumination duration, frequency, and intensity.
24.

Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology.

blue CRY2/CRY2 human IPSCs U-2 OS Organelle manipulation
Elife, 20 Mar 2019 DOI: 10.7554/elife.39578 Link to full text
Abstract: Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.
25.

Engineering strategy and vector library for the rapid generation of modular light-controlled protein-protein interactions.

blue CrLOV1 CRY2/CRY2 NcWC1-LOV RsLOV VfAU1-LOV HEK293 Cell death
bioRxiv, 20 Mar 2019 DOI: 10.1101/583369 Link to full text
Abstract: Optogenetics enables the spatio-temporally precise control of cell and animal behaviour. Many optogenetic tools are driven by light-controlled protein-protein-interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPI to new target proteins is challenging because it is difficult to predict whether one the many available LSDs will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
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