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 71 results
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1.

Substratum stiffness regulates Erk signaling dynamics through receptor-level control.

blue CRY2/CRY2 iLID MCF10A Signaling cascade control
Cell Rep, 28 Dec 2021 DOI: 10.1016/j.celrep.2021.110181 Link to full text
Abstract: The EGFR/Erk pathway is triggered by extracellular ligand stimulation, leading to stimulus-dependent dynamics of pathway activity. Although mechanical properties of the microenvironment also affect Erk activity, their effects on Erk signaling dynamics are poorly understood. Here, we characterize how the stiffness of the underlying substratum affects Erk signaling dynamics in mammary epithelial cells. We find that soft microenvironments attenuate Erk signaling, both at steady state and in response to epidermal growth factor (EGF) stimulation. Optogenetic manipulation at multiple signaling nodes reveals that intracellular signal transmission is largely unaffected by substratum stiffness. Instead, we find that soft microenvironments decrease EGF receptor (EGFR) expression and alter the amount and spatial distribution of EGF binding at cell membranes. Our data demonstrate that the mechanical microenvironment tunes Erk signaling dynamics via receptor-ligand interactions, underscoring how multiple microenvironmental signals are jointly processed through a highly conserved pathway that regulates tissue development, homeostasis, and disease progression.
2.

Stress ball morphogenesis: How the lizard builds its lung.

blue CRY2/CRY2 C2C12 Immediate control of second messengers
Sci Adv, 22 Dec 2021 DOI: 10.1126/sciadv.abk0161 Link to full text
Abstract: The function of the lung is closely coupled to its structural anatomy, which varies greatly across vertebrates. Although architecturally simple, a complex pattern of airflow is thought to be achieved in the lizard lung due to its cavernous central lumen and honeycomb-shaped wall. We find that the wall of the lizard lung is generated from an initially smooth epithelial sheet, which is pushed through holes in a hexagonal smooth muscle meshwork by forces from fluid pressure, similar to a stress ball. Combining transcriptomics with time-lapse imaging reveals that the hexagonal meshwork self-assembles in response to circumferential and axial stresses downstream of pressure. A computational model predicts the pressure-driven changes in epithelial topology, which we probe using optogenetically driven contraction of 3D-printed engineered muscle. These results reveal the physical principles used to sculpt the unusual architecture of the lizard lung, which could be exploited as a novel strategy to engineer tissues.
3.

Temperature-responsive optogenetic probes of cell signaling.

blue BcLOV4 CRY2/CRY2 iLID HEK293T NIH/3T3 Schneider 2 zebrafish in vivo Signaling cascade control
Nat Chem Biol, 22 Dec 2021 DOI: 10.1038/s41589-021-00917-0 Link to full text
Abstract: We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.
4.

Formation of nuclear condensates by the Mediator complex subunit Med15 in mammalian cells.

blue CRY2/CRY2 NIH/3T3 Organelle manipulation
BMC Biol, 17 Nov 2021 DOI: 10.1186/s12915-021-01178-y Link to full text
Abstract: The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood.
5.

Aberrant Phase Separation of FUS Leads to Lysosome Sequestering and Acidification.

blue CRY2/CRY2 HEK293 Organelle manipulation
Front Cell Dev Biol, 22 Oct 2021 DOI: 10.3389/fcell.2021.716919 Link to full text
Abstract: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro. ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention.
6.

Wnt signaling rescues amyloid beta induced stem cell loss.

blue CRY2/CRY2 D. melanogaster in vivo Signaling cascade control Developmental processes
bioRxiv, 6 Sep 2021 DOI: 10.1101/2021.09.06.459094 Link to full text
Abstract: Previously, we established an optogenetic model to induce Amyloid-β intracellular oligomerization to model distinct disease etiologies (Lim et al. 2020). Here we examine the effect of Wnt signaling on Amyloid in this model. We observe that Wnt activation rescues the detrimental effects of Amyloid expression and oligomerization. We analyze the gene expression changes downstream of Wnt that contribute to this rescue and find changes in aging related genes, protein misfolding, metabolism and inflammation. We propose that Wnt expression reduces inflammation through repression of Toll activating factors and confirm that chronic Toll activation reduces lifespan. We propose that the protective effect observed for Lithium treatment functions at least in part through Wnt activation and inhibition of inflammation.
7.

SATB1 undergoes isoform-specific phase transitions in T cells.

blue CRY2/CRY2 NIH/3T3 Organelle manipulation
bioRxiv, 11 Aug 2021 DOI: 10.1101/2021.08.11.455932 Link to full text
Abstract: Intracellular space is demarcated into functional membraneless organelles and nuclear bodies via the process of phase separation. Phase transitions are involved in many functions linked to such bodies as well as in gene expression regulation and other cellular processes. In this work we describe how the genome organizer SATB1 utilizes its prion-like domains to undergo phase transitions. We have identified two SATB1 isoforms with distinct biophysical behavior and showed how phosphorylation and interaction with nuclear RNA, impact their phase transitions. Moreover, we show that SATB1 is associated with transcription and splicing, both of which evinced deregulation in Satb1 knockout mice. Thus, the tight regulation of different SATB1 isoforms levels and their post-translational modifications are imperative for SATB1’s physiological roles in T cell development while their deregulation may be linked to disorders such as cancer.
8.

A Non-Canonical Raf Function Is Required for Dorsal-Ventral Patterning During Drosophila Embryogenesis.

blue CRY2/CRY2 iLID D. melanogaster in vivo Developmental processes
bioRxiv, 29 Jul 2021 DOI: 10.1101/2021.07.29.454294 Link to full text
Abstract: Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.
9.

Optogenetic actuator/biosensor circuits for large-scale interrogation of ERK dynamics identify sources of MAPK signaling robustness.

blue CRY2/CRY2 iLID NIH/3T3 Signaling cascade control
bioRxiv, 27 Jul 2021 DOI: 10.1101/2021.07.27.453955 Link to full text
Abstract: Measurements of single-cell ERK activity dynamics provide unique insights in the MAPK network topology. We built genetic circuits consisting of optogenetic actuators activating ERK from different nodes within the MAPK network together with an ERK biosensor to measure single-cell ERK dynamics. Evaluating ERK dynamics induced by different temporal optogenetic inputs, in response to a large number of perturbations, shows that the MAPK network is robust to downregulation of most of its nodes. This robustness emerges in part because of the ERK-RSK2-SOS negative feedback. Bypassing this feedback, by direct activation of the RAS/RAF/MEK/ERK submodule, or by RSK2 perturbation, breaks MAPK network robustness. Targeting the RSK2-mediated feedback in a ErbB2-dependent oncogenic signaling model greatly sensitizes ERK to MEK inhibition, allowing efficient ERK activity shutdown within a cell population. Thus, the RSK2-mediated negative feedback is a weak node of the MAPK network whose perturbation enables potent inhibition of ERK.
10.

An optogenetic proximity labeling approach to probe the composition of inducible biomolecular condensates in cultured cells.

blue CRY2/CRY2 HEK293
STAR Protoc, 22 Jul 2021 DOI: 10.1016/j.xpro.2021.100677 Link to full text
Abstract: Inducible biomolecular condensates play fundamental roles in cellular responses to intracellular and environmental cues. Knowledge about their composition is crucial to understand the functions that arise specifically from the assembly of condensates. This protocol combines an optogenetic and an efficient proximity labeling approach to analyze protein modifications driven by protein condensation in cultured cells. Low endogenous biotin level ensures sharp signals. For complete details on the use and execution of this protocol, please refer to Frattini et al. (2021).
11.

Spatiotemporal sensitivity of mesoderm specification to FGFR signalling in the Drosophila embryo.

blue CRY2/CRY2 D. melanogaster in vivo Signaling cascade control Developmental processes
Sci Rep, 8 Jul 2021 DOI: 10.1038/s41598-021-93512-1 Link to full text
Abstract: Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Although Htl has been extensively studied, the dynamics of its action are poorly understood after the initial phases of mesoderm formation and spreading. To begin to address this challenge, we have developed an optogenetic version of the FGFR Heartless in Drosophila (Opto-htl). Opto-htl enables us to activate the FGFR pathway in selective spatial (~ 35 μm section from one of the lateral sides of the embryo) and temporal domains (ranging from 40 min to 14 h) during embryogenesis. Importantly, the effects can be tuned by the intensity of light-activation, making this approach significantly more flexible than other genetic approaches. We performed controlled perturbations to the FGFR pathway to define the contribution of Htl signalling to the formation of the developing embryonic heart and somatic muscles. We find a direct correlation between Htl signalling dosage and number of Tinman-positive heart cells specified. Opto-htl activation favours the specification of Tinman positive cardioblasts and eliminates Eve-positive DA1 muscles. This effect is seen to increase progressively with increasing light intensity. Therefore, fine tuning of phenotypic responses to varied Htl signalling dosage can be achieved more conveniently than with other genetic approaches. Overall, Opto-htl is a powerful new tool for dissecting the role of FGFR signalling during development.
12.

TOR signaling regulates liquid phase separation of the SMN complex governing snRNP biogenesis.

blue CRY2/CRY2 HeLa Signaling cascade control Organelle manipulation
Cell Rep, 22 Jun 2021 DOI: 10.1016/j.celrep.2021.109277 Link to full text
Abstract: The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase β-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.
13.

Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch.

blue CRY2/CRY2 iLID HEK293T Jurkat NIH/3T3 SYF Signaling cascade control Organelle manipulation
Cell Rep, 22 Jun 2021 DOI: 10.1016/j.celrep.2021.109280 Link to full text
Abstract: Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.
14.

Robustness of epithelial sealing is an emerging property of local ERK feedback driven by cell elimination.

blue CRY2/CRY2 D. melanogaster in vivo Signaling cascade control Cell death
Dev Cell, 28 May 2021 DOI: 10.1016/j.devcel.2021.05.006 Link to full text
Abstract: What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.
15.

Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development.

blue CRY2/CIB1 CRY2/CRY2 BHK-21 HEK293T Xenopus in vivo Signaling cascade control Developmental processes
J Mol Biol, 19 May 2021 DOI: 10.1016/j.jmb.2021.167050 Link to full text
Abstract: Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.
16.

Optogenetic-induced multimerization of the dopamine transporter increases uptake and trafficking to the plasma membrane.

blue CRY2/CRY2 HEK293 SH-SY5Y Control of vesicular transport
J Biol Chem, 17 May 2021 DOI: 10.1016/j.jbc.2021.100787 Link to full text
Abstract: The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine (METH) and cocaine (COC), have been reported to induce the formation of DAT multimeric complexes in vivo and in vitro. The interpretation of DAT multimer function has been primarily in the context of compounds that induce structural and functional modifications of DAT, complicating the understanding of the significance of DAT multimers. To examine multimerization in the absence of DAT ligands as well as in their presence, we developed a novel, optogenetic fusion chimera of cryptochrome 2 and DAT with a mCherry fluorescent reporter (Cry2-DAT). Using blue light to induce Cry2-DAT multimeric protein complex formation, we were able to simultaneously test the functional contributions of DAT multimerization in the absence or presence of substrates or inhibitors with high spatiotemporal precision. We found that blue light-stimulated Cry2-DAT multimers significantly increased IDT307 uptake and MFZ 9-18 binding in the absence of ligands as well as after METH and nomifensine (NOM) treatment. Blue light induced Cry2-DAT multimerization increased colocalization with recycling endosomal marker Rab11 and had decreased presence in Rab5-positive early endosomes and Rab7-positive late endosomes. Our data suggest that the increased uptake and binding results from induced and rapid trafficking of DAT multimers to the plasma membrane. Our data suggest that DAT multimers may function to help maintain DA homeostasis.
17.

Optogenetic Control of Non-Apoptotic Cell Death.

blue cpLOV2 cpLOVTRAP CRY2/CRY2 LOVTRAP 786-O B16-F0 E. coli HEK293T HeLa Jurkat Signaling cascade control Cell death
Adv Biology, 6 May 2021 DOI: 10.1002/advs.202100424 Link to full text
Abstract: Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.
18.

Optogenetic control of calcium influx in mammalian cells.

blue AsLOV2 CRY2/CRY2 HEK293T HeLa
Methods Enzymol, 16 Mar 2021 DOI: 10.1016/bs.mie.2021.02.010 Link to full text
Abstract: Optogenetics combines optics and genetics to enable non-invasive interrogation of cell physiology at an unprecedented high spatiotemporal resolution. Here, we introduce Opto-CRAC as a set of genetically-encoded calcium actuators (GECAs) engineered from the calcium release-activated calcium (CRAC) channel, which has been tailored for optical control of calcium entry and calcium-dependent physiological responses in non-excitable cells and tissues. We describe a detailed protocol for applying Opto-CRAC as an optogenetic tool to achieve photo-tunable control over intracellular calcium signals and calcium-dependent gene expression in mammalian cells.
19.

Multiple Sclerosis-Associated hnRNPA1 Mutations Alter hnRNPA1 Dynamics and Influence Stress Granule Formation.

blue CRY2/CRY2 HEK293T Control of cytoskeleton / cell motility / cell shape Organelle manipulation
Int J Mol Sci, 12 Mar 2021 DOI: 10.3390/ijms22062909 Link to full text
Abstract: Evidence indicates that dysfunctional heterogeneous ribonucleoprotein A1 (hnRNPA1; A1) contributes to the pathogenesis of neurodegeneration in multiple sclerosis. Understanding molecular mechanisms of neurodegeneration in multiple sclerosis may result in novel therapies that attenuate neurodegeneration, thereby improving the lives of MS patients with multiple sclerosis. Using an in vitro, blue light induced, optogenetic protein expression system containing the optogene Cryptochrome 2 and a fluorescent mCherry reporter, we examined the effects of multiple sclerosis-associated somatic A1 mutations (P275S and F281L) in A1 localization, cluster kinetics and stress granule formation in real-time. We show that A1 mutations caused cytoplasmic mislocalization, and significantly altered the kinetics of A1 cluster formation/dissociation, and the quantity and size of clusters. A1 mutations also caused stress granule formation to occur more quickly and frequently in response to blue light stimulation. This study establishes a live cell optogenetics imaging system to probe localization and association characteristics of A1. It also demonstrates that somatic mutations in A1 alter its function and promote stress granule formation, which supports the hypothesis that A1 dysfunction may exacerbate neurodegeneration in multiple sclerosis.
20.

TopBP1 assembles nuclear condensates to switch on ATR signaling.

blue CRY2/CRY2 HEK293 Signaling cascade control
Mol Cell, 16 Jan 2021 DOI: 10.1016/j.molcel.2020.12.049 Link to full text
Abstract: ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.
21.

Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.

blue red CRY2/CIB1 CRY2/CRY2 PhyB/PIF6 HEK293 mouse in vivo U-2 OS Transgene expression
Sci Adv, 1 Jan 2021 DOI: 10.1126/sciadv.abd3568 Link to full text
Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.
22.

Engineering Supramolecular Organizing Centers for Optogenetic Control of Innate Immune Responses.

blue CRY2/CRY2 LOVTRAP HEK293T HeLa RAW264.7 THP-1
Adv Biol, 30 Dec 2020 DOI: 10.1002/adbi.202000147 Link to full text
Abstract: The spatiotemporal organization of oligomeric protein complexes, such as the supramolecular organizing centers (SMOCs) made of MyDDosome and MAVSome, is essential for transcriptional activation of host inflammatory responses and immunometabolism. Light‐inducible assembly of MyDDosome and MAVSome is presented herein to induce activation of nuclear factor‐kB and type‐I interferons. Engineering of SMOCs and the downstream transcription factor permits programmable and customized innate immune operations in a light‐dependent manner. These synthetic molecular tools will likely enable optical and user‐defined modulation of innate immunity at a high spatiotemporal resolution to facilitate mechanistic studies of distinct modes of innate immune activations and potential intervention of immune disorders and cancer.
23.

Use of Optogenetic Amyloid-β to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans.

blue CRY2/CRY2 C. elegans in vivo D. melanogaster in vivo zebrafish in vivo
Bio Protoc, 5 Dec 2020 DOI: 10.21769/bioprotoc.3856 Link to full text
Abstract: Alzheimer's Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. We developed an optogenetic method to induce Aβ aggregation and tested its biological influence in three model organisms-D. melanogaster, C. elegans and D. rerio. We generated a fluorescently labeled, optogenetic Aβ peptide that oligomerizes rapidly in vivo in the presence of blue light in all organisms. Here, we detail the procedures for expressing this fusion protein in animal models, investigating the effects on the nervous system using time lapse light-sheet microscopy, and performing metabolic assays to measure changes due to intracellular Aβ aggregation. This method, employing optogenetics to study the pathology of AD, allows spatial and temporal control in vivo that cannot be achieved by any other method at present.
24.

Spatio-temporal Control of ERK Pulse Frequency Coordinates Fate Decisions during Mammary Acinar Morphogenesis.

blue CRY2/CIB1 CRY2/CRY2 MCF10A Signaling cascade control Cell differentiation Cell death
bioRxiv, 21 Nov 2020 DOI: 10.1101/2020.11.20.387167 Link to full text
Abstract: The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the frequency of ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated ERK waves emerge across multiple cells of an acinus, correlating with high and low ERK pulse frequency in outer surviving and inner dying cells respectively. A PIK3CA H1047R mutation, commonly observed in breast cancer, increases ERK pulse frequency and inner cell survival, causing loss of lumen formation. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Thus, fate decisions during acinar morphogenesis are fine-tuned by different spatio-temporal coordination modalities of ERK pulse frequency.
25.

Spatiotemporal sensitivity of embryonic heart specification to FGFR signaling in Drosophila.

blue CRY2/CRY2 D. melanogaster in vivo Signaling cascade control Developmental processes
bioRxiv, 19 Nov 2020 DOI: 10.1101/2020.11.16.384123 Link to full text
Abstract: Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Htl is involved in shaping the mesoderm at both early and later stages during embryogenesis. How Htl expression levels and timing of signaling affect mesoderm morphogenesis after spreading remains elusive. We have developed an optogenetic tool (Opto-htl) to control the activation of Htl signaling with precise spatiotemporal resolution in vivo. We find that the embryo is most sensitive to Htl over-activation within a developmental window of ~4 hours ranging from late stage 10 until early stage 13, which corresponds to early stages of heart morphogenesis. Opto-htl restores heart cells in htl mutants upon light activation, independent of its role in early mesoderm shaping events. We also successfully generated spatially distinct regions of Htl activity in the mesoderm using light-sheet microscopy. The developing tissue was unable to correct for the ensuing asymmetries in cell fate. Overall, Opto-htl is a powerful tool for studying spatiotemporal regulation of Htl signaling during embryogenesis.
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