DOCSLIB.ORG

Development and Characterization of Human Ipsc-Derived Neurons for Drug Discovery Applications

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Development and Characterization of Human Ipsc-Derived Neurons for Drug Discovery Applications Development and Characterization of Human iPSC-derived Neurons for Drug Discovery Applications Lucas Chase1, Monica Strathman1, Jeff Grinager1, David Majewski1, Regina Whitemarsh2, Sabine Pellett2, Oksana Sirenko3, Jayne Hesley3, Penny Tavormina3, Casey Stankewicz1, Matt George1, Ning Liu1, Nathan Meyer1, Matthew Riley1, Xuezhu Feng1, Eric Johnson2, Wen Bo Wang1 and Brad Swanson1 1Cellular Dynamics International, Inc., Madison, WI 53711; 2Department of Bacteriology, University of Wisconsin, Madison, WI 53706; 3Molecular Devices, Sunnyvale, CA 94089 Target Target Compound Lead Preclinical Clinical Identification Validation Screening Optimization Trials Trials Abstract Phenotype Characterization Electrophysiology High Content Image-based Assays Cell-based Assays A. B. The human brain represents a complex organ that has A. Evoked Action Potential B. Spontaneous Action Potential A. B. consistently been proven difficult to model in vitro. Current models A. B. 30 20 20 including primary rodent tissue and immortalized cell lines have 10 10 0 0mV served as mainstays in both academic research and the 0 0mV ubulin t -10 - -10 pharmaceutical industry. These models, while providing a means -20 III -20 for numerous landmark discoveries, have suffered from various -30 -30 10mV10mV -40 10mV Figure 10. iCell Neurons display an expected sensitivity to known compounds. iCell Class Class 1sec 5msec 1sec -40 issues including biological relevance, reproducibility and -50 Neurons were cultured for 7-14 days post-thaw on PLO/Laminin pre-coated 96-well plates -60 scalability. Considerable efforts have been made specifically -50 and exposed to a dilution series of (A) staurosporine and (B) kainic acid. Viability (as ® -60 measured using cellular ATP content) was determined using the CellTiter-Glo within the pharmaceutical industry to reduce late-stage drug Nestin Luminescent Cell Viability Assay (Promega). attrition through the development of more relevant in vitro human Class III -tubulin /Nestin/ Hoechst Figure 7. High content image analysis. (A) An example overlay image of post-thaw iCell Neurons stained with class III -tubulin (green) and DAPI stain (blue) using the Figure 5. iCell Neurons exhibit neuron-like action potentials. Action potential tracings ® ® model systems. One area given significant attention has been the Figure 2. A highly pure neuronal population. iCell Neurons represent a highly pure Molecular Devices ImageXpress Micro system and MetaXpress analysis software. BoNT Toxigenicity Testing recorded from a single iCell Neuron using a whole-cell patch clamp methodology. (A) A population as demonstrated by (A) flow cytometry (Day 1 post-thaw) and (B) Magnification = 10x objective. (B) Neurite outgrowth analysis using the Neurite Outgrowth development of platforms than can enable the modeling of human representative evoked action potential from post-thaw day 11 neurons. Evoked action immunocytochemistry (Day 7 post-thaw) for class III -tubulin (positive; neuronal marker) module of MetaXpress Software. potentials from these cells display an average resting membrane potential of -46mV as early A. B. degenerative (e.g. Alzheimer’s and Parkinson’s disease) and and nestin (negative, neural stem/progenitor cell marker). as 9 days post-thaw. (B) Representative spontaneous action potentials from a post-thaw day genetic (i.e. Huntington's disease and muscular dystrophy) Day 5 14 neuron. All action potentials demonstrate an overshoot of the depolarization phase above A. B. Day 10 10 Day 15 0mV and an undershoot of the repolarization phase below baseline before correction to Day 20 diseases as well as neurotoxicity. The recent discovery of induced A. B. Total Outgrowth Adult Human Brain steady-state. pluripotent stem cells (iPSCs) not only overcomes the ethical and 100 1 30000 90 logistical issues associated with human embryonic stem cells, but 80 0.1 70 20000 also provides a flexible platform for generating various 60 antimycin A 0.01 50 + antimycin A staurosporine A. Na Channel Current (I ) – Tetrodotoxin Inhibition 40 differentiated cell types from diseased individuals. Using this Na staurosporine mitomycin C 0.001 10000 30 ExpressionRelative GAPDH) (vs. mitomycin C MK571 Total outgrowth Total 20 platform, we have developed a highly consistent and scalable Voltage (mV) MK571 significant cells growth 0.0001 I 10 STX1A STX1B VAMP1 VAMP2 VAMP3 SV2A SV2B SV2C SNAP25 SYT1 SYT2 Na -50 -30 -10 10 30 50 70 % 0 0 protocol to differentiate and cryopreserve purified human iPSC- 200 0.001 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 100 1000 100 ® Concentration, uM Concentration, uM Control Concentration (mM) Concentration (mM) derived neurons, called iCell Neurons. Phenotypically, these 0 4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2 Total neurite % cells with4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2 BoNT/A BoNT/B -100 10nM TTX Antimycin A (Antimycin A: Concentration vs Mean... 2.48e+04 4.63 13.3 2.25e+03 0.936 outgrowth Antimycinoutgrowth A (Antimycin A: Concentration vs Mean... 90.9 28.6 14.5 16.4 0.987 C. -200 Staurosporin (Staurosporin: Concentration vs Mea... 2.6e+04 2.26 0.943 1.63e+03 0.983 Staurosporin (Staurosporin: Concentration vs Mea... 85.3 1.47 1.77 12.3 0.978 cells are >90% pure as measured using flow cytometry for vGAT / vGLUT2 MAP2 / GABA / Hoechst Compound IC50 (µM) MK IC50(MK: Concentration (µM) vs MeanValue) 90 29.3 3.94 12 0.982 30nM TTX MK (MK: Concentration vs MeanValue) 2.67e+04 2.27 3.95 -532 0.988 -300 antimycin A 13 Mitomycin14 C (Mitomycin A: Concentration vs Mean... 87.9 27.4 0.667 16.1 0.996 Mitomycin C (Mitomycin A: Concentration vs Mean... 2.67e+04 3.53 0.732 1.06e+03 0.992 __________ -400 100nm TTX __________ staurosporine 0.9 1.7 presence of the neuronal marker class III beta tubulin (TuJ1) and (pA) Current Weighting: Fixed Weighting: Fixed Figure 3. iCell Neurons display characteristic subtype protein expression. Post-thaw -500 300nM TTX mitomycin C 0.7 0.7 absence of the progenitor marker nestin. Within 24hrs of thawing, 150pA -600 C. MK571 4.1 3.9 iCell Neurons were plated in iCell Neurons Maintenance Medium with iCell Neurons 1ms -700 these neurons display a typical neuronal morphology including a Supplement on poly-L-ornithine/laminin-coated tissue culture plates and immunostained on -800 0.01mM 1mM 10mM day 14 for (A) the synaptic markers vGAT (vesicular GABA transporter) and vGLUT2 dense network of neurites. Detailed phenotypic analyses reveal (vesicular glutamate transporter 2), markers of GABAergic and Glutamatergic neurons, respectively; and (B) MAP2 (microtubule-associated protein 2) and GABA (gamma- that these neurons are comprised of a mix of predominantly + aminobutyric acid). Magnification = 20x objective. B. K Channel Current (IK) – Tetraethylammonium Inhibition GABAergic and Glutamatergic subtypes as measured at both the BoNT/C BoNT/E gene expression and protein levels and form characteristic IK 800 synaptic connections. Functionally, these cells reveal typical 700 Regional Gene Expression 600 Control 10.000000 electrophysiological characteristics as measured using single-cell 500 0.1mM TEA 400 1.000000 0.3mM TEA patch clamp to detect both spontaneous and evoked action 300 0.100000 1mM TEA Current (pA) Current 200 Figure 8. High content image-based assay for neurite outgrowth. (A and B) iCell (vs. GAPDH) (vs. 0.010000 potentials as well as functional ion channels. Finally, when applied 100 3mM TEA Neurons treated with increasing concentrations of antimycin A, staurosporine, mitomycin 0.001000 0 C and MK571 were stained for nuclei (DAPI) and class III -tubulin and analyzed using the to high throughput applications, including cytotoxicity assays, iCell -50 -40 -30 -20 -10 0 10 20 30 40 0.000100 Voltage (mV) Molecular Devices ImageXpress Micro system and MetaXpress software. The resultant Figure 11. BoNT target characterization and toxigenicity testing. (A) TaqMan gene Neurons reveal characteristic pharmacological responses to Expression Relative 0.000010 dose response curves to cytotoxic compounds for 2 parameters: (A) total outgrowth and expression assays were used to detect Botulinum neurotoxin (BoNT) receptor and target 0.000001 (B) % cells with significant growth, were generated. (C) Images of cells treated with known toxic compounds. The results demonstrate not only a LHX2 DACH1 FOXG1 OTX2 GBX2 EN1 PAX2 OLIG2 HOXB4 protein expression in iCell Neurons cultured for 5-20 days post-thaw. Adult human brain was increasing concentrations of mitomycin C and stained with class III -tubulin. Magnification used as a positive control. (B) Protein expression of BoNT receptors and target proteins was novel cell model for use in various academic and pharmaceutical + C. Ca Channel Current (I ) – Nifedipine Inhibition = 20x objective. analyzed via Western blot for iCell Neurons cultured for 4-21 days post-thaw. Rat spinal cord Subtype Gene Expression Ca applications, but they also support the use of the iPSC technology B. Control cells (RSC) were used as a positive control. (C) iCell Neurons (4 or 7 days post-thaw) and rat as a platform capable of generating neurons against diverse 10.000000 I A. spinal cord cells were exposed to serial dilutions of BoNT/A, /B, /C, or /E for 48 hrs. Cell 1.000000 Ca Voltage (mV) lysates were analyzed for respective SNARE target protein cleavage by Western blot. Data genetic backgrounds. 0.100000 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 from three Western blots were quantified by densitometry and dose-response curves were (vs. GAPDH) (vs. 50 0.010000 plotted
Load more

Recommended publications
  • VAMP3 and VAMP8 Regulate the Development and Functionality of 5 Parasitophorous Vacuoles Housing Leishmania Amazonensis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.09.195032; this version posted July 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 4 VAMP3 and VAMP8 regulate the development and functionality of 5 parasitophorous vacuoles housing Leishmania amazonensis 6 7 8 Olivier Séguin1, Linh Thuy Mai1, Sidney W. Whiteheart2, Simona Stäger1, Albert Descoteaux1* 9 10 11 12 1Institut national de la recherche scientifique, Centre Armand-Frappier Santé Biotechnologie, 13 Laval, Québec, Canada 14 15 2Department of Molecular and Cellular Biochemistry, University of Kentucky College of 16 Medicine, Lexington, Kentucky, United States of America 17 18 19 *Corresponding author: 20 E-mail: [email protected] 21 22 23 24 Short title: SNAREs and Leishmania-harboring communal parasitophorous vacuoles 25 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.09.195032; this version posted July 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 26 ABSTRACT 27 28 To colonize mammalian phagocytic cells, the parasite Leishmania remodels phagosomes into 29 parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular 30 and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are 31 poorly understood.
    [Show full text]
  • Targeted Genes and Methodology Details for Neuromuscular Genetic Panels
    Targeted Genes and Methodology Details for Neuromuscular Genetic Panels Reference transcripts based on build GRCh37 (hg19) interrogated by Neuromuscular Genetic Panels Next-generation sequencing (NGS) and/or Sanger sequencing is performed Motor Neuron Disease Panel to test for the presence of a mutation in these genes. Gene GenBank Accession Number Regions of homology, high GC-rich content, and repetitive sequences may ALS2 NM_020919 not provide accurate sequence. Therefore, all reported alterations detected ANG NM_001145 by NGS are confirmed by an independent reference method based on laboratory developed criteria. However, this does not rule out the possibility CHMP2B NM_014043 of a false-negative result in these regions. ERBB4 NM_005235 Sanger sequencing is used to confirm alterations detected by NGS when FIG4 NM_014845 appropriate.(Unpublished Mayo method) FUS NM_004960 HNRNPA1 NM_031157 OPTN NM_021980 PFN1 NM_005022 SETX NM_015046 SIGMAR1 NM_005866 SOD1 NM_000454 SQSTM1 NM_003900 TARDBP NM_007375 UBQLN2 NM_013444 VAPB NM_004738 VCP NM_007126 ©2018 Mayo Foundation for Medical Education and Research Page 1 of 14 MC4091-83rev1018 Muscular Dystrophy Panel Muscular Dystrophy Panel Gene GenBank Accession Number Gene GenBank Accession Number ACTA1 NM_001100 LMNA NM_170707 ANO5 NM_213599 LPIN1 NM_145693 B3GALNT2 NM_152490 MATR3 NM_199189 B4GAT1 NM_006876 MYH2 NM_017534 BAG3 NM_004281 MYH7 NM_000257 BIN1 NM_139343 MYOT NM_006790 BVES NM_007073 NEB NM_004543 CAPN3 NM_000070 PLEC NM_000445 CAV3 NM_033337 POMGNT1 NM_017739 CAVIN1 NM_012232 POMGNT2
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Defining the Kv2.1–Syntaxin Molecular Interaction Identifies a First-In-Class Small Molecule Neuroprotectant
    Defining the Kv2.1–syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant Chung-Yang Yeha,b,1, Zhaofeng Yec,d,1, Aubin Moutale, Shivani Gaura,b, Amanda M. Hentonf,g, Stylianos Kouvarosf,g, Jami L. Salomana, Karen A. Hartnett-Scotta,b, Thanos Tzounopoulosa,f,g, Rajesh Khannae, Elias Aizenmana,b,g,2, and Carlos J. Camachoc,2 aDepartment of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; bPittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; cDepartment of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; dSchool of Medicine, Tsinghua University, Beijing 100871, China; eDepartment of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724; fDepartment of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; and gPittsburgh Hearing Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 Edited by Lily Yeh Jan, University of California, San Francisco, CA, and approved June 19, 2019 (received for review February 27, 2019) + The neuronal cell death-promoting loss of cytoplasmic K follow- (13). The Kv2.1-dependent cell death pathway is normally initiated ing injury is mediated by an increase in Kv2.1 potassium channels in by the oxidative liberation of zinc from intracellular metal-binding the plasma membrane. This phenomenon relies on Kv2.1 binding to proteins (14), leading to the sequential phosphorylation of syntaxin 1A via 9 amino acids within the channel intrinsically disor- Kv2.1 residues Y124 and S800 by Src and p38 kinases, respectively dered C terminus. Preventing this interaction with a cell and blood- (15–17).
    [Show full text]
  • Mapping Protein Interactions of Sodium Channel Nav1.7 Using 2 Epitope-Tagged Gene Targeted Mice
    bioRxiv preprint doi: https://doi.org/10.1101/118497; this version posted March 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Mapping protein interactions of sodium channel NaV1.7 using 2 epitope-tagged gene targeted mice 3 Alexandros H. Kanellopoulos1†, Jennifer Koenig1, Honglei Huang2, Martina Pyrski3, 4 Queensta Millet1, Stephane Lolignier1, Toru Morohashi1, Samuel J. Gossage1, Maude 5 Jay1, John Linley1, Georgios Baskozos4, Benedikt Kessler2, James J. Cox1, Frank 6 Zufall3, John N. Wood1* and Jing Zhao1†** 7 1Molecular Nociception Group, WIBR, University College London, Gower Street, 8 London WC1E 6BT, UK. 9 2Mass Spectrometry Laboratory, Target Discovery Institute, University of Oxford, The 10 Old Road Campus, Oxford, OX3 7FZ, UK. 11 3Center for Integrative Physiology and Molecular Medicine, Saarland University, 12 Kirrbergerstrasse, Bldg. 48, 66421 Homburg, Germany. 13 4Division of Bioscience, University College London, Gower Street, London WC1E 6BT, UK.14 15 †These authors contributed equally to directing this work. 16 *Correspondence author. Tel: +44 207 6796 954; E-mail: [email protected] 17 **Corresponding author: Tel: +44 207 6790 959; E-mail: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/118497; this version posted March 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 18 Abstract 19 The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. 20 Besides action potential propagation, NaV1.7 regulates neurotransmitter release, 21 integrates depolarizing inputs over long periods and regulates transcription.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Familial Hyperkalemic Hypertension
    Disease of the Month Familial Hyperkalemic Hypertension Juliette Hadchouel,* Ce´line Delaloy,* Se´bastien Faure´,† Jean-Michel Achard,† and Xavier Jeunemaitre* *Colle`ge de France, Paris; INSERM U36, Paris; AP-HP, Department of Genetics, Hoˆpital Europe´en Georges Pompidou; University Paris-Descartes, Faculty of Medicine, Paris, France; and †Division of Nephrology and Department of Physiology, Limoges University Hospital, Limoges, France J Am Soc Nephrol 17: 208–217, 2006. doi: 10.1681/ASN.2005030314 amilial hyperkalemic hypertension (FHHt) syndrome biochemical abnormalities and age or BP, which seemed to (1,2), also known as Gordon syndrome (3) or pseudohy- depend primarily on age in affected individuals (11). This F poaldosteronism type 2 (4), is a rare inherited form of phenotypic variability, associated with sensitivity to thia- low-renin hypertension associated with hyperkalemia and hy- zides—which are widely used in hypertension—and with a low perchloremic metabolic acidosis in patients with a normal GFR probability of this rare disease’s being recognized by most (OMIM no. 145260). This monogenic form of arterial hyperten- doctors may have led to an underestimation of its frequency. sion has excited new interest since the discovery of a new, The mode of inheritance of the disease is consistent with unsuspected molecular pathway that is responsible for both the autosomal dominant transmission in most, if not all, of the biochemical abnormalities and the increase in BP observed. pedigrees reported. However, we have identified two families Genetic analysis has led to the identification of mutations in in which the parents of the affected individuals were first two genes that belong to a new family of kinases, the WNK cousins, suggesting possible autosomal recessive transmission.
    [Show full text]
  • Fluorescence Lifetime Imaging Microscopy Reveals Rerouting Of
    RESEARCH ARTICLE Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation Danie¨ lle Rianne Jose´ Verboogen, Natalia Gonza´ lez Mancha, Martin ter Beest, Geert van den Bogaart* Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands Abstract SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Fo¨ rster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood- derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution. DOI: 10.7554/eLife.23525.001 *For correspondence: geert. [email protected] Introduction Competing interests: The One of the central paradigms in cell biology is that all intracellular membrane fusion, except for authors declare that no mitochondrial fusion, is catalyzed by soluble NSF (N-ethylmaleimide-sensitive fusion protein) attach- competing interests exist. ment protein receptor (SNARE) proteins (Hong, 2005; Jahn and Scheller, 2006).
    [Show full text]
  • Human VAMP3 Suppresses Or Negatively Regulates Bax Induced Apoptosis in Yeast
    biomedicines Article Human VAMP3 Suppresses or Negatively Regulates Bax Induced Apoptosis in Yeast Damilare D. Akintade 1,2,* and Bhabatosh Chaudhuri 2 1 School of Life Sciences, Medical School, University of Nottingham, Nottingham NG7 2UH, UK 2 Leicester School of Pharmacy, De Montfort University, Leicester LE1 9BH, UK; [email protected] * Correspondence: [email protected] Abstract: Apoptosis is an essential process that is regulated genetically and could lead to a serious disease condition if not well controlled. Bax is one of the main proapoptotic proteins and actively involved in programmed cell death. It has been suggested that Bax induced apoptosis in yeast could be obstructed by enhancing vesicular membrane trafficking. Plasma membrane proteins and lipid oxidation were reduced by a vesicle-associated membrane protein (VAMP) when expressed in yeast, suggesting its potential role in repairing membranes. Membrane integrity is crucial, as the loss of membrane integrity will result in the leakage of ions from mitochondria, and ultimately cell death due to overproduction of reactive oxygen species (ROS). Expression of Arabidopsis’ VAMP has been linked to antiapoptosis activity. Since plant VAMP has been associated with antiapoptotic activities, this study investigates the possible participation of human VAMP3 in blocking human Bax mediated apoptosis. Some novel genes were identified to rescue Bax’s proapoptotic effects, in a yeast-based human hippocampal cDNA library screen. VAMP3 (a gene code for proteins involved in protein secretion) gene was chosen for further study to confirm its role in inhibiting apoptosis. VAMP3 was coexpressed with a chromosomally integrated Bax gene expression cassette driven by the GAL1 promoter.
    [Show full text]
  • Mapping Protein Interactions of Sodium Channel
    Published online: January 15, 2018 Resource Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice Alexandros H Kanellopoulos1,†, Jennifer Koenig1,†, Honglei Huang2, Martina Pyrski3 , Queensta Millet1, Stéphane Lolignier1,4, Toru Morohashi1, Samuel J Gossage1, Maude Jay1, John E Linley1,5, Georgios Baskozos6 , Benedikt M Kessler2, James J Cox1, Annette C Dolphin7, Frank Zufall3, John N Wood1,* & Jing Zhao1,** Abstract Introduction The voltage-gated sodium channel NaV1.7 plays a critical role in Pain is a major clinical problem. A 2012 National Health Interview pain pathways. We generated an epitope-tagged NaV1.7 mouse Survey (NHIS) in the United States revealed that 25.3 million adults that showed normal pain behaviours to identify channel-inter- (11.2%) suffered from daily (chronic) pain and 23.4 million acting proteins. Analysis of NaV1.7 complexes affinity-purified (10.3%) reported severe pain within a previous 3-month period under native conditions by mass spectrometry revealed 267 (Nahin, 2015). Many types of chronic pain are difficult to treat as proteins associated with Nav1.7 in vivo. The sodium channel b3 most available drugs have limited efficacy and can cause side (Scn3b), rather than the b1 subunit, complexes with Nav1.7, and effects. There is thus a huge unmet need for novel analgesics we demonstrate an interaction between collapsing-response medi- (Woodcock, 2009). Recent human and animal genetic studies have ator protein (Crmp2) and Nav1.7, through which the analgesic drug indicated that the voltage-gated sodium channel (VGSC) NaV1.7 lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein plays a crucial role in pain signalling (Nassar et al, 2004; Cox et al, interactors including membrane-trafficking protein synaptotag- 2006; Dib-Hajj et al, 2013), highlighting NaV1.7 as a promising drug min-2 (Syt2), L-type amino acid transporter 1 (Lat1) and trans- target for development of novel analgesics (Habib et al, 2015; membrane P24-trafficking protein 10 (Tmed10) together with Emery et al, 2016).
    [Show full text]
  • Genes and Symptoms of Schizophrenia: Modifiers, Networks, and Interactions in Complex Disease
    Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 Genes and Symptoms of Schizophrenia: Modifiers, Networks, and Interactions in Complex Disease Sarah Bergen Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medical Genetics Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/1940 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Genes and Symptoms of Schizophrenia: Modifiers, Networks, and Interactions in Complex Disease A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University by: Sarah E. Bergen B.A., Macalester College, 2000 M.S., University of Pittsburgh, 2004 Director: Kenneth S. Kendler, M.D. Distinguished Professor, Departments of Psychiatry and Human and Molecular Genetics Virginia Commonwealth University Richmond, VA September, 2009 Acknowledgements First, I want to thank my parents whose unconditional love and only slightly conditional support has carried me through every bump and rough patch on my journey through grad school (and life). None of my work would have been accomplished without all you have done for me, and I am profoundly grateful. I have been extremely fortunate in my experiences here to not only have been quite productive scientifically, but also to have enjoyed the graduate school experience. This is almost entirely due to the brilliant and kind people I have had the opportunity to work with.
    [Show full text]
  • Calcium Control of Neurotransmitter Release
    Downloaded from http://cshperspectives.cshlp.org/ on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Calcium Control of Neurotransmitter Release Thomas C. Su¨dhof Department of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305 Correspondence: [email protected] Upon entering a presynaptic terminal, an action potential opens Ca2þ channels, and transiently increases the local Ca2þ concentration at the presynaptic active zone. Ca2þ then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca2þ. Synaptotagmins bind Ca2þ via two C2-domains, and transduce the Ca2þ signal into a nanomechanical activation of the membrane fusion machinery; this acti- vation is mediated by the Ca2þ-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The con- served function of synaptotagmins and complexins operates generally in most, if not all, Ca2þ-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exo- cytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release. ynaptic transmission is initiated when an in the brainstem (Fig. 1B; reviewed in Mein- Saction potential invades a nerve terminal, renken et al. 2003). Overall, these high-resolution opening Ca2þ channels, which gate a highly electrophysiological studies on neurotrans- localized, transient increase in intracellular mitter release revealed that a presynaptic action Ca2þ at the active zone (Fig.
    [Show full text]