DOCSLIB.ORG

Differential Expression of Claudins in Retinas During Normal Development and the Angiogenesis of Oxygen- Induced Retinopathy

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Differential Expression of Claudins in Retinas During Normal Development and the Angiogenesis of Oxygen- Induced Retinopathy Retinal Cell Biology Differential Expression of Claudins in Retinas during Normal Development and the Angiogenesis of Oxygen- Induced Retinopathy Yan Luo,*,1,2 Wei Xiao,1,2 Xiaobo Zhu,1 Yani Mao,3 Xialin Liu,1 Xiaoyun Chen,1 Juan Huang,1 Shibo Tang,*,1 and Lawrence J. Rizzolo4 PURPOSE. Angiogenesis accompanies several retinal pathologies angiogenesis induced by oxygen formed a leaky barrier due to that impair the inner blood–retinal barrier. Claudins are key the mislocalization of these claudins. Studies of the mecha- structural and functional proteins of the barrier. This study nisms that regulate the intracellular distribution of claudins compared the expression of claudins during the normal angio- may lead to new therapeutic approaches for retinal vascular genesis of development with that of oxygen-induced retinop- disease. (Invest Ophthalmol Vis Sci. 2011;52:7556–7564) DOI: athy. 10.1167/iovs.11-7185 METHODS. Real-time PCR was used to monitor mRNA from postnatal day 8 (P8) to P21 in normal mice and oxygen-induced he microenvironment of the retina is separated from the retinopathy (OIR) mice. Protein expression was monitored by Tcirculation by the blood–retinal barrier. This barrier in- immunoblotting and immunofluorescence. Isolectin B4 was cludes the outer blood–retinal barrier formed by the retinal used to identify blood vessels and occludin was used to identify pigment epithelium and the inner blood–retinal barrier (iBRB) tight junctions. Neovascularization and permeability were formed by retinal vessel endothelial cells. The iBRB controls monitored using FITC–dextran and Evans blue. cellular and molecular trafficking between the blood and inter- stitium of the neural retina. Disruption of the iBRB occurs in a RESULTS. The mRNA of claudin-1, -2, -3, -4, -5, -12, -22, and -23 number of ocular fundus diseases, such as diabetic retinopa- was developmentally regulated, but only claudin-1, -2, and -5 thy1 and retinopathy of prematurity.2 were found in the tight junctions of retinal vessels. OIR in- The barrier function of the iBRB depends on the integrity of duced the formation of leaky neovascular vessels. The mRNA the tight junctions that seal the space between adjacent endo- and protein of claudin-2 and -5 were overexpressed, whereas thelial cells. This semiselective seal retards the diffusion of claudin-1 and occludin were unaffected. Despite their overex- solutes across these paracellular spaces. Tight junctions also pression, each claudin was distributed throughout the cell, divide the plasma membrane into apical and basolateral mem- especially in the neovascular tufts. Occludin was retained at brane domains and maintain cell polarity.3–5 The numerous the lateral membranes but exhibited a punctate distribution. tight junction proteins include transmembrane and scaffold CONCLUSIONS. Claudin-1, -2, and -5 are the most prominent proteins. Transmembrane proteins include claudins, junctional claudins of the inner blood–retinal barrier. The pathologic adhesion molecules, occludin, and tricellulin.6 Claudins form the tight junctional strands that are observed by electron microscopy and determine the permeability and 7–9 1 selectivity of the strands. Claudins are 20- to 27-kDa tetras- From the State Key Laboratory of Ophthalmology, Zhongshan pan proteins with a short cytoplasmic N-terminus, two extra- Ophthalmic Center, Sun Yat-sen University, Guangzhou, P.R. China; 10 3 cellular loops, and a C-terminal cytoplasmic domain. At least the Department of Ophthalmology, Guangzhou Women and Chil- 11 dren’s Medical Center, Guangzhou, P.R. China; and the 4Department of 24 family members have been identified. Each tissue ex- Surgery, Department of Ophthalmology and Visual Sciences, Yale Uni- presses its own subset of claudins, according to the physiologic 12–16 versity School of Medicine, New Haven, Connecticut. role of that tissue. In the eye, tissue-specific expression 2These authors contributed equally to the work presented here has been reported for the lens, cornea, and conjunctiva.17,18 and should therefore be regarded as equivalent authors. The expression profile and function of the claudins have Supported in part by National Natural Science Foundation of not been reported for the iBRB. This study examines the mRNA China Grants 30872819 (YL) and 81070747 (XL), Program for New and protein expression levels of claudin family members dur- Century Excellent Talents in University Grant NCET-09-0809 (YL), ing the normal formation of the iBRB and the hyperstimulated Fundamental Research Funds of State Key Laboratory Grant CX-03 retinal angiogenesis of oxygen-induced retinopathy (OIR). (YL), Science and Technology Projects in Guangdong Province Grant 2009B030801024 (YL), and Bureau of Science, Technology and Infor- With the retinas of normal development and OIR, we could mation of Guangzhou Grant 2008J1-C011 (YM). begin to address the questions of the expression and distribu- Submitted for publication January 7, 2011; revised July 9, 2011; tion of claudins during retinal angiogenesis. accepted August 11, 2011. Disclosure: Y. Luo, None; W. Xiao, None; X. Zhu, None; Y. Mao, None; X. Liu, None; X. Chen, None; J. Huang, None; S. Tang, None; METHODS L.J. Rizzolo, None *Each of the following is a corresponding author: Yan Luo, State Animals Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, P. R. China All the studies adhered to the ARVO Statement for the Use of Animals [email protected]. in Ophthalmic and Vision Research and were approved by the Ethics Shibo Tang, State Key Laboratory of Ophthalmology, Zhongshan Oph- Committee in Animal and Human Experimentation of Sun Yat-sen thalmic Center, Sun Yat-sen University, Guangzhou, 510060, P. R. University, Guangzhou, China. C57BL/6J mice were purchased from China; [email protected]. Southern Medical University, Guangzhou, China. Mouse pups and their Investigative Ophthalmology & Visual Science, September 2011, Vol. 52, No. 10 7556 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from jov.arvojournals.org on 09/29/2021 IOVS, September 2011, Vol. 52, No. 10 Claudins in Retinal Angiogenesis 7557 TABLE 1. Real-Time PCR Primers Used in This Study Gene Accession No. Primers Product Size (bp) 18S XR034450 5Ј-TTCCGATAACGAACGAGACTCT-3Ј 99 RNA 5Ј-TGGCTGAACGCCACTTGTC-3Ј Cldn-1 NM016674 5Ј-TCTACGAGGGACTGTGGATG-3Ј 84 5Ј-TCAGATTCAGCTAGGAGTCG-3Ј Cldn-2 NM016675 5Ј-GGCTGTTAGGCTCATCCAT-3Ј 66 5Ј-TGGCACCAACATAGGAACTC-3Ј Cldn-3 NM009902 5Ј-AAGCCGAATGGACAAAGAA-3Ј 72 5Ј-CTGGCAAGTAGCTGCAGTG-3Ј Cldn-4 NM009903 5Ј-CGCTACTCTTGCCATTACG-3Ј 71 5Ј-ACTCAGCACACCATGACTTG-3Ј Cldn-5 NM013805 5Ј-GTGGAACGCTCAGATTTCAT-3Ј 97 5Ј-TGGACATTAAGGCAGCATCT-3Ј Cldn-6 NM018777 5Ј-CATTACATGGCCTGCTATTC-3Ј 75 5Ј-CACATAATTCTTGGTGGGATATT-3Ј Cldn-7 NM016887 5Ј-AGGGTCTGCTCTGGTCCTT-3Ј 76 5Ј-GTACGCAGCTTTGCTTTCA-3Ј Cldn-8 NM018778 5Ј-GCCGGCATCATCTTCTTCAT-3Ј 100 5Ј-CATCCACCAGTGGGTTGTAG-3Ј Cldn-9 NM020293 5Ј-GTCACACTTTGAGCGTCCC-3Ј 82 5Ј-CCTCTTATCCAGTCCCGAAG-3Ј Cldn-10 NM021386 5Ј-CCCAGAATGGGCTACACATA-3Ј 74 5Ј-CCTTCTCCGCCTTGATACTT-3Ј Cldn-11 NM021386 5Ј-TCTTGGTTCCTGTATGTGCC-3Ј 63 5Ј-CGTACAGCGAGTAGCCAAAG-3Ј Cldn-12 NM022890 5Ј-GTCCTCTCCTTTCTGGCAAC-3Ј 105 5Ј-ATGTCGATTTCAATGGCAGA-3Ј Cldn-13 NM020504 5Ј-TAGTGTTGGCCTTCTTGAGC-3Ј 83 5Ј-AGCCAAGCAATGGGTTAAAG-3Ј Cldn-14 NM019500 5Ј-GCTCCTAGGCTTCCTGCTTA-3Ј 169 5Ј-CTGGTAGATGCCTGTGCTGT-3Ј Cldn-15 NM021719 5Ј-CAGCTTCGGTAAATATGCCA-3Ј 76 5Ј-CAGTGGGACAAGAAATGGTG-3Ј Cldn-16 NM053241 5Ј-GCCATATTCTCCACTGGGTT-3Ј 70 5Ј-AGTCATCAGCGTTCACCATC-3Ј Cldn-17 NM181490 5Ј-TCGTTCTGATTCCAGTGTCC-3Ј 95 5Ј-TCCTCCAAGTTCTCGCTTCT-3Ј Cldn-18 NM019815 5Ј-GACCGTTCAGACCAGGTACA-3Ј 96 5Ј-GCGATGCACATCATCACTC-3Ј Cldn-19 NM153105 5Ј-ACCAGAATGAGGACCAGGAT-3Ј 75 5Ј-TCCTTCAGCAAATACGTTGG-3Ј Cldn-20* XM888581 5Ј-CAGCTCCTTGCTTTCATCCTG-3Ј 354 5Ј-CAGACTCCTCCAGCAAAGGAA-3Ј Cldn-20† XM888581 5Ј-TCCAGCTCCTTGCTTTCA-3Ј 105 5Ј-ATGTTGGGTCCTGCGTAT-3Ј Cldn-22 NM029383 5Ј-GCTCCTGCAGCCTCGAGTCACTATG-3Ј 81 5Ј-TGGATTGGCTTGCTTCAGCTCCA-3Ј Cldn-23 NM027998 5Ј-ACAGGGACACCAGCAAGCTCAA-3Ј 178 5Ј-AGGTCAGAGTCACAGGGCAACGAA-3Ј Occludin NM008756 5Ј-AGACCCAAGAGCAGCCAAAG-3Ј 61 5Ј-GGAAGCGATGAAGCAGAAGG-3Ј All primers were designed using commercial software (Primer Premier 5.0; PREMIER Biosoft Inter- national). * Primers were used for RT-PCR. † Primers were used for real-time PCR. mothers were housed in special pathogen-free conditions at the animal Aldrich, St. Louis, MO). DNase I–treated RNA (1 ␮g) was then con- laboratory center of Zhongshan Ophthalmic Center, Guangzhou, verted to cDNA using reverse transcriptase (Takara, Siga, Japan). cDNA China. The number of pups present in each litter was about the same samples were aliquoted and stored at Ϫ80°C. (n ϭ 8–12); 23 litters were analyzed in this study. RNA Isolation and cDNA Preparation Nonquantitative PCR and Quantitative Real-Time PCR After mice were anesthetized by an intraperitoneal (IP) injection of 10% chloral hydrate (2.5 mL/kg), retinas of postnatal day 8 (P8), P11, Primers for claudins, occludin, and 18S rRNA (Table 1) were designed P13, P15, P18, and P21 mice, together with kidney and brain of using commercial software (Primer Premier 5.0; PREMIER Biosoft In- 6-week-old mice, were collected. Total RNA was extracted using a ternational, Palo Alto, CA). Specificity of the primer sequences was commercial reagent (TRIzol; Invitrogen, Carlsbad, CA) according to examined using the National Center for Biotechnology Information the manufacturer’s instructions. Potential contamination by genomic Basic Local Alignment Search Tool module.
Load more

Recommended publications
  • Molecular Dynamics Simulations of Tight Junction Proteins
    Molecular Dynamics Simulations of Tight Junction Proteins Eleni Fitsiou This dissertation is submitted for the degree of Doctor of Philosophy August 2020 Department of Chemistry I would like to dedicate this dissertation to my husband Antonios and sons Dimitrios and Ioannis. ii “Wisdom begins in wonder” Socrates iii Declaration I, Eleni Fitsiou, declare that this thesis titled ‘Molecular Dynamics Simulations of Tight Junction Proteins’ has not been submitted in support of an application for another degree at this or any other university. It is the result of my own work and includes nothing that is the outcome of work done in collaboration except where specifically indicated. Where I have quoted from the work of others, the source is always given. Lancaster University, UK iv Abstract Tight junctions (TJs) are specialised cell-cell structures that serve primarily as a barrier to molecular transport through the intercellular space between the cells. The claudin family of proteins are the main structural and functional components of the TJ strands that circumscribe the cells. The detailed molecular organisation at the TJs is not entirely resolved, being relatively inaccessible by current experimental methods. Here, we have employed molecular dynamics simulations using both atomistic and coarse-grained models to investigate the TJ structure formed by claudin-1 using self-assembly coupled with free energy calculations and enhanced sampling techniques. A feature of the studies is that the self-assembly simulations have been carried out using atomistic detail (a first) by simulating only the extracellular domains of claudin-1 in an implied membrane. The results show that the cis-interaction can occur in the absence of trans-interacting partners and that a claudin dimer is the smallest stable unit.
    [Show full text]
  • Tight Junction Structure and Function Revisited
    Trends in Cell Biology Review Tight Junction Structure and Function Revisited Tetsuhisa Otani 1,2,3,*,@ and Mikio Furuse1,2,3 Tight junctions (TJs) are intercellular junctions critical for building the epithelial Highlights barrier and maintaining epithelial polarity. The claudin family of membrane pro- Tight junction strand formation and teins play central roles in TJ structure and function. However, recent findings membrane apposition formation are have uncovered claudin-independent aspects of TJ structure and function, and differentially regulated. additional players including junctional adhesion molecules (JAMs), membrane Claudins form charge-selective small lipids, phase separation of the zonula occludens (ZO) family of scaffolding pores, while junctional adhesion mole- proteins, and mechanical force have been shown to play important roles in TJ cules regulate the formation of size- structure and function. In this review, we discuss how these new findings have selective large pores. the potential to transform our understanding of TJ structure and function, and Tight junction proteins regulate epithelial how the intricate network of TJ proteins and membrane lipids dynamically polarity, although how tight junctions interact to drive TJ assembly. form a membrane fence remains unclear. Tight Junctions Regulate Epithelial Barrier and Polarity Tight junction associated membrane proteins regulate tight junction assembly – Tight junctions (TJs) are epithelial intercellular junctions located at the most apical region of cell in conjunction with zonula occludens cell contacts. TJs are structurally defined by electron microscopy. On ultrathin sections, TJs protein phase separation, membrane appear as a region with close apposition of adjacent plasma membranes where adjacent plasma lipids, mechanical force, and polarity signaling proteins.
    [Show full text]
  • Adherens Junctions, Desmosomes and Tight Junctions in Epidermal Barrier Function Johanna M
    14 The Open Dermatology Journal, 2010, 4, 14-20 Open Access Adherens Junctions, Desmosomes and Tight Junctions in Epidermal Barrier Function Johanna M. Brandner1,§, Marek Haftek*,2,§ and Carien M. Niessen3,§ 1Department of Dermatology and Venerology, University Hospital Hamburg-Eppendorf, Hamburg, Germany 2University of Lyon, EA4169 Normal and Pathological Functions of Skin Barrier, E. Herriot Hospital, Lyon, France 3Department of Dermatology, Center for Molecular Medicine, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Germany Abstract: The skin is an indispensable barrier which protects the body from the uncontrolled loss of water and solutes as well as from chemical and physical assaults and the invasion of pathogens. In recent years several studies have suggested an important role of intercellular junctions for the barrier function of the epidermis. In this review we summarize our knowledge of the impact of adherens junctions, (corneo)-desmosomes and tight junctions on barrier function of the skin. Keywords: Cadherins, catenins, claudins, cell polarity, stratum corneum, skin diseases. INTRODUCTION ADHERENS JUNCTIONS The stratifying epidermis of the skin physically separates Adherens junctions are intercellular structures that couple the organism from its environment and serves as its first line intercellular adhesion to the cytoskeleton thereby creating a of structural and functional defense against dehydration, transcellular network that coordinate the behavior of a chemical substances, physical insults and micro-organisms. population of cells. Adherens junctions are dynamic entities The living cell layers of the epidermis are crucial in the and also function as signal platforms that regulate formation and maintenance of the barrier on two different cytoskeletal dynamics and cell polarity.
    [Show full text]
  • Tight Junctions: from Simple Barriers to Multifunctional Molecular Gates
    Tight Junctions: From simple barriers to multifunctional molecular gates Ceniz Zihni1, Clare Mills1, Karl Matter1# and Maria S Balda1# Department of Cell Biology UCL Institute of Ophthalmology University College London London EC1V 9EL, UK # Address for correspondence UCL Institute of Ophthalmology University College London Bath Street London EC1V 9EL United Kingdom Tel – 44 20 7608 4014/6861 Fax – 44 20 7608 4034 E-mail: [email protected]/[email protected] Main body of text: 5983 words 1 Epithelia and endothelia separate different tissue compartments and protect multicellular organisms form the outside world. This requires the formation of tight junctions, selective gates that control paracellular diffusion of ions and solutes. Tight junctions also form the border between the apical and basolateral plasma membrane domains and are linked to the machinery that controls apicobasal polarization. Additionally, signalling networks that guide diverse cell behaviours and functions are connected to tight junctions, transmitting information to and from the cytoskeleton, nucleus and different cell adhesion complexes. Here, we discuss recent advances in our understanding of the molecular architecture and cellular functions of tight junctions. Microscopists in the 19th century described the paracellular space between neighbouring cells within an epithelial sheet to be sealed by a “terminal bar”, a structure later resolved by electron microscopy into a composite of distinct cell-cell junctions that is now called the epithelial junctional complex and is formed by tight junctions, adherens junctions and desmosomes1,2. As the former two junctions are more tightly associated and often reside at the apical end of the lateral membrane, they are often referred to as the apical junctional complex (however, in endothelia, tight junctions and adherens junctions can be intercalated) (Fig.
    [Show full text]
  • Actin-Related Protein2/3 Complex Regulates Tight Junctions and Terminal Differentiation to Promote Epidermal Barrier Formation
    Actin-related protein2/3 complex regulates tight junctions and terminal differentiation to promote epidermal barrier formation Kang Zhoua,1, Andrew Muroyamaa,1, Julie Underwooda, Rebecca Leyleka, Samriddha Raya, Scott H. Soderlinga,b, and Terry Lechlera,c,2 Departments of aCell Biology, bNeurobiology, and cDermatology, Duke University Medical Center, Durham, NC 27710 Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved August 19, 2013 (received for review May 3, 2013) The epidermis provides an essential seal from the external gastrulation in Caenorhabditis elegans embryos(11,12).In environment and retains fluids within the body. To form an Drosophila, the Arp2/3 complex plays roles in myoblast fusion, effective barrier, cells in the epidermis must form tight junctions ring canal expansion, adherens junction formation, Notch sig- and terminally differentiate into cornified envelopes. Here, we naling, and bristle development (6, 13, 14). demonstrate that the branched actin nucleator, the actin-related The functions of the Arp2/3 complex in intact tissues during protein (Arp)2/3 complex, is unexpectedly required for both these mammalian development and homeostasis have only begun to be activities. Loss of the ArpC3 subunit of the Arp2/3 complex addressed. Loss of the Arp2/3 complex globally results in early resulted in minimal changes in the morphogenesis and architec- lethality at the blastocyst stage in mice (15). In the mouse oocyte, ture of this stratified squamous epithelium, but resulted in the Arp2/3 complex is required for asymmetric cell division profound defects in its physiology. Mutant embryos did not through positioning of the mitotic spindle (16).
    [Show full text]
  • The Density of Small Tight Junction Pores Varies Among Cell Types and Is Increased by Expression of Claudin-2
    298298 Research Article The density of small tight junction pores varies among cell types and is increased by expression of claudin-2 Christina M. Van Itallie1,*, Jennifer Holmes2, Arlene Bridges3, Jody L. Gookin4, Maria R. Coccaro4, William Proctor3, Oscar R. Colegio5 and James M. Anderson2 1Department of Medicine, 2Cell and Molecular Physiology and 3School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA 4Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC 27695, USA 5Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510, USA *Author for correspondence (e-mail: [email protected]) Accepted 4 November 2007 Journal of Cell Science 121, 298-305 Published by The Company of Biologists 2008 doi:10.1242/jcs.021485 Summary Epithelial tight junctions contain size- and charge-selective cell types and is not necessarily related to electrical resistance. pores that control the paracellular movement of charged and Expression of claudin-2 results in a selective increase in pore noncharged solutes. Claudins influence the charge selectivity number but not size and has no effect on the permeability of and electrical resistance of junctions, but there is no direct PEGs that are larger than the pores; however, neither evidence describing pore composition or whether pore size or knockdown of claudin-2 nor overexpression of several other density differs among cell types. To characterize paracellular claudins altered either the number of small pores or their size. pores independent of influences from charge selectivity, we We speculate that permeability of all small solutes is profiled the ‘apparent permeabilities’ (Papp) of a continuous proportional to pore number but that small electrolytes are series of noncharged polyethylene glycols (PEGs) across subject to further selectivity by the profile of claudins monolayers of five different epithelial cell lines and porcine expressed, explaining the dissociation between the Papp for ileum.
    [Show full text]
  • Relocalization of Cell Adhesion Molecules During Neoplastic Transformation of Human Fibroblasts
    INTERNATIONAL JOURNAL OF ONCOLOGY 39: 1199-1204, 2011 Relocalization of cell adhesion molecules during neoplastic transformation of human fibroblasts CRISTINA BELGIOVINE, ILARIA CHIODI and CHIARA MONDELLO Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, Italy Received May 6, 2011; Accepted June 10, 2011 DOI: 10.3892/ijo.2011.1119 Abstract. Studying neoplastic transformation of telomerase cell-cell contacts (1,2). Cadherins are transmembrane glyco- immortalized human fibroblasts (cen3tel), we found that the proteins mediating homotypic cell-cell adhesion via their transition from normal to tumorigenic cells was associated extracellular domain. Through their cytoplasmic domain, with the loss of growth contact inhibition, the acquisition of an they bind to catenins, which mediate the connection with the epithelial-like morphology and a change in actin organization, actin cytoskeleton. Different types of cadherins are expressed from stress fibers to cortical bundles. We show here that these in different cell types; e.g. N-cadherin is typically expressed variations were paralleled by an increase in N-cadherin expres- in mesenchymal cells, such as fibroblasts, while E-cadherin sion and relocalization of different adhesion molecules, such participates in the formation of adherens junctions in cells of as N-cadherin, α-catenin, p-120 and β-catenin. These proteins epithelial origin. The role of E-cadherin and β-catenin in the presented a clear membrane localization in tumorigenic cells development and progression of tumors of epithelial origin compared to a more diffuse, cytoplasmic distribution in is well documented (3). In particular, loosening of cell-cell primary fibroblasts and non-tumorigenic immortalized cells, contacts because of loss of E-cadherin expression and nuclear suggesting that tumorigenic cells could form strong cell-cell accumulation of β-catenin are hallmarks of the epithelial- contacts and cell contacts did not induce growth inhibition.
    [Show full text]
  • Effect of Graphene and Graphene Oxide on Airway Barrier and Differential Phosphorylation of Proteins in Tight and Adherens Junction Pathways
    nanomaterials Article Effect of Graphene and Graphene Oxide on Airway Barrier and Differential Phosphorylation of Proteins in Tight and Adherens Junction Pathways Sofie Van Den Broucke 1, Jeroen A. J. Vanoirbeek 2 , Rita Derua 3, Peter H. M. Hoet 1,2,* and Manosij Ghosh 1,2,* 1 Centre for Environment and Health, Department of Public Health and Primary Care, KU Leuven, 3000 Leuven, Belgium; vandenbroucke.sofi[email protected] 2 Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE), KU Leuven, 3000 Leuven, Belgium; [email protected] 3 Laboratory of Protein Phosphorylation and Proteomics, KU Leuven, 3000 Leuven, Belgium; [email protected] * Correspondence: [email protected] (P.H.M.H.); [email protected] (M.G.) Abstract: Via inhalation we are continuously exposed to environmental and occupational irritants which can induce adverse health effects, such as irritant-induced asthma (IIA). The airway epithelium forms the first barrier encountered by these agents. We investigated the effect of environmental and occupational irritants on the airway epithelial barrier in vitro. The airway epithelial barrier was mimicked using a coculture model, consisting of bronchial epithelial cells (16HBE) and monocytes (THP-1) seeded on the apical side of a permeable support, and human lung microvascular endothelial cells (HLMVEC) grown on the basal side. Upon exposure to graphene (G) and graphene oxide Citation: Van Den Broucke, S.; (GO) in a suspension with fetal calf serum (FCS), ammonium persulfate (AP), sodium persulfate Vanoirbeek, J.A.J.; Derua, R.; Hoet, (SP) and hypochlorite (ClO−), the transepithelial electrical resistance (TEER) and flux of fluorescent P.H.M.; Ghosh, M.
    [Show full text]
  • Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions
    bioRxiv preprint doi: https://doi.org/10.1101/589580; this version posted March 26, 2019. 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. Phase separation of zonula occludens proteins drives formation of tight junctions Oliver Beutel1, Riccardo Maraspini1, Karina Pombo-Garcia1, Cécilie Martin-Lemaitre1 and Alf Honigmann1* 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany * Correspondence: [email protected] Abstract: Tight junctions are cell adhesion complexes that seal tissues and are involved in cell polarity and signalling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands is dependent on the two membrane associated scaffolding proteins ZO1 and ZO2. To understand how ZO proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight junction proteins including adhesion receptors, cytoskeletal adapters and transcription factors. Our results suggest that an active phase transition of ZO proteins into a condensed membrane bound compartment drives claudin polymerization and coalescence of a continuous tight junction belt. Introduction: Tight junctions are cell-cell adhesion complexes that regulate para-cellular flux of solutes and prevent pathogen entry across epithelial and endothelial cell layers including the blood brain barrier (Anderson and Van Itallie, 2009; Citi, 2018).
    [Show full text]
  • Regional Differences in Tight Junction Protein Expression in the Blood–DRG Barrier and Their Alterations After Nerve Traumatic
    International Journal of Molecular Sciences Article Regional Differences in Tight Junction Protein Expression in the Blood–DRG Barrier and Their Alterations after Nerve Traumatic Injury in Rats Thomas J. Lux 1 , Xiawei Hu 1, Adel Ben-Kraiem 1, Robert Blum 2, 1, , 1, , Jeremy Tsung-Chieh Chen * y and Heike L. Rittner * y 1 Department of Anaesthesiology, University Hospital of Wuerzburg, 97074 Wuerzburg, Germany; [email protected] (T.J.L.); [email protected] (X.H.); [email protected] (A.B.-K.) 2 Institute of Clinical Neurobiology, University Hospital of Wuerzburg, 94074 Wuerzburg, Germany; [email protected] * Correspondence: [email protected] (J.T.-C.C.); [email protected] (H.L.R.); Tel.: +49-931-170-7870047 (H.L.R.) These authors contributed equally to this work. y Received: 9 December 2019; Accepted: 28 December 2019; Published: 31 December 2019 Abstract: The nervous system is shielded by special barriers. Nerve injury results in blood–nerve barrier breakdown with downregulation of certain tight junction proteins accompanying the painful neuropathic phenotype. The dorsal root ganglion (DRG) consists of a neuron-rich region (NRR, somata of somatosensory and nociceptive neurons) and a fibre-rich region (FRR), and their putative epi-/perineurium (EPN). Here, we analysed blood–DRG barrier (BDB) properties in these physiologically distinct regions in Wistar rats after chronic constriction injury (CCI). Cldn5, Cldn12, and Tjp1 (rats) mRNA were downregulated 1 week after traumatic nerve injury. Claudin-1 immunoreactivity (IR) found in the EPN, claudin-19-IR in the FRR, and ZO-1-IR in FRR-EPN were unaltered after CCI.
    [Show full text]
  • Chapter 7 Cell Adhesion and the Extracellular Matrix
    Chapter 7 Cell Adhesion and the Extracellular Matrix © 2020 Elsevier Inc. All rights reserved. Figure 7–1. Types of cell adhesion molecules. © 2020 Elsevier Inc. All rights reserved. 2 Figure 7–2. Structural features of selectins and PSGL-1. (Adapted from Kappelmayer, Nagy. Biomed Res Int 2017, https://doi.org/10.1155/2017/6138145, by permission with added colors.) © 2020 Elsevier Inc. All rights reserved. 3 Figure 7–3. Integrin activation and clustering. (A) Domain structure of integrin subunits, (B) activation and clustering of integrins. © 2020 Elsevier Inc. All rights reserved. 4 Figure 7–4. Hierarchical organization of integrin clusters in migrating cell. Image of a mouse embryonic fibroblast spread on fibronectin-coated substrate for 30 min. dSTORM imaging of endogenous paxillin conjugated (labeled with paxillin antibody, BD biosciences) to antibody tagged with AlexaFlour-647 (antimouse secondary antibody conjugated to AlexaFlour-647). Gray scale shows the TIRF image, whereas false color image shows the reconstructed superresolution image. Color code (top left corner, from left to right) indicates increasing intensity of molecules. Encircled in dotted lines from left- to right-cell edge, nascent adhesions, maturing adhesions, mature adhesions. Scale bar 2 μm. (Adapted from Changede, Sheetz. BioEssays 2016;39:1, 1600123, by permission.) © 2020 Elsevier Inc. All rights reserved. 5 Figure 7–5. Intercellular junctions and the junctional complex. Simple epithelial cells have a junctional complex at their apicolateral borders. The components are the tight junction, the adherens junction, and the desmosome. The tight and adherens junctions are zonular, extending right around the cells, whereas desmosomes are punctate. Desmosomes are also present beneath the junctional complex, as is another punctate junction, the gap junction.
    [Show full text]
  • Tight Junction Proteins Expression and Modulation in Immune Cells and Multiple Sclerosis
    Mandel et al. JCMM-03-2011-057.R1 Tight Junction Proteins Expression and Modulation in Immune Cells and Multiple Sclerosis Ilana Mandel, PhD 1, Tamar Paperna, PhD 1, Lea Glass-Marmor, PhD 1, Anat Volkowich, MSc1, Samih Badarny, MD1,2, Ilya Schwartz, MD2, Pnina Vardi, MD3,4 , Ilana Koren, MD3, Ariel Miller, MD, PhD1,5* 1Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa; 2Movement Disorders clinic, Carmel Medical Center, Haifa; 3Pediatric Endocrinology and Diabetes Center, Armon Clalit Health Services clinic, Haifa; 4Felsenstein Medical Research Center (FMRC), Faculty of Medicine, Tel Aviv University; 5Division of Neuroimmunology & Multiple Sclerosis Center, Carmel Medical Center, Haifa, Israel This is an Accepted Article that has been peer-reviewed and approved for publication in the Journal of Cellular and Molecular Medicine, but has yet to undergo copy-editing and proof correction. Please cite this article as an “Accepted Article”; doi: 10.1111/j.1582-4934.2011.01380.x 1 Mandel et al. JCMM-03-2011-057.R1 *Correspondence: Ariel Miller, M.D., Ph.D., Division of Neuroimmunology& Multiple Sclerosis Center, Carmel Medical Center, 7 Michal St., Haifa, 34362, Israel. Phone: +972-4-8250851; Fax: +972-4-8250909 E-mail: [email protected] -or- [email protected] Abstract The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood-brain-barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation, and modulation associated with autoimmune disease states and therapies.
    [Show full text]