ZO-1 mRNA and Protein Expression during Tight Junction Assembly in Caco-2 Cells

We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions i...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:The Journal of cell biology 1989-09, Vol.109 (3), p.1047-1056
Hauptverfasser: Anderson, James Melvin, Van Itallie, Christina M., Peterson, Michelle D., Stevenson, Bruce R., Carew, Elizabeth A., Mooseker, Mark S.
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 1056
container_issue 3
container_start_page 1047
container_title The Journal of cell biology
container_volume 109
creator Anderson, James Melvin
Van Itallie, Christina M.
Peterson, Michelle D.
Stevenson, Bruce R.
Carew, Elizabeth A.
Mooseker, Mark S.
description We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of ∼7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/ Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/ Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at ∼10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.
doi_str_mv 10.1083/jcb.109.3.1047
format Article
fullrecord <record><control><sourceid>jstor_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2115763</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><jstor_id>1613560</jstor_id><sourcerecordid>1613560</sourcerecordid><originalsourceid>FETCH-LOGICAL-c4477-92c7dd58c1f1e1857221de24704d0692dcaca5f2f55a0bb549fae1e9530abdb13</originalsourceid><addsrcrecordid>eNqFkUtvEzEUhS1EVdLAlhVIXqDuJvj6ObNBiqIWqCqCUNmwsTy2J3U0j9SeQe2_xyFRgBUb-8rn87m-Pgi9BrIAUrL3W1vnolqwvHL1DM1AcFKUwMlzNCOEQlEJKl6gi5S2hGSEs3N0TqUileAztP6xLgB3374ssekd_hqH0YceXz3uok8pDD12Uwz9Bt-Fzf2Ib6bejvvTZUq-q9snnOGVsUNB8cq3bXqJzhrTJv_quM_R9-uru9Wn4nb98fNqeVtYzpUqKmqVc6K00ICHUihKwXnKFeGOyIo6a6wRDW2EMKSuBa8a48FXghFTuxrYHH04-O6muvPO-n6MptW7GDoTn_Rggv5X6cO93gw_NQUQSrJscHk0iMPD5NOou5BsHsH0fpiSVhUoBlT-FwTBpGK_HRcH0MYhpeib02uA6H1WOmeVi0ozvc8qX3j79wwn_BhO1t8ddZOsaZtoehvSCZOSijL_yBy9OWDbNA7xT1MJTEjCfgGCaqUy</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>15367363</pqid></control><display><type>article</type><title>ZO-1 mRNA and Protein Expression during Tight Junction Assembly in Caco-2 Cells</title><source>MEDLINE</source><source>EZB-FREE-00999 freely available EZB journals</source><source>Alma/SFX Local Collection</source><creator>Anderson, James Melvin ; Van Itallie, Christina M. ; Peterson, Michelle D. ; Stevenson, Bruce R. ; Carew, Elizabeth A. ; Mooseker, Mark S.</creator><creatorcontrib>Anderson, James Melvin ; Van Itallie, Christina M. ; Peterson, Michelle D. ; Stevenson, Bruce R. ; Carew, Elizabeth A. ; Mooseker, Mark S.</creatorcontrib><description>We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of ∼7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/ Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/ Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at ∼10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.</description><identifier>ISSN: 0021-9525</identifier><identifier>EISSN: 1540-8140</identifier><identifier>DOI: 10.1083/jcb.109.3.1047</identifier><identifier>PMID: 2670954</identifier><identifier>CODEN: JCLBA3</identifier><language>eng</language><publisher>New York, NY: Rockefeller University Press</publisher><subject>Animals ; Biological and medical sciences ; Blotting, Northern ; Caco 2 cells ; Cell Line ; Cell lines ; Cells ; Complementary DNA ; Culture Techniques - methods ; Cultured cells ; DNA - genetics ; DNA - isolation &amp; purification ; Epithelial cells ; Fluorescent Antibody Technique ; Fundamental and applied biological sciences. Psychology ; Gene expression ; Humans ; Immunoblotting ; Intercellular Junctions - physiology ; Intercellular Junctions - ultrastructure ; Kidney cells ; Liver - metabolism ; Liver - ultrastructure ; Membrane Proteins - analysis ; Membrane Proteins - genetics ; Messenger RNA ; Molecular and cellular biology ; Molecular genetics ; Nucleic Acid Hybridization ; Phosphoproteins - analysis ; Phosphoproteins - genetics ; Protein Biosynthesis ; Rats ; RNA ; RNA, Messenger - genetics ; Tight junctions ; Transcription, Genetic ; Zonula Occludens-1 Protein</subject><ispartof>The Journal of cell biology, 1989-09, Vol.109 (3), p.1047-1056</ispartof><rights>Copyright 1989 The Rockefeller University Press</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4477-92c7dd58c1f1e1857221de24704d0692dcaca5f2f55a0bb549fae1e9530abdb13</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&amp;idt=6625895$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2670954$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Anderson, James Melvin</creatorcontrib><creatorcontrib>Van Itallie, Christina M.</creatorcontrib><creatorcontrib>Peterson, Michelle D.</creatorcontrib><creatorcontrib>Stevenson, Bruce R.</creatorcontrib><creatorcontrib>Carew, Elizabeth A.</creatorcontrib><creatorcontrib>Mooseker, Mark S.</creatorcontrib><title>ZO-1 mRNA and Protein Expression during Tight Junction Assembly in Caco-2 Cells</title><title>The Journal of cell biology</title><addtitle>J Cell Biol</addtitle><description>We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of ∼7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/ Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/ Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at ∼10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Blotting, Northern</subject><subject>Caco 2 cells</subject><subject>Cell Line</subject><subject>Cell lines</subject><subject>Cells</subject><subject>Complementary DNA</subject><subject>Culture Techniques - methods</subject><subject>Cultured cells</subject><subject>DNA - genetics</subject><subject>DNA - isolation &amp; purification</subject><subject>Epithelial cells</subject><subject>Fluorescent Antibody Technique</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene expression</subject><subject>Humans</subject><subject>Immunoblotting</subject><subject>Intercellular Junctions - physiology</subject><subject>Intercellular Junctions - ultrastructure</subject><subject>Kidney cells</subject><subject>Liver - metabolism</subject><subject>Liver - ultrastructure</subject><subject>Membrane Proteins - analysis</subject><subject>Membrane Proteins - genetics</subject><subject>Messenger RNA</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Nucleic Acid Hybridization</subject><subject>Phosphoproteins - analysis</subject><subject>Phosphoproteins - genetics</subject><subject>Protein Biosynthesis</subject><subject>Rats</subject><subject>RNA</subject><subject>RNA, Messenger - genetics</subject><subject>Tight junctions</subject><subject>Transcription, Genetic</subject><subject>Zonula Occludens-1 Protein</subject><issn>0021-9525</issn><issn>1540-8140</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtvEzEUhS1EVdLAlhVIXqDuJvj6ObNBiqIWqCqCUNmwsTy2J3U0j9SeQe2_xyFRgBUb-8rn87m-Pgi9BrIAUrL3W1vnolqwvHL1DM1AcFKUwMlzNCOEQlEJKl6gi5S2hGSEs3N0TqUileAztP6xLgB3374ssekd_hqH0YceXz3uok8pDD12Uwz9Bt-Fzf2Ib6bejvvTZUq-q9snnOGVsUNB8cq3bXqJzhrTJv_quM_R9-uru9Wn4nb98fNqeVtYzpUqKmqVc6K00ICHUihKwXnKFeGOyIo6a6wRDW2EMKSuBa8a48FXghFTuxrYHH04-O6muvPO-n6MptW7GDoTn_Rggv5X6cO93gw_NQUQSrJscHk0iMPD5NOou5BsHsH0fpiSVhUoBlT-FwTBpGK_HRcH0MYhpeib02uA6H1WOmeVi0ozvc8qX3j79wwn_BhO1t8ddZOsaZtoehvSCZOSijL_yBy9OWDbNA7xT1MJTEjCfgGCaqUy</recordid><startdate>19890901</startdate><enddate>19890901</enddate><creator>Anderson, James Melvin</creator><creator>Van Itallie, Christina M.</creator><creator>Peterson, Michelle D.</creator><creator>Stevenson, Bruce R.</creator><creator>Carew, Elizabeth A.</creator><creator>Mooseker, Mark S.</creator><general>Rockefeller University Press</general><general>The Rockefeller University Press</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FD</scope><scope>FR3</scope><scope>M7Z</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19890901</creationdate><title>ZO-1 mRNA and Protein Expression during Tight Junction Assembly in Caco-2 Cells</title><author>Anderson, James Melvin ; Van Itallie, Christina M. ; Peterson, Michelle D. ; Stevenson, Bruce R. ; Carew, Elizabeth A. ; Mooseker, Mark S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4477-92c7dd58c1f1e1857221de24704d0692dcaca5f2f55a0bb549fae1e9530abdb13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Blotting, Northern</topic><topic>Caco 2 cells</topic><topic>Cell Line</topic><topic>Cell lines</topic><topic>Cells</topic><topic>Complementary DNA</topic><topic>Culture Techniques - methods</topic><topic>Cultured cells</topic><topic>DNA - genetics</topic><topic>DNA - isolation &amp; purification</topic><topic>Epithelial cells</topic><topic>Fluorescent Antibody Technique</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene expression</topic><topic>Humans</topic><topic>Immunoblotting</topic><topic>Intercellular Junctions - physiology</topic><topic>Intercellular Junctions - ultrastructure</topic><topic>Kidney cells</topic><topic>Liver - metabolism</topic><topic>Liver - ultrastructure</topic><topic>Membrane Proteins - analysis</topic><topic>Membrane Proteins - genetics</topic><topic>Messenger RNA</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Nucleic Acid Hybridization</topic><topic>Phosphoproteins - analysis</topic><topic>Phosphoproteins - genetics</topic><topic>Protein Biosynthesis</topic><topic>Rats</topic><topic>RNA</topic><topic>RNA, Messenger - genetics</topic><topic>Tight junctions</topic><topic>Transcription, Genetic</topic><topic>Zonula Occludens-1 Protein</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Anderson, James Melvin</creatorcontrib><creatorcontrib>Van Itallie, Christina M.</creatorcontrib><creatorcontrib>Peterson, Michelle D.</creatorcontrib><creatorcontrib>Stevenson, Bruce R.</creatorcontrib><creatorcontrib>Carew, Elizabeth A.</creatorcontrib><creatorcontrib>Mooseker, Mark S.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 1</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of cell biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Anderson, James Melvin</au><au>Van Itallie, Christina M.</au><au>Peterson, Michelle D.</au><au>Stevenson, Bruce R.</au><au>Carew, Elizabeth A.</au><au>Mooseker, Mark S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>ZO-1 mRNA and Protein Expression during Tight Junction Assembly in Caco-2 Cells</atitle><jtitle>The Journal of cell biology</jtitle><addtitle>J Cell Biol</addtitle><date>1989-09-01</date><risdate>1989</risdate><volume>109</volume><issue>3</issue><spage>1047</spage><epage>1056</epage><pages>1047-1056</pages><issn>0021-9525</issn><eissn>1540-8140</eissn><coden>JCLBA3</coden><abstract>We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of ∼7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/ Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/ Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at ∼10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.</abstract><cop>New York, NY</cop><pub>Rockefeller University Press</pub><pmid>2670954</pmid><doi>10.1083/jcb.109.3.1047</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 0021-9525
ispartof The Journal of cell biology, 1989-09, Vol.109 (3), p.1047-1056
issn 0021-9525
1540-8140
language eng
recordid cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2115763
source MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection
subjects Animals
Biological and medical sciences
Blotting, Northern
Caco 2 cells
Cell Line
Cell lines
Cells
Complementary DNA
Culture Techniques - methods
Cultured cells
DNA - genetics
DNA - isolation & purification
Epithelial cells
Fluorescent Antibody Technique
Fundamental and applied biological sciences. Psychology
Gene expression
Humans
Immunoblotting
Intercellular Junctions - physiology
Intercellular Junctions - ultrastructure
Kidney cells
Liver - metabolism
Liver - ultrastructure
Membrane Proteins - analysis
Membrane Proteins - genetics
Messenger RNA
Molecular and cellular biology
Molecular genetics
Nucleic Acid Hybridization
Phosphoproteins - analysis
Phosphoproteins - genetics
Protein Biosynthesis
Rats
RNA
RNA, Messenger - genetics
Tight junctions
Transcription, Genetic
Zonula Occludens-1 Protein
title ZO-1 mRNA and Protein Expression during Tight Junction Assembly in Caco-2 Cells
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-24T17%3A27%3A56IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-jstor_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=ZO-1%20mRNA%20and%20Protein%20Expression%20during%20Tight%20Junction%20Assembly%20in%20Caco-2%20Cells&rft.jtitle=The%20Journal%20of%20cell%20biology&rft.au=Anderson,%20James%20Melvin&rft.date=1989-09-01&rft.volume=109&rft.issue=3&rft.spage=1047&rft.epage=1056&rft.pages=1047-1056&rft.issn=0021-9525&rft.eissn=1540-8140&rft.coden=JCLBA3&rft_id=info:doi/10.1083/jcb.109.3.1047&rft_dat=%3Cjstor_pubme%3E1613560%3C/jstor_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=15367363&rft_id=info:pmid/2670954&rft_jstor_id=1613560&rfr_iscdi=true