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Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development.

ABSTRACT: ZO-1, ZO-2, and ZO-3 are closely related scaffolding proteins that link tight junction (TJ) transmembrane proteins such as claudins, junctional adhesion molecules, and occludin to the actin cytoskeleton. Even though the zonula occludens (ZO) proteins are among the first TJ proteins to have been identified and have undergone extensive biochemical analysis, little is known about the physiological roles of individual ZO proteins in different tissues or during vertebrate development. Here, we show that ZO-3 knockout mice lack an obvious phenotype. In contrast, embryos deficient for ZO-2 die shortly after implantation due to an arrest in early gastrulation. ZO-2(-)(/)(-) embryos show decreased proliferation at embryonic day 6.5 (E6.5) and increased apoptosis at E7.5 compared to wild-type embryos. The asymmetric distribution of prominin and E-cadherin to the apical and lateral plasma membrane domains, respectively, is maintained in cells of ZO-2(-)(/)(-) embryos. However, the architecture of the apical junctional complex is altered, and paracellular permeability of a low-molecular-weight tracer is increased in ZO-2(-/-) embryos. Leaky TJs and, given the association of ZO-2 with connexins and several transcription factors, effects on gap junctions and gene expression, respectively, are likely causes for embryonic lethality. Thus, ZO-2 is required for mouse embryonic development, but ZO-3 is dispensable. This is to our knowledge the first report showing that an individual ZO protein plays a nonredundant and critical role in mammalian development.

SUBMITTER: Xu J

PROVIDER: S-EPMC2258782 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development.

Xu Jianliang J Kausalya P Jaya PJ Phua Dominic C Y DC Ali Safiah Mohamed SM Hossain Zakir Z Hunziker Walter W

Molecular and cellular biology 20080102 5

ZO-1, ZO-2, and ZO-3 are closely related scaffolding proteins that link tight junction (TJ) transmembrane proteins such as claudins, junctional adhesion molecules, and occludin to the actin cytoskeleton. Even though the zonula occludens (ZO) proteins are among the first TJ proteins to have been identified and have undergone extensive biochemical analysis, little is known about the physiological roles of individual ZO proteins in different tissues or during vertebrate development. Here, we show t ...[more]

PMID: 18172007

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Project description:Gap junctions between glial cells in mammalian CNS are known to contain several connexins (Cx), including Cx26, Cx30 and Cx43 at astrocyte-to-astrocyte junctions, and Cx29 and Cx32 on the oligodendrocyte side of astrocyte-to-oligodendrocyte junctions. Recent reports indicating that oligodendrocytes also express Cx47 prompted the present studies of Cx47 localization and relationships to other glial connexins in mouse CNS. In view of the increasing number of connexins reported to interact directly with the scaffolding protein zonula occludens-1 (ZO-1), we investigated ZO-1 expression and Cx47/ZO-1 interaction capabilities in brain, spinal cord and Cx47-transfected HeLa cells. From counts of over 9000 oligodendrocytes labeled by immunofluorescence in various brain regions, virtually all of these cells were found to express Cx29, Cx32 and Cx47. Oligodendrocyte somata displayed robust Cx47-immunopositive puncta that were co-localized with punctate labeling for Cx32 and Cx43. By freeze-fracture replica immunogold labeling, Cx47 was abundant on the oligodendrocyte-side of oligodendrocyte/astrocyte gap junctions. By immunofluorescence, labeling for Cx47 along myelinated fibers was sparse in most brain regions, whereas Cx29 and Cx32 were previously found to be concentrated along these fibers. By immunogold labeling, Cx47 was found in numerous small gap junctions linking myelin to astrocytes, but not within deeper layers of myelin. Brain subcellular fractionation revealed a lack of Cx47 enrichment in myelin fractions, which nevertheless contained an enrichment of Cx32 and Cx29. Oligodendrocytes were immunopositive for ZO-1, and displayed almost total Cx47/ZO-1 co-localization. ZO-1 was found to co-immunoprecipitate with Cx47, and pull-down assays indicated binding of Cx47 to the second PDZ domain of ZO-1. Our results indicate widespread expression of Cx47 by oligodendrocytes, but with a distribution pattern in relative levels inverse to the abundance of Cx29 in myelin and paucity of Cx29 in oligodendrocyte somata. Further, our findings suggest a scaffolding and/or regulatory role of ZO-1 at the oligodendrocyte side of astrocyte-to-oligodendrocyte gap junctions.

Project description:Most gap junctions between neurons in mammalian retina contain abundant connexin36, often in association with the scaffolding protein zonula occludens-1. We now investigate co-association of connexin36, zonula occludens-1, zonula occludens-2 and Y-box transcription factor 3 (zonula occludens-1-associated nucleic acid-binding protein) in mouse and rat retina. By immunoblotting, zonula occludens-1-associated nucleic acid-binding protein and zonula occludens-2 were both detected in retina, and zonula occludens-2 in retina was found to co-immunoprecipitate with connexin36. By immunofluorescence, the four proteins appeared as puncta distributed in the plexiform layers. In the inner plexiform layer, most connexin36-puncta were co-localized with zonula occludens-1, and many were co-localized with zonula occludens-1-associated nucleic acid-binding protein. Moreover, zonula occludens-1-associated nucleic acid-binding protein was often co-localized with zonula occludens-1. Nearly all zonula occludens-2-puncta were positive for connexin36, zonula occludens-1 and zonula occludens-1-associated nucleic acid-binding protein. In the outer plexiform layer, connexin36 was also often co-localized with zonula occludens-1-associated nucleic acid-binding protein. In connexin36 knockout mice, labeling of zonula occludens-1 was slightly reduced in the inner plexiform layer, zonula occludens-1-associated nucleic acid-binding protein was decreased in the outer plexiform layer, and both zonula occludens-1-associated nucleic acid-binding protein and zonula occludens-2 were markedly decreased in the inner sublamina of the inner plexiform layer, whereas zonula occludens-1, zonula occludens-2 and zonula occludens-1-associated nucleic acid-binding protein puncta persisted and remained co-localized in the outer sublamina of the inner plexiform layer. By freeze-fracture replica immunogold labeling, connexin36 was found to be co-localized with zonula occludens-2 within individual neuronal gap junctions. In addition, zonula occludens-1-associated nucleic acid-binding protein was abundant in a portion of ultrastructurally-defined gap junctions throughout the inner plexiform layer, and some of these junctions contained both connexin36 and zonula occludens-1-associated nucleic acid-binding protein. These distinct patterns of connexin36 association with zonula occludens-1, zonula occludens-2 and zonula occludens-1-associated nucleic acid-binding protein in different sublaminae of retina, and differential responses of these proteins to connexin36 gene deletion suggest differential regulatory and scaffolding roles of these gap junction accessory proteins. Further, the persistence of a subpopulation of zonula occludens-1/zonula occludens-2/zonula occludens-1-associated nucleic acid-binding protein co-localized puncta in the outer part of the inner plexiform layer of connexin36 knockout mice suggests close association of these proteins with other structures in retina, possibly including gap junctions composed of an as-yet-unidentified connexin.

Project description:BackgroundWe previously showed that zonula occludens toxin (Zot) encoded by Campylobacter concisus zot (808T) gene has the potential to initiate inflammatory bowel disease. This Zot protein caused prolonged intestinal epithelial barrier damage, induced intestinal epithelial and macrophage production of tumor necrosis factor-α and enhanced the responses of macrophages to other microbes. In order to understand the potential virulence of Zot proteins in other Campylobacter species, in this study we examined their presence, similarities, motifs and prophages.MethodsThe presence of Zot proteins in Campylobacter species was examined by searching for the Zot family domain in multiple protein databases. Walker A and Walker B motifs in Zot proteins were identified using protein sequence alignment. A phylogenetic tree based on Campylobacter zot genes was constructed using maximum-likelihood method. Campylobacter Zot proteins were compared using protein sequence alignment. The zot-containing prophages in Campylobacter species were identified and compared with known prophage proteins and other viral proteins using protein sequence alignment and protein BLAST.ResultsTwelve Zot proteins were found in nine Campylobacter species/subspecies. Among these Campylobacter species, three species had two Zot proteins and the remaining six species/subspecies had one Zot protein. Walker A and Walker B motifs and a transmembrane domain were found in all identified Campylobacter Zot proteins. The twelve Campylobacter zot genes from the nine Campylobacter species/subspecies formed two clusters. The ZotCampyType_1 proteins encoded by Cluster 1 Campylobacter zot genes showed high similarities to each other. However, ZotCampyType_2 proteins encoded by Cluster 2 Campylobacter zot genes were more diverse. Furthermore, the zot-containing Campylobacter prophages were identified.ConclusionThis study reports the identification of two types of Campylobacter Zot proteins. The high similarities of ZotCampyType_1 proteins suggest that they are likely to have similar virulence. ZotCampyType_2 proteins are less similar to each other and their virulent properties, if any, remain to be examined individually.

Dataset Information

Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development.

Publications

Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development.

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