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Scott D Grayowen - One of the best experts on this subject based on the ideXlab platform.

  • specific binding to differentially expressed human carcinoembryonic antigen related cell adhesion molecules determines the outcome of neisseria gonorrhoeae infections along the female reproductive tract
    Infection and Immunity, 2018
    Co-Authors: Epshita A Islam, Varun C Anipindi, Ian P Francis, Yazdan Shaikdasthagirisaheb, Nelly Leung, Anna Sintsova, Mohsen Amin, Charu Kaushic, Lee M Wetzler, Scott D Grayowen
    Abstract:

    The gonococcal Opa proteins are an antigenically variable family of surface adhesins that bind human CEACAM1, CEACAM3, CEACAM5 and/or CEACAM6, cell surface glycoproteins that are differentially expressed on a broad spectrum of human cells and tissues. While presumed to be important for infection, the significance of various Opa-CEACAM mediated cellular interactions in the context of the genital tract has remained unclear. Here, we observe that CEACAM1 and CEACAM5 are differentially expressed on epithelia lining the upper and lower portions of the human female genital tract, respectively. Using transgenic mouse lines expressing human CEACAMs in a manner that reflects this differential pattern, we consider the impact of Opa-CEACAM interactions during uncomplicated lower genital tract infections versus during pelvic inflammatory disease. Our results demonstrate that Opa-CEACAM5 binding on vaginal epithelia facilitates long term colonization of the lower genital tract, while Opa protein binding to CEACAM1 on uterine epithelia enhances gonococcal association and penetration into these tissues. While these Opa-dependent interactions with CEACAM-expressing epithelial surfaces promote infection, Opa binding by neutrophil-expressed CEACAMs counterbalances this by facilitating more effective gonococcal clearance. Furthermore, during uterine infections, CEACAM-dependent tissue invasion aggravates disease pathology by increasing the acute inflammatory response. Together, these findings demonstrate that the outcome of infection are determined by both the cell type-specific expression of human CEACAMs and the CEACAM-specificity of the Opa variants expressed, which combine to determine the level of gonococcal association with the genital mucosa versus the extent of CEACAM-dependent inflammation and gonococcal clearance by neutrophils.

  • characterization of ceacam1 and lipid raft nanoclustering association and structure by dstorm and homo fret imaging
    Biophysical Journal, 2016
    Co-Authors: Amine Driouchi, Maximilano Giuliani, Scott D Grayowen
    Abstract:

    The advent of super-resolution microscopy has revealed that most membrane proteins cluster at nanoscale lengths. Clustering and compartmentalization of membrane proteins is partially caused by lipid rafts that exist at small spatial (10-100 nm) and temporal scales, that are not perceptible using classic fluorescence microscopy (1,2). Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are cell surface glycoproteins involved in homo- and hetero-philic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. Here, we investigate the association of lipid rafts with CEACAMs using direct stochastic optical reconstruction microscopy (dSTORM). We have previously demonstrated using live cell TIRF-homoFRET-dSTORM that CEACAM1 can exist as monomers and oligomers at the cell membrane, with the monomers being predominantly organized in clustered regions and the dimers in more diffuse regions. We super-resolved CEACAM1 and lipid rafts using an anti-GFP labeled nanobody and a labeled Cholera Toxin subunit B, respectively. This approach allows us to obtain super-resolved images from which coordinates with a localization precision of 20 nm can be derived. Subsequent clustering analyses allowed us to quantify the degree of association between lipid rafts and CEACAM1. Moreover, correlating homo-FRET CEACAM data with these super-resolved maps allowed us to elucidate CEACAM monomer/oligomer distribution and association with lipid rafts. Our results show that both CEACAM1 and lipid rafts exhibit nano and micro-sized clusters of various shapes and partial spatial colocalization. This reinforces the body of work suggesting that lipid rafts are necessary to support cell adhesion, pathogen binding and intercellular signalling.

  • selection for a ceacam receptor specific binding phenotype during neisseria gonorrhoeae infection of the human genital tract
    Infection and Immunity, 2015
    Co-Authors: Anna Sintsova, Henry K Wong, Mumtaz Virji, Kelly S Macdonald, Rupert Kaul, Scott D Grayowen
    Abstract:

    ABSTRACT Infections by Neisseria gonorrhoeae are increasingly common, are often caused by antibiotic-resistant strains, and can result in serious and lasting sequelae, prompting the reemergence of gonococcal disease as a leading global health concern. N. gonorrhoeae is a human-restricted pathogen that primarily colonizes urogenital mucosal surfaces. Disease progression varies greatly between the sexes: men usually present with symptomatic infection characterized by a painful purulent urethral discharge, while in women, the infection is often asymptomatic, with the most severe pathology occurring when the bacteria ascend from the lower genital tract into the uterus and fallopian tubes. Classical clinical studies demonstrated that clinically infectious strains uniformly express Opa adhesins; however, their specificities were unknown at the time. While in vitro studies have since identified CEACAM proteins as the primary target of Opa proteins, the gonococcal specificity for this human family of receptors has not been addressed in the context of natural infection. In this study, we characterize a collection of low-passage-number clinical-specimen-derived N. gonorrhoeae isolates for Opa expression and assess their CEACAM-binding profiles. We report marked in vivo selection for expression of phase-variable Opa proteins that bind CEACAM1 and CEACAM5 but selection against expression of Opa variants that bind to the neutrophil-restricted decoy receptor CEACAM3. This is the first study showing phenotypic selection for distinct CEACAM-binding phenotypes in vivo, and it supports the opposing functions of CEACAMs that facilitate infection versus driving inflammation within the genital tract.

  • dynamic changes in the monomer dimer equilibrium of the intercellular adhesion molecule ceacam1 revealed by live cell tirf based fret microscopy
    Biophysical Journal, 2009
    Co-Authors: John Oreopoulos, Scott D Grayowen, Hannah S W Lee, Jonathan V Rocheleau, Christopher M Yip
    Abstract:

    The carcinoembryonic-antigen-related cell-adhesion molecule (CEACAM) family of proteins has been implicated in various intercellular-adhesion and intracellular signaling-mediated effects that govern the growth and differentiation of normal and cancerous cells. Recent studies also show that members of the CEACAM family play an important role in modulating the immune responses associated with infection, inflammation and cancer. At the epithelial cell surface, CEACAM1 (also known as BGP, C-CAM and CD66a) is believed to exist as a cis-homodimer. The dimeric state is thought to regulate the ability of the receptor to recruit signaling molecules, including SRC-family kinases and the tyrosine phosphatase SRC homology 2 (SH2)-domain-containing protein tyrosine phosphatase 1 (SHP1). To characterize the dynamics and monomer-dimer equilibrium of CEACAM1, we have applied a live-cell combinatorial microscopy imaging approach. Time-lapse total internal reflection fluorescence (TIRF) imaging of HELA cells overexpressing cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-CEACAM1 constructs revealed a heterogeneous distribution of bright, micron-sized jagged structures that rapidly changed shape and diffused across the cell surface. Homo- and hetero-Forster resonance energy transfer (FRET) imaging techniques (acceptor photobleaching, sensitized emission, and polarization anisotropy) were then used in combination with TIRF microscopy to determine that these jagged structures are likely aggregates of monomeric CEACAM1 proteins while the rest of the membrane contains lower concentrations of dimeric CEACAM1. This combinatorial microscopy strategy not only compliments traditional biochemical assays, but also provides new quantitative insights into spatial-temporal dynamics of this important membrane protein. We are exploiting these insights to further understand the various immune responses associated with the CEACAM family.

  • ceacam1 dynamics during neisseria gonorrhoeae suppression of cd4 t lymphocyte activation
    Journal of Immunology, 2008
    Co-Authors: Mario A. Ostrowski, Scott D Grayowen
    Abstract:

    Neisseria gonorrhoeae colony opacity-associated (Opa) proteins bind to human carcinoembryonic antigen cellular adhesion molecules (CEACAM) found on host cells including T lymphocytes. Opa binding to CEACAM1 suppresses the activation of CD4+ T cells in response to a variety of stimuli. In this study, we use primary human CD4+ T cells isolated from peripheral blood to define the molecular events occurring subsequent to Opa-CEACAM1 binding. We establish that, in contrast to other cell types, T cells do not engulf N. gonorrhoeae upon CEACAM1 binding. Instead, the bacteria recruit CEACAM1 from intracellular stores and maintain it on the T cell surface. Upon TCR ligation, the co-engaged CEACAM1 becomes phosphorylated on tyrosine residues within the ITIMs apparent in the cytoplasmic domain. This allows the recruitment and subsequent activation of the src homology domain 2-containing tyrosine phosphatases SHP-1 and SHP-2 at the site of bacterial attachment, which prevents the normal tyrosine phosphorylation of the CD3ζ-chain and ZAP-70 kinase in response to TCR engagement. Combined, this dynamic response allows the bacteria to effectively harness the coinhibitory function of CEACAM1 to suppress the adaptive immune response at its earliest step.

Bernhard B Singer - One of the best experts on this subject based on the ideXlab platform.

  • Fusobacterium spp. target human CEACAM1 via the trimeric autotransporter adhesin CbpF
    Taylor & Francis Group, 2019
    Co-Authors: Matthew L. Brewer, Bernhard B Singer, Mumtaz Virji, David Dymock, Leo R. Brady, Darryl J. Hill
    Abstract:

    Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis are pathogenic bacteria adapted to reside on human respiratory mucosal epithelia. One common feature of these species is their ability to target members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, especially CEACAM1, which is achieved via structurally distinct ligands expressed by each species. Beside respiratory epithelial cells, cells at the dentogingival junction express high levels of CEACAM1. It is possible that bacterial species resident within the oral cavity also utilise CEACAM1 for colonisation and invasion of gingival tissues. From a screen of 59 isolates from the human oral cavity representing 49 bacterial species, we identified strains from Fusobacterium bound to CEACAM1. Of the Fusobacterium species tested, the CEACAM1-binding property was exhibited by F. nucleatum (Fn) and F. vincentii (Fv) but not F. polymorphum (Fp) or F. animalis (Fa) strains tested. These studies identified that CEACAM adhesion was mediated using a trimeric autotransporter adhesin (TAA) for which no function has thus far been defined. We therefore propose the name CEACAM binding protein of Fusobacterium (CbpF). CbpF was identified to be present in the majority of unspeciated Fusobacterium isolates confirming a subset of Fusobacterium spp. are able to target human CEACAM1

  • Soluble CEACAM1 and CEACAM6 are differently expressed in blood serum of pregnant women during normal pregnancy.
    American journal of reproductive immunology (New York N.Y. : 1989), 2017
    Co-Authors: Pawel Mach, Alexandra Gellhaus, Sebastian Prager, Thomas Moore, Gunther Wennemuth, Rainer Kimmig, Angela Köninger, Bernhard B Singer
    Abstract:

    Problem CEACAM1 and CEACAM6 belong to the carcinoembryonic antigen (CEA) family and may play an immune-modulatory role during pregnancy. The aim of the study was to determine the blood serum levels of soluble CEACAM1 and CEACAM6 over the course of pregnancy and postpartum. Method of study CEACAM1 and CEACAM6 levels were determined with customized in-house Sandwich-enzyme-linked immunosorbent assay (ELISA) systems. The study population (n=125) was divided into four groups according to the pregnancy trimester and postpartum. Additionally, samples of non-pregnant women (n=14) were analyzed. Results Serum levels of CEACAM1 in healthy pregnant women were much lower than in non-pregnant women, a difference not seen for CEACAM6. Comparison between the trimesters and postpartum revealed a significant difference in CEACAM1 serum levels. The highest CEACAM1 levels were detected in third trimester. These levels were statistically significantly different from the CEACAM1 levels in first trimester and second trimester. The lowest levels were observed in the second trimester. Postpartum CEACAM1 serum concentrations were slightly lower than in the third trimester, but higher than in the first trimester and significantly higher compared to levels in the second trimester. Conclusion Decreased concentration of CEACAM1 during the pregnancy suggests its regulatory role in the immune tolerance during the course of pregnancy.

  • Binding of Candida albicans to Human CEACAM1 and CEACAM6 Modulates the Inflammatory Response of Intestinal Epithelial Cells
    mBio, 2017
    Co-Authors: Esther Klaile, Kerstin A Heyl, Mario M Muller, Miriam R. Schäfer, Ann-katrin Clauder, Sabina Feer, Magdalena Stock, Tilman E. Klassert, Peter F. Zipfel, Bernhard B Singer
    Abstract:

    Candida albicans colonizes human mucosa, including the gastrointestinal tract, as a commensal. In immunocompromised patients, C. albicans can breach the intestinal epithelial barrier and cause fatal invasive infections. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM5 (CEA), and CEACAM6 (CD66c) are immunomodulatory receptors expressed on human mucosa and are recruited by bacterial and viral pathogens. Here we show for the first time that a fungal pathogen (i.e., C. albicans) also binds directly to the extracellular domain of human CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Binding was specific for human CEACAMs and mediated by the N-terminal IgV-like domain. In enterocytic C2BBe1 cells, C. albicans caused a transient tyrosine phosphorylation of CEACAM1 and induced higher expression of membrane-bound CEACAM1 and soluble CEACAM6. Lack of the CEACAM1 receptor after short hairpin RNA (shRNA) knockdown abolished CXCL8 (interleukin-8) secretion by C2BBe1 cells in response to C. albicans In CEACAM1-competent cells, the addition of recombinant soluble CEACAM6 reduced the C. albicans-induced CXCL8 secretion.IMPORTANCE The present study demonstrates for the first time that fungal pathogens can be recognized by at least four members of the immunomodulatory CEACAM receptor family: CEACAM1, -3, -5, and -6. Three of the four receptors (i.e., CEACAM1, -5, and -6) are expressed in mucosal cells of the intestinal tract, where they are implicated in immunomodulation and control of tissue homeostasis. Importantly, the interaction of the major fungal pathogen in humans Candida albicans with CEACAM1 and CEACAM6 resulted in an altered epithelial immune response. With respect to the broad impact of CEACAM receptors on various aspects of the innate and the adaptive immune responses, in particular epithelial, neutrophil, and T cell behavior, understanding the role of CEACAMs in the host response to fungal pathogens might help to improve management of superficial and systemic fungal infections.

  • soluble ceacam8 interacts with ceacam1 inhibiting tlr2 triggered immune responses
    PLOS ONE, 2014
    Co-Authors: Bernhard B Singer, Annina Heinrich, Frauke Schreiber, Ramona Bindingliermann, Luis Carlos Berrocalalmanza, Kerstin A Heyl, Mario M Muller, Janine Zweigner, Andreas Weimann, Hortense Slevogt
    Abstract:

    Lower respiratory tract bacterial infections are characterized by neutrophilic inflammation in the airways. The carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 8 is expressed in and released by human granulocytes. Our study demonstrates that human granulocytes release CEACAM8 in response to bacterial DNA in a TLR9-dependent manner. Individuals with a high percentage of bronchial lavage fluid (BALF) granulocytes were more likely to have detectable levels of released CEACAM8 in the BALF than those with a normal granulocyte count. Soluble, recombinant CEACAM8-Fc binds to CEACAM1 expressed on human airway epithelium. Application of CEACAM8-Fc to CEACAM1-positive human pulmonary epithelial cells resulted in reduced TLR2-dependent inflammatory responses. These inhibitory effects were accompanied by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and by recruitment of the phosphatase SHP-1, which could negatively regulate Toll-like receptor 2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. Our results suggest a new mechanism by which granulocytes reduce pro-inflammatory immune responses in human airways via secretion of CEACAM8 in neutrophil-driven bacterial infections.

  • carcinoembryonic antigen cea related cell adhesion molecules are co expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non typable haemophilus influenzae moraxella catarrhalis tlr3 and type i and ii int
    Respiratory Research, 2013
    Co-Authors: Esther Klaile, Annina Heinrich, Kerstin A Heyl, Mario M Muller, Tilman E. Klassert, Inka Scheffrahn, Hendrik Dienemann, Christiane Grunewald, Robert Bals, Bernhard B Singer
    Abstract:

    Background The carcinoembryonic antigen (CEA)-related cell adhesion molecules CEACAM1 (BGP, CD66a), CEACAM5 (CEA, CD66e) and CEACAM6 (NCA, CD66c) are expressed in human lung. They play a role in innate and adaptive immunity and are targets for various bacterial and viral adhesins. Two pathogens that colonize the normally sterile lower respiratory tract in patients with chronic obstructive pulmonary disease (COPD) are non-typable Haemophilus influenzae (NTHI) and Moraxella catarrhalis. Both pathogens bind to CEACAMs and elicit a variety of cellular reactions, including bacterial internalization, cell adhesion and apoptosis.

Scott D. Gray-owen - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of CEACAM1 Structure and Dynamics by Correlated homo-FRET and dSTORM Imaging
    Biophysical Journal, 2015
    Co-Authors: Amine Driouchi, Scott D. Gray-owen
    Abstract:

    Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are involved in intercellular interactions that control cellular growth, differentiation, transformation, tumourigenesis, inflammation and infection. Previous studies of the CEACAM family have provided compelling evidence for the existence of a complex equilibrium of monomers and dimers (or oligomers) and have identified key factors that appear to influence the equilibrium. To date, how CEACAMs behave, interact, organize and self-associate at the cell membrane remains poorly understood. We have previously demonstrated using live cell TIRF-homoFRET imaging that a heterogeneous population of CEACAM1 monomers and oligomers exist throughout the cell membrane but that the population was skewed towards the monomeric form in actin-rich domains. However these homo-FRET maps are diffraction-limited and do not represent the true spatial distribution or dynamics of CEACAM1. In order to address these questions, we have applied direct stochastic optical reconstruction microscopy (dSTORM), to directly map the structure, distribution and oligomeric states of CEACAM1. We also applied two colour dSTORM imaging of actin filaments and intracellular CEACAM1 in order to further characterize the oligomeric form of CEACAM1 during transport from the cytosol to the cell membrane. These powerful approaches will enable us to elucidate the spatial distribution of CEACAM1, and aid in identifying correlations between CEACAM1 self-association, molecular structure, and function.

  • Monitoring the CEACAM1 Monomer and Dimer Distribution upon Cell-Substrate Contact
    Biophysical Journal, 2013
    Co-Authors: Kelsey J. Downie, Jocelyn R. Lo, Scott D. Gray-owen
    Abstract:

    The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is an intercellular adhesion molecule that plays a key role in processes related to cell growth, differentiation, and infection in normal and cancerous cells. Expressed on the cell surface, its extracellular domain participates in interactions with CEACAMs on neighbouring cells, as well as interacting with Opa proteins expressed on Neisseria gonorrhoeae and Neisseria meningitides in order to facilitate bacterial entry. While CEACAM1 is known to exist in both monomeric and dimeric states that are heterogeneously distributed at the cell surface, which form participates in the various inter-cellular and bacterial interactions remains a mystery. Resolving this uncertainty is fundamental to understanding the role of the receptor, as the monomer-dimer status of CEACAM1 has been shown to differentially affect its ability to bind certain downstream signalling molecules. We are using live cell fluorescence and homogeneous Forster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization microscopy (TIRFPM) and confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of EYFP-labeled CEACAM1. Cells expressing the labeled receptor are monitored as they come into contact with CEACAM1-engaging surfaces, such as other cells or glass dishes that have been patterned with Opa+ Neisseria gonorrhoeae. These approaches will allow us to directly examine the initial stages of CEACAM1 association with CEACAM1-interacting proteins and will answer critical questions regarding the nature of these interactions. Such insights are essential for understanding the nature of CEACAM1 signalling, function, and regulation, and ultimately targeting its functions for purposes such as cancer therapy or the treatment of infection.

  • Phosphatidylinositol 3-Kinases in Carcinoembryonic Antigen-related Cellular Adhesion Molecule-mediated Internalization ofNeisseria gonorrhoeae
    Journal of Biological Chemistry, 2003
    Co-Authors: James W. Booth, David Telio, Edward H. Liao, Tsuyoshi Matsuo, Shannon E. Mccaw, Sergio Grinstein, Scott D. Gray-owen
    Abstract:

    Abstract Neisseria gonorrhoeae can be internalized by mammalian cells through interactions between bacterial opacity-associated (Opa) adhesins and members of the humancarcinoembryonic antigen-relatedcellular adhesion molecule (CEACAM) family. We examined the role of phosphatidylinositol 3-kinases (PI3Ks) in gonococcal invasion of epithelial cell lines expressing either CEACAM1 or CEACAM3. CEACAM3-mediated internalization, but not that mediated by CEACAM1, was accompanied by localized and transient accumulation of the class I PI3K product phosphatidylinositol 3,4,5-trisphosphate at sites of bacterial engulfment. Inhibition of phosphatidylinositol 3-kinases reduced CEACAM3-mediated uptake but, paradoxically, led to an increase in intracellular survival of bacteria internalized via either CEACAM1 or CEACAM3, suggesting additional roles for PI3K products. Consistent with this finding, the class III PI3K product phosphatidylinositol 3-phosphate accumulated and persisted in the membrane of gonococcal phagosomes after internalization. Inhibition of PI3K blocked phagosomal acquisition of the late endosomal marker lysosome-associated membrane protein 2 and reduced phagosomal acidification. Inhibiting phagosomal acidification with concanamycin A also increased survival of intracellular gonococci. These results suggest two modes of action of phosphatidylinositol 3-kinases during internalization of gonococci: synthesis of phosphatidylinositol 3,4,5-trisphosphate is important for CEACAM3-mediated uptake, while phosphatidylinositol 3-phosphate is needed for phagosomal maturation and acidification, which are required for optimal bacterial killing.

Hiroshi Iida - One of the best experts on this subject based on the ideXlab platform.

  • Identification of CEACAM6 as an Intermediate Filament-Associated Protein Expressed in Sertoli Cells of Rat Testis
    Biology of reproduction, 2011
    Co-Authors: Hitoshi Kurio, Hiroshi Hatsuda, Emi Murayama, Takane Kaneko, Hiroshi Iida
    Abstract:

    CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6 gene) has recently been isolated by differential display followed by RT-PCR and DNA sequence analyses. CEACAM6 is a member of an immunoglobulin superfamily and encodes a protein of 266 amino acid residues possessing one immunoglobulin (Ig)-like domain. RT-PCR analysis showed that CEACAM6 was dominantly expressed in rat testis and its expression level prominently increased after 6 wk of postnatal development in testis. Immunohistochemical analyses using the anti-CEACAM6 antibody revealed that CEACAM6 colocalized with intermediate filaments (vimentin) in Sertoli cells and interstitial cells. The association between CEACAM6 and vimentin was observed throughout postnatal development in rat testis. Transfection experiments performed in COS-7 cells suggested that overexpression of CEACAM6 brought about aggregation of vimentin filament around nuclei with which CEACAM6 colocalized and that the N-terminus region of CEACAM6, including the Ig-like domain, seemed to be required for association with vimentin filaments. Interaction between CEACAM6 and vimentin in rat testis and transfected COS-7 cells was confirmed by immunoprecipitation. Our observations strongly suggested that CEACAM6 might be a novel intermediate filament-associated protein involved in regulation of vimentin architecture in Sertoli cells. CEACAM6, cytoskeleton, Sertoli cells, testis, vimentin

  • Intron Retention Generates a Novel Isoform of CEACAM6 That May Act as an Adhesion Molecule in the Ectoplasmic Specialization Structures Between Spermatids and Sertoli Cells in Rat Testis
    Biology of reproduction, 2008
    Co-Authors: Hitoshi Kurio, Emi Murayama, Takane Kaneko, Yosaburo Shibata, Tetsuichiro Inai, Hiroshi Iida
    Abstract:

    By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigenrelated cell adhesion molecule 6 (CEACAM6) and its novel spliced variant CEACAM6-Long (CEACAM6-L) from rat testis. CEACAM6-L mRNA was generated by retention of 67 nucleotide-length third intron in CEACAM6 gene. CEACAM6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that CEACAM6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII–VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis. gamete biology, gametogenesis, Sertoli cells, sperm, testis

Hitoshi Kurio - One of the best experts on this subject based on the ideXlab platform.

  • Identification of CEACAM6 as an Intermediate Filament-Associated Protein Expressed in Sertoli Cells of Rat Testis
    Biology of reproduction, 2011
    Co-Authors: Hitoshi Kurio, Hiroshi Hatsuda, Emi Murayama, Takane Kaneko, Hiroshi Iida
    Abstract:

    CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6 gene) has recently been isolated by differential display followed by RT-PCR and DNA sequence analyses. CEACAM6 is a member of an immunoglobulin superfamily and encodes a protein of 266 amino acid residues possessing one immunoglobulin (Ig)-like domain. RT-PCR analysis showed that CEACAM6 was dominantly expressed in rat testis and its expression level prominently increased after 6 wk of postnatal development in testis. Immunohistochemical analyses using the anti-CEACAM6 antibody revealed that CEACAM6 colocalized with intermediate filaments (vimentin) in Sertoli cells and interstitial cells. The association between CEACAM6 and vimentin was observed throughout postnatal development in rat testis. Transfection experiments performed in COS-7 cells suggested that overexpression of CEACAM6 brought about aggregation of vimentin filament around nuclei with which CEACAM6 colocalized and that the N-terminus region of CEACAM6, including the Ig-like domain, seemed to be required for association with vimentin filaments. Interaction between CEACAM6 and vimentin in rat testis and transfected COS-7 cells was confirmed by immunoprecipitation. Our observations strongly suggested that CEACAM6 might be a novel intermediate filament-associated protein involved in regulation of vimentin architecture in Sertoli cells. CEACAM6, cytoskeleton, Sertoli cells, testis, vimentin

  • Intron Retention Generates a Novel Isoform of CEACAM6 That May Act as an Adhesion Molecule in the Ectoplasmic Specialization Structures Between Spermatids and Sertoli Cells in Rat Testis
    Biology of reproduction, 2008
    Co-Authors: Hitoshi Kurio, Emi Murayama, Takane Kaneko, Yosaburo Shibata, Tetsuichiro Inai, Hiroshi Iida
    Abstract:

    By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigenrelated cell adhesion molecule 6 (CEACAM6) and its novel spliced variant CEACAM6-Long (CEACAM6-L) from rat testis. CEACAM6-L mRNA was generated by retention of 67 nucleotide-length third intron in CEACAM6 gene. CEACAM6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that CEACAM6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII–VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis. gamete biology, gametogenesis, Sertoli cells, sperm, testis