Biomedical subjects

Klaus Ebnet

Publications and source records attributed to Klaus Ebnet.

11 recordsLinked to original sources

PubMed2006

JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration.

Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.

Amino Acid Sequence↗

PubMed2006

A distinct PAR complex associates physically with VE-cadherin in vertebrate endothelial cells.

A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Junctional adhesion molecule-a participates in the formation of apico-basal polarity through different domains.

Junctional adhesion molecule (JAM)-A is an integral membrane protein at tight junctions of epithelial cells which associates with the cell polarity protein PAR-3. Here, we demonstrate that downregulation of JAM-A impairs the ability of MDCK II cells to form cysts in a three-dimensional matrix indicating the requirement of JAM-A for the development of apico-basal polarity. To define the regions of JAM-A important for this function, we have generated MDCK II cell lines stably expressing inducible JAM-A mutants. Mutants of JAM-A which were designed to mislocalize strongly impaired the development of cysts and the formation of functional tight junctions. Surprisingly, similar mutants that lacked the PDZ domain-binding motif at the C-terminus were still impaired in apico-basal polarity formation suggesting that additional regions within the cytoplasmic tail of JAM-A are important for the function of JAM-A. A JAM-A mutant lacking the first Ig-like domain necessary for homophilic binding localized to cell-cell contacts similar to wild-type JAM-A. However, despite this same localization, this mutant interfered with cell polarity and tight junction formation. Together our findings suggest an important role for JAM-A in the development of apico-basal polarity in epithelial cells and identify regions in JAM-A which are critical for this role.

Amino Acid Motifs↗

PubMed2005

Inhibitory effect of naked neural BC1 RNA or BC200 RNA on eukaryotic in vitro translation systems is reversed by poly(A)-binding protein (PABP).

Regulated protein biosynthesis in dendrites of neurons might be a key mechanism underlying learning and memory. Neuronal dendritic BC1 RNA and BC200 RNA and similar small untranslated RNAs inhibit protein translation in vitro systems, such as rabbit reticulocyte lysate. Likewise, co-transfection of these RNAs with reporter mRNA suppressed translation levels in HeLa cells. The oligo(A)-rich region of all active small RNAs were identified as the RNA domains chiefly responsible for the inhibitory effects. Addition of recombinant human poly(A)-binding protein (PABP) significantly compensated the inhibitory effect of the small oligo(A)-rich RNA. In vivo, all BC1 RNA appears to be complexed with PABP. Nevertheless, in the micro-environment of dendritic spines of neuronal cells, BC1 RNPs or BC200 RNPs might mediate regulatory functions by differential interactions with locally limited PABP and/or directly or indirectly, with other translation initiation factors.

Animals↗

PubMed2005

Association of Csk to VE-cadherin and inhibition of cell proliferation.

Vascular endothelial cadherin (VE-cadherin) mediates contact inhibition of cell growth in quiescent endothelial cell layers. Searching for proteins that could be involved in VE-cadherin signaling, we found the cytosolic C-terminal Src kinase (Csk), a negative regulator of Src family kinases. We show that Csk binds via its SH2 domain to the phosphorylated tyrosine 685 of VE-cadherin. VE-cadherin recruits Csk to cell contacts and both proteins can be co-precipitated from cell lysates of transfected cells and endothelial cells. Association of VE-cadherin and Csk in endothelial cells increased with increasing cell density. CHO cells expressing the tyrosine replacement mutant VE-cadherin-Y685F grow to higher cell densities than cells expressing wild-type VE-cadherin. Overexpression of Csk in these cells under an inducible promoter inhibits cell proliferation in the presence and absence of VE-cadherin, but not in the presence of VE-cadherin-Y685F. Reduction of Csk expression by RNA interference enhances endothelial cell proliferation. Our results suggest that the phosphorylated tyrosine residue 685 of VE-cadherin and probably the binding of Csk to this site are involved in inhibition of cell growth triggered by cell density.

Animals↗

PubMed2004

Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts.

Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell-cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Spermatid differentiation requires the assembly of a cell polarity complex downstream of junctional adhesion molecule-C.

During spermatogenesis in the mammalian testis, stem cells (spermatogonia) differentiate into spermatocytes, which subsequently undergo two consecutive meiotic divisions to give rise to haploid spermatids. These cells are initially round but progressively elongate, condense their nuclei, acquire flagellar and acrosomal structures, and shed a significant amount of their cytoplasm to form spermatozoa (the sperm cells) in a developmental cascade termed spermiogenesis. Defects in these processes will lead to a lack of mature sperm cells (azoospermia), which is a major cause of male infertility in the human population. Here we report that a cell-surface protein of the immunoglobulin superfamily, junctional adhesion molecule-C (JAM-C), is critically required for the differentiation of round spermatids into spermatozoa in mice. We found that Jam-C is essential for the polarization of round spermatids, a function that we attribute to its role in the assembly of a cell polarity complex.

Animals↗

PubMed2004

Junctional adhesion molecules (JAMs): more molecules with dual functions?

Junctional adhesion molecules (JAMs) are members of an immunoglobulin subfamily expressed by leukocytes and platelets as well as by epithelial and endothelial cells, in which they localize to cell-cell contacts and are specifically enriched at tight junctions. The recent identification of extracellular ligands and intracellular binding proteins for JAMs suggests two functions for JAMs. JAMs associate through their extracellular domains with the leukocyte beta2 integrins LFA-1 and Mac-1 as well as with the beta1 integrin alpha4beta1. All three integrins are involved in the regulation of leukocyte-endothelial cell interactions. Through their cytoplasmic domains, JAMs directly associate with various tight junction-associated proteins including ZO-1, AF-6, MUPP1 and the cell polarity protein PAR-3. PAR-3 is part of a ternary protein complex that contains PAR-3, atypical protein kinase C and PAR-6. This complex is highly conserved through evolution and is involved in the regulation of cell polarity in organisms from Caenorhabditis elegans and Drosophila to vertebrates. These findings point to dual functions for JAMs: they appear to regulate both leukocyte/platelet/endothelial cell interactions in the immune system and tight junction formation in epithelial and endothelial cells during the acquisition of cell polarity.

Animals↗

PubMed2003

The junctional adhesion molecule (JAM) family members JAM-2 and JAM-3 associate with the cell polarity protein PAR-3: a possible role for JAMs in endothelial cell polarity.

Tight junctions play a central role in the establishment of cell polarity in vertebrate endothelial and epithelial cells. A ternary protein complex consisting of the cell polarity proteins PAR-3 and PAR-6 and the atypical protein kinase C localizes at tight junctions and is crucial for tight junction formation. We have recently shown that PAR-3 directly associates with the junctional adhesion molecule (JAM), which suggests that the ternary complex is targeted to tight junctions of epithelial cells through PAR-3 binding to JAM. The expression of JAM-related proteins by endothelial cells prompted us to test whether recruitment of the ternary complex in endothelial cells can occur through binding to JAM-2, JAM-3, endothelial cell-selective adhesion molecule (ESAM) or coxsackie- and adenovirus receptor (CAR). Here we show that the two JAM-related proteins JAM-2 and JAM-3 directly associate with PAR-3. The association between PAR-3 and JAM-2/-3 is mediated through the first PDZ domain of PAR-3. In agreement with the predominant expression of JAM-2 and JAM-3 in endothelial cells, we found that PAR-3 is expressed by endothelial cells in vivo and is localized at cell contacts of cultured endothelial cells. PAR-3 associates with JAM-2/-3 but not with the JAM-related Ig-superfamily members ESAM or CAR. In addition, we show that the tight junction-associated protein ZO-1 associates with JAM-2/-3 in a PDZ domain-dependent manner. Using ectopic expression of JAM-2 in CHO cells, we show that the junctional localization of JAM-2 is regulated by serine phosphorylation and that its clustering at cell-cell contacts recruits endogenous PAR-3 and ZO-1. Our findings suggest that JAM-2 affects endothelial cell junctions by its regulated clustering at intercellular contacts, and they support a role for JAM-2, and possibly JAM-3, in tight junction formation of endothelial cells.

Adaptor Proteins, Signal Transducing↗

PubMed2002

aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization.

We have previously shown that aPKC interacts with cell polarity proteins PAR-3 and PAR-6 and plays an indispensable role in cell polarization in the C. elegans one-cell embryo as well as in mammalian epithelial cells. Here, to clarify the molecular basis underlying this aPKC function in mammalian epithelial cells, we analyzed the localization of aPKC and PAR-3 during the cell repolarization process accompanied by wound healing of MTD1-A epithelial cells. Immunofluorescence analysis revealed that PAR-3 and aPKClambda translocate to cell-cell contact regions later than the formation of the primordial spot-like adherens junctions (AJs) containing E-cadherin and ZO-1. Comparison with three tight junction (TJ) membrane proteins, JAM, occludin and claudin-1, further indicates that aPKClambda is one of the last TJ components to be recruited. Consistently, the expression of a dominant-negative mutant of aPKClambda (aPKClambdakn) in wound healing cells does not inhibit the formation of the spot-like AJs; rather, it blocks their development into belt-like AJs. These persistent spot-like AJs in aPKClambda-expressing cells contain all TJ membrane proteins and PAR-3, indicating that aPKC kinase activity is not required for their translocation to these premature junctional complexes but is indispensable for their further differentiation into belt-like AJs and TJs. Cortical bundle formation is also blocked at the intermediate step where fine actin bundles emanating from premature cortical bundles link the persistent spot-like AJs at apical tips of columnar cells. These results suggest that aPKC contributes to the establishment of epithelial cell polarity by promoting the transition of fibroblastic junctional structures into epithelia-specific asymmetric ones.

Actin Cytoskeleton↗

PubMed2002

A transmembrane tight junction protein selectively expressed on endothelial cells and platelets.

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.

Animals↗