Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF-and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation. INTRODUCTIONThe acquisition of polarity in epithelial cells is a fundamental mechanism crucial for the development of multicellular organisms. This process involves transmembrane proteins acting as cell surface organizers that nucleate cortical proteins, thus providing a scaffold for the building of junctional complexes that physically separate the apical surface from the basolateral domain of the plasma membrane (Knust, 2000;Tepass et al., 2001). Sorting of newly synthesized proteins is then necessary to maintain the polarized distribution of apical and basolateral proteins Nelson and Yeaman, 2001). Cell surface organizers such as integrins and cadherins have been identified in mammalian epithelial cells (Yeaman et al., 1999), but in the last few years, genetics in Drosophila proved to be instrumental in opening new perspectives on protein complexes involved in epithelial polarity (Muller, 2000). Among such complexes, the Crumbs complex stands out because it contains an apical transmembrane protein crucial for cell morphogenesis (Rashbass and Skaer, 2000;Tepass et al., 2001). Crumbs is a large (2139-aa) glycoprotein with an extracellular domain containing 30 EGF-like repeats and four laminin A-like G domains, a transmembrane domain, and a short cytoplasmic domain of 37 amino acids (Tepass et al., 1990). Expression of crb starts at gastrulation, and the protein is accumulated at the subapical region (marginal zone) of ectodermal epithelial cells where it colocalizes with two other gene products involved in epithelial polarity, Stardust (sdt) and Discs lost (dlt). These three genes, crb, s...

Members of the LAP protein family, LET-413 inCaenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2 0 -deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.

dCrumbs is an apical organizer crucial for the maintenance of epithelial polarity in Drosophila (1). It is known that dCrumbs interacts with Discs lost (Dlt), a protein with four PDZ (PSD95/Discs Large/ZO-1) domains (2), and Stardust (Sdt), a protein of the MAGUK (membrane-associated guanylate kinase) family (3, 4). We have searched for potential homologs of Dlt in human epithelial cells and characterized one of them in intestinal epithelial cells. Human INAD-like (hINADl) contains 8 PDZ domains, is concentrated in tight junctions, and is also found at the apical plasma membrane. Overexpression of hINADl disrupted the tight junctions localization of ZO-1 and 3. We also identified a partial cDNA coding the transmembrane and cytoplasmic domains of a new human crumbs (CRB3) expressed in Caco-2 cells. This CRB3 was able to interact through its C-terminal end with the N-terminal domain of hINADl. Taken together, the data indicate that hINADl is likely to represent a Dlt homolog in mammalian epithelial cells and might be involved in regulating the integrity of tight junctions. We thus propose to rename hINADl PATJ for protein associated to tight junctions.

Objectives-During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration.Among molecules at the interendothelial junction, CD146 is involved in cell-cell cohesion and permeability, but its role in monocyte transmigration is unknown. Methods and Results-TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes.Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes. Conclusions-Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation. Key Words: endothelial cells Ⅲ inflammation Ⅲ cytokines Ⅲ human Ⅲ adhesion molecules T he endothelial junctions play a fundamental role in endothelial integrity, vascular permeability, and cellular traffic. 1 At least 2 types of cell-to-cell junctional structures have been identified in the endothelium: adherens junctions (AJ) and tight junctions (TJ). 2 These junctions are tightly regulated structures composed of several adhesion molecules interacting with cytoskeletal proteins. 3 Among the adhesive molecules, endothelial VE-cadherin 4 is localized in AJ and junctional adhesion molecule (JAM) 5 in TJ, whereas other molecules such as PECAM-1/CD31 (platelet endothelial cell adhesion molecule-1) and CD99 are not restricted to 1 type of junctional structure. 6 The inflammatory response is characterized by leukocyte infiltration from the circulation toward the tissues, after a multistep process in which proinflammatory endothelial activation results in increased vascular permeability and then in leukocyte adhesion and transmigration. 7 Endothelial activation is mediated by several inflammatory cytokines. Among them, TNF␣ increases the expression of cell adhesion molecules like ICAM-1 or VCAM-1 and induces the redistribution of junctional adhesion molecules such as PECAM-1, JAM, VE-cadherin, and CD99 which, in turn, promote the transendothelial migration of leukocytes. 8 We have previously shown that CD146 (S-Endo1 Ag) is a component of the endothelial junction localized outside defined junctional structures. 9 CD146, also referred to as MUC18, is a member of the immunoglobulin superfamily (IgSF) constitutively expressed in all typ...

The Crumbs complex that also contains the cortical proteins Stardust and DPATJ (a homologue of PATJ), is crucial for the building of epithelial monolayers in Drosophila. Although loss of function of the Crumbs or Stardust genes prevents the stabilization of a belt of adherens junctions at the apico-lateral border of the cells, no phenotype has been described for the Dpatj gene and its role in epithelial morphogenesis and polarity remains unknown. We have produced downregulated PATJ stable lines of Caco2 to clarify its role in epithelial morphogenesis. In PATJ knockdown cells, Pals1 (a Stardust homologue) is no longer associated with tight junctions whereas Crumbs3 (Crb3) is accumulated into a compartment spatially close to the apical membrane and related to early endosomes. Furthermore, occludin and ZO-3, two proteins of tight junctions are mislocalized on the lateral membrane indicating that PATJ plays a novel role in the building of tight junctions by providing a link between their lateral and apical components. Thus, PATJ stabilizes the Crb3 complex and regulates the spatial concentration of several components at the border between the apical and lateral domains.

Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal diseases including Leber's congenital amaurosis (LCA) and retinitis pigmentosa type 12. The CRB1 transmembrane protein localizes at a subapical region (SAR) above intercellular adherens junctions between photoreceptor and Müller glia (MG) cells. We demonstrate that the Crb1-/- phenotype, as shown in Crb1-/- mice, is accelerated and intensified in primary retina cultures. Immuno-electron microscopy showed strong Crb1 immunoreactivity at the SAR in MG cells but barely in photoreceptor cells, whereas Crb2, Crb3, Patj, Pals1 and Mupp1 were present in both cell types. Human CRB1, introduced in MG cells in Crb1-/- primary retinas, was targeted to the SAR. RNA interference-induced silencing of the Crb1-interacting-protein Pals1 (protein associated with Lin7; Mpp5) in MG cells resulted in loss of Crb1, Crb2, Mupp1 and Veli3 protein localization and partial loss of Crb3. We conclude that Pals1 is required for correct localization of Crb family members and its interactors at the SAR of polarized MG cells.

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Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. We previously described Erbin as a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. We show here that Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.