Apico-basal elongation requires a drebrin-E-EB3 complex in columnar human epithelial cells.

Although columnar epithelial cells are known to acquire an elongated shape, the mechanisms involved in this morphological feature have not yet been completely elucidated. Using columnar human intestinal Caco2 cells, it was established here that the levels of drebrin E, an actin-binding protein, increase in the terminal web both in vitro and in vivo during the formation of the apical domain. Drebrin E depletion was found to impair cell compaction and elongation processes in the monolayer without affecting cell polarity or the formation of tight junctions. Decreasing the drebrin E levels disrupted the normal subapical F-actin-myosin-IIB-ßII-spectrin network and the apical accumulation of EB3, a microtubule-plus-end-binding protein. Decreasing the EB3 levels resulted in a similar elongation phenotype to that resulting from depletion of drebrin E, without affecting cell compaction processes or the pattern of distribution of F-actin-myosin-IIB. In addition, EB3, myosin IIB and ßII spectrin were found to form a drebrin-E-dependent complex. Taken together, these data suggest that this complex connects the F-actin and microtubule networks apically during epithelial cell morphogenesis, while drebrin E also contributes to stabilizing the actin-based terminal web.

CD146 and its soluble form regulate monocyte transendothelial migration.

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.

PSD95beta regulates plasma membrane Ca2+ pump localization at the photoreceptor synapse.

At the presynaptic plasma membrane of the photoreceptor the correct localization of the calcium extruder, plasma membrane Ca2+-ATPase (PMCA), is determined by a unique protein complex. Here, the role of two proteins within the complex; membrane palmitoylated protein 4 (MPP4) and postsynaptic density protein 95 (PSD95) is investigated in more details, using Mpp4 and Psd95 mutant mice. MPP4 deficiency results in the loss of both PMCA and PSD95 from the photoreceptor synapse. Truncation of the C-terminal part of MPP4 leads to a loss of PSD95 and mislocalization of PMCA, while truncation of the C-terminal part of PSD95 did not affect the localization of the complex members. Lentivirus-mediated molecular replacement strategy was used to selectively express either PSD95alpha or PSD95beta in wild type or Mpp4 mutant primary retinal explants. Silencing of the Psd95 gene resulted in the loss of presynaptic MPP4 and PMCA1. The plasma membrane localization of MPP4 and PMCA1 could be restored by the expression of PSD95beta. We conclude that both scaffold proteins PSD95beta and MPP4 are essential for the modulation of PMCA levels at the presynaptic plasma membrane and thereby influence the photoreceptor synaptic calcium handling.

Vesicular trafficking and secretion of matrix metalloproteinases-2, -9 and tissue inhibitor of metalloproteinases-1 in neuronal cells.

Matrix metalloproteinases (MMPs) are endopeptidases that cleave matrix, soluble and membrane-bound proteins and are regulated by their endogenous inhibitors the tissue inhibitors of MMPs (TIMPs). Nothing is known about MMP/TIMP trafficking and secretion in neuronal cells. We focussed our attention on the gelatinases MMP-2 and MMP-9, and their inhibitor TIMP-1. MMPs and TIMP-1 fused to GFP were expressed in N2a neuroblastoma and primary neuronal cells to study trafficking and secretion using real time video-microscopy, imaging, electron microscopy and biochemical approaches. We show that MMPs and TIMP-1 are secreted in 160-200 nm vesicles in a Golgi-dependent pathway. These vesicles distribute along microtubules and microfilaments, co-localise differentially with the molecular motors kinesin and myosin Va and undergo both anterograde and retrograde trafficking. MMP-9 retrograde transport involves the dynein/dynactin molecular motor. In hippocampal neurons, MMP-2 and MMP-9 vesicles are preferentially distributed in the somato-dendritic compartment and are found in dendritic spines. Non-transfected hippocampal neurons also demonstrate vesicular secretion of MMP-2 in both its pro- and active forms and gelatinolytic activity localised within dendritic spines. Our results show differential trafficking of MMP and TIMP-1-containing vesicles in neuronal cells and suggest that these vesicles could play a role in neuronal and synaptic plasticity.

CRB3 binds directly to Par6 and regulates the morphogenesis of the tight junctions in mammalian epithelial cells.

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.

Junctional recruitment of mammalian Scribble relies on E-cadherin engagement.

Members of the LAP protein family, LET-413 in Caenorhabditis 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'-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.

[Role of Crumbs proteins in the control of epithelial cell and photoreceptor morphogenesis]. / Rôle des protéines Crumbs dans le contrôle de la morphogenèse des cellules épithéliales et des photorécepteurs.

Degeneration of retina can have many causes and among the genes involved, CRB1 has been shown to be associated with Retinitis pigmentosa (RP) group 12 and Leber congenital amaurosis (LCA), two dramatic pathologies in young patients. CRB1 belongs to a family of genes conserved from Caenorhabditis elegans to human. In Drosophila melanogaster, for example, crb is essential both for the formation of the adherens junctions in epithelial cells of ectodermal origin during gastrulation and for the morphogenesis of photoreceptors in the eye. Crumbs is a transmembrane protein with a short cytoplasmic domain that interacts with scaffold proteins, Stardust and Discs lost, and with the apical cytoskeleton made of moesin and betaheavy-spectrin. The extracellular domain of Crumbs is essential for its function in photoreceptors but so far there are no known proteins interacting with it. In human, there are three known crb homologues, CRB1, 2 and 3, and CRB1 is expressed in the retina and localizes to the adherens junctions of the rods. Based on the model drawn from Drosophila, CRB1 could be involved in maintaining the morphology of rods to ensure a normal function of the retina. This is supported by the fact that the homologues of the known partners of Crumbs are also conserved in human and expressed in the retina. Understanding the precise molecular mechanism by which CRB1 acts will help to find new therapies for patients suffering from RP12 and LCA.

Hook2 is involved in the morphogenesis of the primary cilium.

Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localizes at the Golgi apparatus and centrosome/basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilizes pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP::Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation.

Crumbs proteins in epithelial morphogenesis.

Cell polarity is an essential feature of most eukaryotic cells, especially epithelial cells in multicellular animals. Polarity protein complexes that regulate epithelial organization have been identified. In this review, it is proposed to describe how the Crumbs complex acts in the process of cell polarity and epithelial organization. During the last decade, several partners of Crumbs, an apical transmembrane protein, have been identified and their direct or indirect associations with the cytoplasmic domain of Crumbs have been dissected. In addition, mutants of several of the genes encoding proteins belonging to the Crumbs network have been obtained in animals ranging from flies to mouse, which have led to a better understanding of their functions in vivo. These functions include polarity axis formation, stabilization of epithelial apico-lateral junctions, photoreceptor organization and ciliogenesis. Since human CRUMBS1 mutations are associated with retina degeneration, it has become essential to define Crumbs network and to understand exactly how this network acts in polarized cells, with a view to developing suitable therapeutic approaches for treating this severe degenerative disease.

Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex.

In human, mutations in tuberous sclerosis complex protein 1 or 2 (TSC1/2 or hamartin/tuberin) cause tuberous sclerosis characterized by the occurrence of multiple hamartomas. On the other hand, mutations in the Crumbs homolog-1 (CRB1) gene cause retinal degeneration diseases including Leber congenital amaurosis and retinitis pigmentosa type 12. Here we report, using a two-hybrid assay, a direct molecular interaction between TSC2 C-terminal part and PDZ 2 and 3 of PATJ, a scaffold member of the Crumbs 3 (CRB 3) complex in human intestinal epithelial cells, Caco2. TSC2 interacts not only with PATJ, but also with the whole CRB 3 complex by GST-pull down assays. In addition, TSC2 co-immunoprecipitates and co-localizes partially with PATJ at the level of the tight junctions. Furthermore, depletion of PATJ from Caco2 cells induces an increase in mammalian Target Of Rapamycin Complex 1 (mTORC1) activity, which is totally inhibited by rapamycin. In contrast, in the same cells, inhibition of phosphoinositol-3 kinase (PI-3K) by wortmannin does not abolish rpS6 phosphorylation. These functional data indicate that the Crumbs complex is a potential regulator of the mTORC1 pathway, cell metabolism and survival through a direct interaction with TSC1/2.

Pals1/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized Muller glia cells.

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.

PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells.

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.

hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells.

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.

The type 3 inositol 1,4,5-trisphosphate receptor is concentrated at the tight junction level in polarized MDCK cells.

The subcellular localization of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ signals is important for the activation of many physiological functions. In epithelial cells the spatial distribution of InsP3 receptor is restricted to specific areas, but little is known about the relationship between the receptor's distribution and cell polarity. To investigate this relationship, the best known polarized cell model, MDCK, was examined. This cell line is characterized by a strong expression of the type 3 InsP3 receptor and the subcellular localization of this receptor was followed during cell polarization using immunofluorescence and confocal analysis. In non-polarized cells, including ras transformed f3 MDCK cells, the type 3 InsP3 receptor was found to co-localize with markers of the endoplasmic reticulum in the cytoplasm. In contrast, in polarized cells, this receptor was mostly distributed at the apex of the lateral plasma membrane with the markers of tight junctions, ZO-1 and occludin. The localization of the type 3 InsP3 receptor in the vicinity of tight junctions was confirmed by immunogold electron microscopy. The culture of MDCK cells in calcium-deprived medium, led to disruption of cell polarity and receptor redistribution in the cytoplasm. Addition of calcium to these deprived cells induced the restoration of polarity and the relocalization of the receptor to the plasma membrane. MDCK cells were stably transfected with a plasmid coding the full-length mouse type 1 InsP3 receptor tagged with EGFP at the C-terminus. The EGFP-tagged type 1 receptor and the endogenous type 3 co-localized in the cytoplasm of non-polarized cells and at the tight junction level of polarized cells. Thus, the localization of InsP3 receptor in MDCK depends on polarity.

The scaffolding domain of caveolin 2 is responsible for its Golgi localization in Caco-2 cells.

In this work, we showed that in Caco-2 cells, a polarized cell line derived from human colon cancer that does not express caveolin 1 (Cav-1), there was no detectable expression of caveolin 2 (Cav-2). When Cav-2 was reintroduced in these cells, it accumulated in the Golgi complex. A chimera, in which the scaffolding domain of Cav-1 was replaced by the one from Cav-2, induced a prominent Golgi staining of Cav-1, strongly indicating that this domain was responsible for the accumulation of Cav-2 in the Golgi complex. Cav-2 was able to interact with Cav-1 in the Golgi complex but this interaction was not sufficient to export it from this compartment. Several chimeras between Cav-1 and 2 were used to show that surface expression of caveolin was necessary but not sufficient to promote caveolae formation. Interestingly, levels of incorporation of the chimeras into Triton insoluble rafts correlated with their ability to trigger caveolae formation raising the possibility that a critical concentration of caveolins to discrete domains of the plasma membrane might be necessary for caveolae formation.

Interaction between Erbin and a Catenin-related protein in epithelial cells.

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.