The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Here, we show that binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. We also show that the mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. We propose that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation.
The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. Here we report that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na(+),K(+)-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na(+),K(+)-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, we show that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. We also find that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity.
The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brushborder formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.
Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.
We and others have shown that phosphatidylinositol 3-kinase (PI3K) is recruited to and activated by E-cadherin engagement. This PI3K activation is essential for adherens junction integrity and intestinal epithelial cell differentiation. Here we provide evidence that hDlg, the homolog of disc-large tumor suppressor, is another key regulator of adherens junction integrity and differentiation in mammalian epithelial cells. We report the following. 1) hDlg co-localizes with E-cadherin, but not with ZO-1, at the sites of cell-cell contact in intestinal epithelial cells. 2) Reduction of hDlg expression levels by RNA i in intestinal cells not only severely alters adherens junction integrity but also prevents the recruitment of p85/PI3K to E-cadherin-mediated cell-cell contact and inhibits sucrase-isomaltase gene expression. 3) PI3K and hDlg are associated with E-cadherin in a common macromolecular complex in living differentiating intestinal cells. 4) This interaction requires the association of hDlg with E-cadherin and with Src homology domain 2 domains of the p85/PI3K subunit. 5) Phosphorylation of hDlg on serine and threonine residues prevents its interaction with the p85 Src homology domain 2 in subconfluent cells, whereas phosphorylation of hDlg on tyrosine residues is essential. We conclude that hDlg may be a determinant in E-cadherin-mediated adhesion and signaling in mammalian epithelial cells.Cadherins mediate cell-cell adhesion through homotypic, calcium-dependent interactions involving their extracellular domains. Classical cadherins have highly conserved cytoplasmic domains that bind ␣-catenin, -catenin, and plakoglobin (1). These complexes link actin microfilaments and other integral membrane proteins, allowing the formation of junctional complexes, the organization of the actin cytoskeleton at junctional complexes, the establishment of membrane domains, and the modulation of signal transduction pathways (1-3). In intestinal epithelium renewal, E-cadherin plays a crucial role in the coordination between cell proliferation, migration, differentiation, and death. Indeed, expression of a dominant-negative N/E-cadherin in villus enterocytes results in the disruption of cell-cell junctions associated with increased epithelial cell migration, alteration of the differentiated phenotype, and increased apoptosis (4). Furthermore, expression of this transgene in the crypt compartment leads to overproliferation (5). The mechanisms by which E-cadherin-mediated cell-cell contact regulates intestinal epithelium homeostasis remain largely undefined. We have demonstrated recently that E-cadherin engagement recruits and activates PI3K 1 (class IA) signaling in intestinal epithelial cells committed to differentiate (3). This activation is required for the association of adherens junction components with the cytoskeleton, thereby inducing morphological and functional differentiation. Indeed, following PI3K inhibition, intestinal epithelial cells exhibit a less cylindrical phenotype, a significant alteration of actin archite...
Summary Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube-size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM) [1–4]. ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ) [5, 6]. Here, we show that apical and basolateral epithelial polarity proteins interact to control tracheal tube-size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins [7, 8], cause an expansion of tracheal tubes, but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes a SJ-associated polarity protein [8, 9]. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb and Scrib operate independently of the Verm pathway. Our data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size.
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