PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein b-Arrestin1. Because b-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42-dependent morphogenic processes through a b-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs b-Arrestin1 membrane localization, b-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by b-Arrestin1 KD or inhibition of b-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of b-Arrestin1, ARHGAP21 and Cdc42.

The PI3 kinase family of lipid kinases promotes cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate. To define targets critical for cancers driven by activation of PI3 kinase, we screened a panel of potent and structurally diverse drug-like molecules that target this enzyme family. Surprisingly, a single agent (PI-103) effected proliferative arrest in glioma cells, despite the ability of many compounds to block PI3 kinase signaling through its downstream effector, Akt. The unique cellular activity of PI-103 was traced directly to its ability to inhibit both PI3 kinase alpha and mTOR. PI-103 showed significant activity in xenografted tumors with no observable toxicity. These data demonstrate an emergent efficacy due to combinatorial inhibition of mTOR and PI3 kinase alpha in malignant glioma.

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical ␤1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a ␤1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of ␤1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane. INTRODUCTIONPolarization of cells is a fundamental process in biology. Epithelial cells polarize into apical and basolateral poles. Much has been learned recently about the mechanisms of epithelial polarization. Three major polarization complexes, the Par3/Par6/atypical protein kinase C (aPKC), Crumbs/ PATJ/Stardust, and Scribble/Discs Large/Lethal Giant Larvae complexes are conserved from Caenorhabditis elegans and Drosophila to mammals and are essential for epithelial polarization (Roh and Margolis, 2003;Macara, 2004). Cell polarization also requires interactions of the cells with each other and the extracellular matrix (ECM), as well as polarized organization of the cytoskeleton and membrane traffic, although how these processes are connected to the three polarization complexes is largely obscure (Vega-Salas et al., 1987;Yeaman et al., 1999;O'Brien et al., 2002;Mostov et al., 2003;Nelson, 2003;Zegers et al., 2003).Formation of epithelial tissues requires that the orientation of polarity of individual epithelial cells be coordinated in space and time. This was underappreciated in studies using cells grown on filter supports, because the filter provides an overriding extrinsic cue to orient the cells with the basolateral surface facing the filter and the apical surface opposite. Building on earlier observations (Wang et al., 1990a), we found that generation of epithelial polarity can be uncoupled from the orientation of that polarity (O'Brien et al., 2001). Madin-Darby canine kidney (MDCK) epithelial cells grown in three-dimensional (3D) collagen gels form cysts, where the apical surface of a polarized monolayer of cells fa...

Food allergy is a pathological, potentially deadly, immune reaction triggered by normally innocuous food protein antigens. The prevalence of food allergies is rising and the standard of care is not optimal, consisting of food-allergen avoidance and treatment of allergen-induced systemic reactions with adrenaline. Thus, accurate diagnosis, prevention and treatment are pressing needs, research into which has been catalysed by technological advances that are enabling a mechanistic understanding of food allergy at the cellular and molecular levels. We discuss the diagnosis and treatment of IgE-mediated food allergy in the context of the immune mechanisms associated with healthy tolerance to common foods, the inflammatory response underlying most food allergies, and immunotherapy-induced desensitization. We highlight promising research advances, therapeutic innovations and the challenges that remain.

Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Here, we ectopically inserted exogenous PtdIns(3,4,5)P3 into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface.

SUMMARY It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. But here we found that self peptide-MHC specific CD8+ T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome encoded SMCY antigen, self-specific T cells exhibited only a three-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors.

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

SUMMARY The formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a β1-integrin/FAK/p190RhoGAP complex to down-regulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCβII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization.