A conserved oligomerization domain in drosophila Bazooka/PAR-3 is important for apical localization and epithelial polarity

The PAR-3/PAR-6/aPKC complex is required to establish polarity in many different cell types, including the C. elegans zygote and epithelial and neuronal cells in Drosophila and mammals. In each context, the components of this complex display a mutually dependent asymmetric cortical localization. PAR-6 is a direct effector of Rho family GTPases and binds to and regulates aPKC. Mammalian PAR-3 (mPar3) can associate with transmembrane proteins and may link the complex to the membrane, but this can account for only part of the requirement for this protein in the complex. Here we investigate the function of a novel conserved domain, CR1, of PAR-3 using computational, biochemical, and genetic approaches. Sequence-structure comparison by FUGUE predicts that CR1 has the same structural fold as a bacterial oligomerization domain. We show that CR1 of the Drosophila homolog, Bazooka (BAZ), mediates oligomerization in vitro and in vivo. Furthermore, deletion of CR1 disrupts BAZ localization in both epithelial cells and the germline and strongly impairs BAZ function in epithelial polarity. These results indicate that this domain is important for the localization and activity of the PAR-3/PAR6/aPKC complex and define a new role for PAR-3 in assembling higher order protein complexes.     

KIBRA: In the brain and beyond

In mammals, the KIBRA locus has been associated with memory performance and cognition by genome-wide single nucleotide polymorphism screening. Genetic studies in Drosophila and human cells have identified KIBRA as a novel regulator of the Hippo signaling pathway, which plays a critical role in human tumorigenesis. Recent studies also indicated that KIBRA is involved in other physiological processes including cell polarity, membrane/vesicular trafficking, mitosis and cell migration. At the biochemical level, KIBRA protein is highly phosphorylated by various kinases in epithelial cells. Here, we discuss the updates concerning the function and regulation of KIBRA in the brain and beyond.     

The PDZ protein discs-large (DLG): the 'Jekyll and Hyde' of the epithelial polarity proteins

Discs-large (DLG) is a multi-PDZ domain-containing protein that belongs to the family of molecular scaffolding proteins known as membrane guanylate kinases or MAGUKs. DLG is a component of the Scribble polarity complex and genetic analyses of DLG in Drosophila have identified a role for the protein in several key biological processes including the regulation of apico-basal polarity of epithelial cells, as well as other polarity processes such as asymmetric cell division and cell invasion. Disturbance of DLG function leads to uncontrolled epithelial cell proliferation and neoplastic transformation, thereby defining DLG as a potential tumour suppressor. However, whether mammalian homologues of DLG (DLG1, DLG2, DLG3 and DLG4) also possess tumour suppressor functions is not known. In this minireview, we focus on the biological functions of DLG1 in human epithelial cells and on how the function of this MAGUK relates to its intracellular location. We examine some of the evidence that implies that DLG has both tumour suppressor and, paradoxically, oncogenic functions depending upon the precise cellular context.     

Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.     

Basolateral targeting by leucine-rich repeat domains in epithelial cells

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure–function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.     

Na,K-ATPase beta-subunit is required for epithelial polarization, suppression of invasion, and cell motility

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an alpha- and beta-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin-mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and beta(1)-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase beta(1)-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin-mediated cell-cell adhesion requires the Na,K-ATPase beta-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the beta(1)-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.     

Proteome identification of binding-partners interacting with cell polarity protein Par3 in Jurkat cells

The evolutionarily conserved cell polarity protein Par3, a scaffold-like PDZreontaining protein, plays a critical role in the establishment and maintenance of epithelial cell polarity. Although the role of Par3 in establishing cell polarity in epithelial cells has been intensively explored, the function of Par3 in hematopoietic cells remains elusive. To address this issue, we generated GST-fusion proteins of Par3 PDZ domains. By combiningthe GST-pull-down approach with liquid chromatography-tandem mass spectrometry, we identified 10 potential novel binding proteins of PDZ domains of Par3 in Jurkat cells (a T-cell line). The interaction of Par3 with three proteins—nuclear transport protein importin-α4 and proteasome activators PA28β and PA28γ—was confirmed using in vitro binding assay, co-immunoprecipitation assay and immunofluorescence microscopy. Our results have the potential to uncover novel functions of the cell polarity protein Par3 in blood cells.     

Molecular networks controlling epithelial cell polarity in development

During embryonic development, polarized epithelial cells are either formed during cleavage or formed from mesenchymal cells. Because the formation of epithelia during embryogenesis has to occur with high fidelity to ensure proper development, embryos allow a functional approach to study epithelial cell polarization in vivo. In particular, genetic model organisms have greatly advanced our understanding of the generation and maintenance of epithelial cell polarity. Many novel and important polarity genes have been identified and characterized in invertebrate systems, like Drosophila melanogaster and Caenorhabditis elegans. With the rapid identification of mammalian homologues of these invertebrate polarity genes, it has become clear that many important protein domains, single proteins and even entire protein complexes are evolutionarily conserved. It is to be expected that the field of epithelial cell polarity is just experiencing the 'top of the iceberg' of a large protein network that is fundamental for the specific adhesive, cell signalling and transport functions of epithelial cells.     

肝细胞极性与膜蛋白分选的分子机制

肝细胞是高度特化的极性上皮细胞,细胞质膜蛋白的分选和极性转运对于肝细胞极性的建立与维持至关重要.首先,膜蛋白在内质网中合成,随后经高尔基体加工修饰,再由反面高尔基体进一步分选,最后通过膜泡运输等不同的机制分别转运到胆汁腔面或窦状隙面,行使其特殊的功能.近些年来,细胞内负责转运的细胞器和主要的分选信号已逐步被揭示.特别是循环内体也被证明参与了胆汁腔面和窦状隙面膜蛋白的极性分选和转运.肝细胞的极性一旦遭到破坏,将会引起胆汁分泌障碍以及其他肝脏功能的损伤,从而可能导致肝脏糖脂代谢紊乱,甚至丧失正常的生理功能.因此,深入研究肝脏细胞极性的形成与维持机制,将为多种肝脏疾病的预防和治疗寻找到新的方向和靶点,具有重要的理论和临床实践意义.     

Phosphorylation-dependent binding of 14-3-3 to the polarity protein Par3 regulates cell polarity in mammalian epithelia

The mammalian homologs of the C. elegans partitioning-defective (Par) proteins have been demonstrated to be necessary for establishment of cell polarity. In mammalian epithelia, the Par3/Par6/aPKC polarity complex is localized to the tight junction and regulates its formation and positioning with respect to basolateral and apical membrane domains. Here we demonstrate a previously undescribed phosphorylation-dependent interaction between a mammalian homolog of the C. elegans polarity protein Par5, 14-3-3, and the tight junction-associated protein Par3. We identify phosphorylated serine 144 as a site of 14-3-3 binding. Expression of a Par3 mutant that contains serine 144 mutated to alanine (S144A) results in defects in epithelial cell polarity. In addition, overexpression of 14-3-3zeta results in a severe disruption of polarity, whereas overexpression of a 14-3-3 mutant that is defective in binding to phosphoproteins has no effect on cell polarity. Together, these data suggest a novel, phosphorylation-dependent mechanism that regulates the function of the Par3/Par6/aPKC polarity complex through 14-3-3 binding.     

PATJ regulates tight junction formation and polarity in mammalian epithelial cells

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.     

Dlg3 trafficking and apical tight junction formation is regulated by nedd4 and nedd4-2 e3 ubiquitin ligases

The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation.     

The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.     

CAS/CSE 1 stimulates E-cadhrin-dependent cell polarity in HT-29 human colon epithelial cells

The establishment and maintenance of epithelial polarity are crucial for tissue organization and function in mammals. Epithelial cadherin (E-cadherin) is expressed in epithelial cell membrane and is important for cell-cell adhesion, intercellular junctions formation, as well as epithelial cell polarization. We report herein that CAS (CAS/CSE 1), the human cellular apoptosis susceptibility protein, interacts with E-cadherin and stimulates polarization of HT-29 human colon epithelial cells. CAS binds with E-cadherin but not with beta-catenin in the immunoprecipitation assays. Interaction of CAS with E-cadherin enhances the formation of E-cadherin/beta-catenin cell-cell adhesive complex. Electron microscopic study demonstrated that CAS overexpression in cells stimulates intercellular junction complex formation. The disorganization of cellular cytoskeleton by cytochalasin D, colchicine, or acrylamide treatment disrupts CAS-stimulated HT-29 cell polarization. CAS-mediated HT-29 cell polarity is also inhibited by antisense E-cadherin DNA expression. Our results indicate that CAS cooperates with E-cadherin and plays a role in the establishment of epithelial cell polarity.     

Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Remodeling Epithelial Cell Organization: Transitions Between Front–Rear and Apical–Basal Polarity

Polarized epithelial cells have a distinctive apical–basal axis of polarity for vectorial transport of ions and solutes across the epithelium. In contrast, migratory mesenchymal cells have a front–rear axis of polarity. During development, mesenchymal cells convert to epithelia by coalescing into aggregates that undergo epithelial differentiation. Signaling networks and protein complexes comprising Rho family GTPases, polarity complexes (Crumbs, PAR, and Scribble), and their downstream effectors, including the cytoskeleton and the endocytic and exocytic vesicle trafficking pathways, together regulate the distributions of plasma membrane and cytoskeletal proteins between front–rear and apical–basal polarity. The challenge is to understand how these regulators and effectors are adapted to regulate symmetry breaking processes that generate cell polarities that are specialized for different cellular activities and functions.     

An Atypical PKC Directly Associates and Colocalizes at the Epithelial Tight Junction with ASIP,a Mammalian Homologue of Caenorhabditis elegans Polarity Protein PAR-3

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCζ and PKCλ have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype–specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKCλ to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.     

glaikit is essential for the formation of epithelial polarity and neuronal development

Epithelial cells have a distinctive polarity based on the restricted distribution of proteins and junctional complexes along an apical-basal axis. Studying the formation of the polarized ectoderm of the Drosophila embryo has identified a number of the molecules that establish this polarity. The Crumbs (Crb) complex is one of three separate complexes that cooperate to control epithelial polarity and the formation of zonula adherens. Here we show that glaikit (gkt), a member of the phospholipase D superfamily, is essential for the formation of epithelial polarity and for neuronal development during Drosophila embryogenesis. In epithelial cells, gkt acts to localize the Crb complex of proteins to the apical lateral membrane. Loss of gkt during neuronal development leads to a severe CNS architecture disruption that is not dependent on the Crb pathway but probably results from the disrupted localization of other membrane proteins. A mutation in the human homolog of gkt causes the neurodegenerative disease spinocerebellar ataxia with neuropathy (SCAN1), making it possible that a failure of membrane protein localization is a cause of this disease.     

Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. We analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways. Surprisingly, we found that none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner.     

Phosphoinositides in cell architecture

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.