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The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithelia. Crb intracellular functions, including its direct binding to PALS1, are mediated by Crb's highly conserved 37-residue cytoplasmic tail. However, the mechanistic basis governing the highly specific Crb-PALS1 complex formation is unclear, as reported interaction between the Crb tail (Crb-CT) and PALS1 PSD-95/DLG/ZO-1 (PDZ) domain is weak and promiscuous. Here we have discovered that the PDZ-Src homolgy 3 (SH3)-Guanylate kinase (GK) tandem of PALS1 binds to Crb-CT with a dissociation constant of 70 nM, which is ∼ 100-fold stronger than the PALS1 PDZ-Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK-Crb-CT complex reveals that PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ-SH3-GK supramodule weaken the PALS1-Crb interaction and compromise PALS1-mediated polarity establishment in Madin-Darby canine kidney (MDCK) cysts. We further show that specific target binding of other members of membrane-associated guanylate kinases (MAGUKs) (e.g., CASK binding to neurexin) also requires the presence of their PDZ-SH3-GK tandems. Show less

The study of animal organ size determination has provided evidence of the existence of organ-intrinsic mechanisms that 'sense' and adjust organ growth. Cell competition, a form of cell interaction that equalizes cell population growth, has been proposed to play a role in organ size regulation. Cell competition involves a cell-context dependent response triggered by perceived differences in cell growth and/or proliferation rates, resulting in apoptosis in growth-disadvantaged cells and compensatory expansion of the more 'fit' cells. The mechanisms that allow cells to compare growth are not yet understood, but a number of genes and pathways have been implicated in cell competition. These include Myc, the members of the Hippo, JAK/STAT and WNT signaling pathways, and the Dlg/Lgl/Scrib and the Crb/Std/PatJ membrane protein complexes. Cell competition was initially characterized in the Drosophila imaginal disc, but several recent studies have shown that cell competition occurs in mouse embryonic stem cells and in the embryonic epiblast, where it plays a role in the regulation of early embryo size. In addition, competition-like behavior has been described in the adult mouse liver and the hematopoietic stem cell compartment. These data indicate that cell competition plays a more universal role in organ size regulation. In addition, as some authors have suggested that similar types of competitive behavior may operate in during tumorigenesis, there may be additional practical reasons for understanding this fundamental process of intercellular communication. Show less

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family. Show less

Patulin is a polyketide-derived mycotoxin produced by numerous filamentous fungi. Among them, Penicillium expansum is by far the most problematic species. This fungus is a destructive phytopathogen capable of growing on fruit, provoking the blue mold decay of apples and producing significant amounts of patulin. The biosynthetic pathway of this mycotoxin is chemically well-characterized, but its genetic bases remain largely unknown with only few characterized genes in less economic relevant species. The present study consisted of the identification and positional organization of the patulin gene cluster in P. expansum strain NRRL 35695. Several amplification reactions were performed with degenerative primers that were designed based on sequences from the orthologous genes available in other species. An improved genome Walking approach was used in order to sequence the remaining adjacent genes of the cluster. RACE-PCR was also carried out from mRNAs to determine the start and stop codons of the coding sequences. The patulin gene cluster in P. expansum consists of 15 genes in the following order: patH, patG, patF, patE, patD, patC, patB, patA, patM, patN, patO, patL, patI, patJ, and patK. These genes share 60-70% of identity with orthologous genes grouped differently, within a putative patulin cluster described in a non-producing strain of Aspergillus clavatus. The kinetics of patulin cluster genes expression was studied under patulin-permissive conditions (natural apple-based medium) and patulin-restrictive conditions (Eagle's minimal essential medium), and demonstrated a significant association between gene expression and patulin production. In conclusion, the sequence of the patulin cluster in P. expansum constitutes a key step for a better understanding of the mechanisms leading to patulin production in this fungus. It will allow the role of each gene to be elucidated, and help to define strategies to reduce patulin production in apple-based products. Show less

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression. Show less

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI1, MAGI2, and MAGI3, MUPP1, 14-3-3zeta, Na/H exchange regulatory factor 1, PTPN13, TIP-2/GIPC, Tip-1, and PATJ. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. However, contribution of degradation of PDZ proteins by E6 to HPV-induced oncogenesis is still controversial. In order to clarify potential roles of molecular interactions between high-risk HPV E6 and one of best characterized PDZ proteins, hDlg in HPV-induced transformation, we used a retroviral infection system to overexpress HPV16 E7 gene alone or together with either HPV16 E6 wild type or E6 mutant gene lacking the PDZ domain-binding motif and investigated the effect of mutating the PDZ domain-binding motif of E6 on the immortalization and differentiation of human foreskin keratinocytes (HFKs) by the high-risk type HPV E6 and E7. Although the PDZ domain-binding motif of E6 was found to be required for the efficient growth of HFKs, it was not necessary for the E6 and E7-induced immortalization of HFKs. Furthermore, the overexpression of E6 and E7 neither induced degradation nor altered cellular localization of hDlg in undifferentiated or differentiated HFKs. These data indicate that the PDZ domain-binding motif of E6 contributes to the efficient cellular growth through mechanisms other than degradation and changes in the subcellular localizations of hDlg. Show less

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton. Show less

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial-like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin-based cell-cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)-based cell-cell adhesion, and cadherin and claudin were recruited to the nectin-3- and JAM-based cell-cell adhesion sites to form AJ-like and TJ-like domains, respectively. This inversed alignment of the AJ-like and TJ-like domains was reversed by complementary expression of CPMs Par-3, atypical protein kinase C, Par-6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico-basal alignment of TJs and AJs in epithelial cells. Show less

The transmembrane proteoglycan NG2 is expressed by oligodendrocyte precursor cells (OPC), which migrate to axons during developmental myelination and remyelinate in the adult after migration to injured sites. Highly invasive glial tumors also express NG2. Despite the fact that NG2 has been implicated in control of OPC migration, its mode of action remains unknown. Here, we show in vitro and in vivo that NG2 controls migration of OPC through the regulation of cell polarity. In stab wounds in adult mice we show that NG2 controls orientation of OPC toward the wound. NG2 stimulates RhoA activity at the cell periphery via the MUPP1/Syx1 signaling pathway, which favors the bipolar shape of migrating OPC and thus directional migration. Upon phosphorylation of Thr-2256, downstream signaling of NG2 switches from RhoA to Rac stimulation. This triggers process outgrowth through regulators of front-rear polarity and we show using a phospho-mimetic form of NG2 that indeed NG2 recruits proteins of the CRB and the PAR polarity complexes to stimulate Rac activity via the GEF Tiam1. Our findings demonstrate that NG2 is a core organizer of Rho GTPase activity and localization in the cell, which controls OPC polarity and directional migration. This work also reveals CRB and PAR polarity complexes as new effectors of NG2 signaling in the establishment of front-rear polarity. Show less

MUPP-1 (multi-PDZ domain protein-1) and PATJ (PALS-1-associated tight junction protein) proteins are closely related scaffold proteins and bind to many common interactors including PALS-1 (protein associated with Lin seven) a member of the Crumbs complex. Our goal is to understand how MUPP-1 and PATJ and their interaction with PALS-1 are regulated in the same cells. We have shown that in MCF10A cells there are at least two different and co-existing complexes, PALS-1/MUPP-1 and PALS-1/PATJ. Surprisingly, MUPP-1 levels inversely correlated with PATJ protein levels by acting on the stabilization of the PATJ/PALS-1 complex. Upon MUPP-1 depletion, the increased amounts of PATJ are in part localized at the migrating front of MCF10A cells and are able to recruit more PAR3 (partition defective 3). All together these data indicate that a precise balance between MUPP-1 and PATJ is achieved in epithelial cells by regulating their association with PALS-1. Show less

The assembly and consolidation of the adherens junctions (AJs) are key events in the establishment of an intact epithelium. However, AJs are further modified to obtain flexibility for cell migration and morphogenetic movements. Intact AJs in turn are a prerequisite for the establishment and maintenance of apical-basal polarity in epithelial cells. In this study, we report that the conserved PDZ (PSD95, Discs large, ZO-1) domain-containing protein PATJ (Pals1-associated tight junction protein) was not per se crucial for the maintenance of apical-basal polarity in Drosophila melanogaster epithelial cells but rather regulated Myosin localization and phosphorylation. PATJ directly bound to the Myosin-binding subunit of Myosin phosphatase and decreased Myosin dephosphorylation, resulting in activated Myosin. Thereby, PATJ supports the stability of the Zonula Adherens. Notably, weakening of AJ in a PATJ mutant epithelium led first to a loss of Myosin from the AJ, subsequently to a disassembly of the AJ, and finally, to a loss of apical-basal polarity and disruption of the tissue. Show less

Patj is described as a core component of the Crumbs complex. Along with the other components, Crumbs and Stardust, Patj has been proposed as essential for epithelial polarity. However, no proper in vivo genetic analysis of Patj function has been performed in any organism. We have generated the first null mutants for Drosophila Patj. These mutants are lethal. However, Patj is not required in all epithelia where the Crumbs complex is essential. Patj is dispensable for ectoderm polarity and embryonic development, whereas more severe defects are observed in the adult follicular epithelium, including mislocalisation of the Crumbs complex from the apical domain, as well as morphogenetic defects. These defects are similar to those observed with crumbs and stardust mutants, although weaker and less frequent. Also, gain-of-function of Crumbs and Patj mutation genetically suppress each other in follicular cells. We also show that the first PDZ domain of Patj associated with the Stardust-binding domain are sufficient to fully rescue both Drosophila viability and Crumbs localisation. We propose that the only crucial function of Patj hinges on the ability of its first two domains to positively regulate the Crumbs complex, defining a new developmental level of regulation of its dynamics. Show less

Epithelial tissue formation and function requires the apical-basal polarization of individual epithelial cells. Apical polarity regulators (APRs) are an evolutionarily conserved group of key factors that govern polarity and several other aspects of epithelial differentiation. APRs compose a diverse set of molecules including a transmembrane protein (Crumbs), a serine/threonine kinase (aPKC), a lipid phosphatase (PTEN), a small GTPase (Cdc42), FERM domain proteins (Moesin, Yurt), and several adaptor or scaffolding proteins (Bazooka/Par3, Par6, Stardust, Patj). These proteins form a dynamic cooperative network that is engaged in negative-feedback regulation with basolateral polarity factors to set up the epithelial apical-basal axis. APRs support the formation of the apical junctional complex and the segregation of the junctional domain from the apical membrane. It is becoming increasingly clear that APRs interact with the cytoskeleton and vesicle trafficking machinery, regulate morphogenesis, and modulate epithelial cell growth and survival. Not surprisingly, APRs have multiple fundamental links to human diseases such as cancer and blindness. Show less

Patj has been characterized as one of the so-called polarity proteins that play essential and conserved roles in regulating cell polarity in many different cell types. Studies of Drosophila and mammalian cells suggest that Patj is required for the apical polarity protein complex Crumbs-Stardust (Pals1 or Mpp5 in mammalian cells) to establish apical-basal polarity. However, owing to the lack of suitable genetic mutants, the exact in vivo function of Patj in regulating apical-basal polarity and development remains to be elucidated. Here, we generated molecularly defined null mutants of Drosophila Patj (dPatj). Our data show conclusively that dPatj only plays supporting and non-essential roles in regulating apical-basal polarity, although such a supporting role may become crucial in cells such as photoreceptors that undergo complex cellular morphogenesis. In addition, our results confirm that dPatj possesses an as yet unidentified function that is essential for pupal development. Show less

In epithelial cells, myosin-II-dependent forces regulate many aspects of animal morphogenesis, such as apical constriction, cell intercalation, cell sorting, and the formation and maintenance of the adherens junction. These forces are mainly generated by the circumferential actomyosin belt, which is composed of F-actin-myosin II bundles located along apical cell-cell junctions. Although several of the molecular pathways regulating the belt have been identified, the precise mechanisms underlying its function are largely unknown. Our recent studies identified Lulu proteins (Lulu1 and Lulu2), FERM-domain-containing molecules, as the regulators of the belt. Lulus activate the circumferential actomyosin belt and thereby induce apical constriction in epithelial cells; conversely, RNAi-mediated Lulu-knockdown results in the severe disorganization of the circumferential actomyosin belt. We also showed that p114RhoGEF is a downstream molecule of Lulu2 in its regulation of the belt; Lulu2 enhances the catalytic activity of p114RhoGEF through a direct interaction and thereby activates the circumferential actomyosin belt. We further identified aPKC and Patj as regulators of Lulu2-p114RhoGEF. In this commentary, we discuss current knowledge of the circumferential actomyosin belt's regulation, focusing on the Lulu2-p114RhoGEF system. Show less

The assembly of supramolecular complexes in multidomain scaffold proteins is crucial for the control of cell polarity. The scaffold protein of protein associated with Lin-7 1 (Pals1) forms a complex with two other scaffold proteins, Pals-associated tight junction protein (Patj) and mammalian homolog-2 of Lin-7 (Mals2), through its tandem Lin-2 and Lin-7 (L27) domains to regulate apical-basal polarity. Here, we report the crystal structure of a 4-L27 domain-containing heterotrimer derived from the tripartite complex Patj/Pals1/Mals2. The heterotrimer consists of two cognate pairs of heterodimeric L27 domains with similar conformations. Structural analysis and biochemical data further show that the dimers assemble mutually independently. Additionally, such mutually independent assembly of the two heterodimers can be observed in another tripartite complex, Disks large homolog 1 (DLG1)/calcium-calmodulin-dependent serine protein kinase (CASK)/Mals2. Our results reveal a novel mechanism for tandem L27 domain-mediated, supramolecular complex assembly with a mutually independent mode. Show less

The Merlin/NF2 tumor suppressor restrains cell growth and tumorigenesis by controlling contact-dependent inhibition of proliferation. We have identified a tight-junction-associated protein complex comprising Merlin, Angiomotin, Patj, and Pals1. We demonstrate that Angiomotin functions downstream of Merlin and upstream of Rich1, a small GTPase Activating Protein, as a positive regulator of Rac1. Merlin, through competitive binding to Angiomotin, releases Rich1 from the Angiomotin-inhibitory complex, allowing Rich1 to inactivate Rac1, ultimately leading to attenuation of Rac1 and Ras-MAPK pathways. Patient-derived Merlin mutants show diminished binding capacities to Angiomotin and are unable to dissociate Rich1 from Angiomotin or inhibit MAPK signaling. Depletion of Angiomotin in Nf2(-/-) Schwann cells attenuates the Ras-MAPK signaling pathway, impedes cellular proliferation in vitro and tumorigenesis in vivo. Show less

Myosin II-driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell-cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. In this paper, we characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain-containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell-cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells. Show less

The kidneys participate in whole-body homeostasis, regulating acid-base balance, electrolyte concentrations, extracellular fluid volume, and regulation of blood pressure. Many of the kidney's functions are accomplished by relatively simple mechanisms of filtration, reabsorption, and secretion, which take place in the nephron. The kidneys generate 140-180 l of primary urine per day, while reabsorbing a large percentage, allowing for only the excretion of approximately 2 l of urine. Within the nephron, the majority of the filtered water and solutes are reabsorbed. This is mainly facilitated by specialized transporters and channels which are localized at different segments of the nephron and asymmetrically localized within the polarized epithelial cells. The asymmetric localization of these transporters and channels is essential for the physiological tasks of the renal tissues. One family of these proteins are the water-permeable aquaporins which are selectively expressed in cells along the nephron and localized at different compartments. Here, we discuss potential molecular links between mechanisms involved in the establishment of cell polarity and the members of the aquaporin family. In the first part of this review, we will focus on aspects of apical cell polarity. In the second part, we will review the motifs identified so far that are involved in aquaporin sorting and point out potential molecular links. Show less

The L27 (LIN-2/LIN-7) domain is a protein-protein interaction module capable of assembling proteins into biologically important complexes. Pals1 contains two L27 domains: the first, L27N, interacts with PATJ, and the second, L27C, interacts with MALS, forming a tripartite complex that plays a crucial role in the establishment and maintenance of cell polarity. To provide a better understanding of the mechanism of assembly of this tripartite complex, four different L27(PATJ)-(L27N,L27C)(Pals1)-L27(MALS) constructs were cloned, expressed, purified and crystallized. Crystals of tripartite complex 1 of L27(PATJ)-(L27N,L27C)(Pals1)-L27(MALS) diffracted to 2.05 Å resolution. These crystals belonged to either space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 145.2, c = 202.5 Å. Assuming the presence of four molecules in the asymmetric unit, a Matthews coefficient of 2.69 Å(3) Da(-1) was calculated, corresponding to a solvent content of 54.25%. Show less

Establishment of epithelial apicobasal polarity is crucial for proper kidney development and function. In recent years, there have been important advances in our understanding of the factors that mediate the initiation of apicobasal polarization. Key among these are the polarity complexes that are evolutionarily conserved from simple organisms to humans. Three of these complexes are discussed in this review: the Crumbs complex, the Par complex, and the Scribble complex. The apical Crumbs complex consists of three proteins, Crumbs, PALS1, and PATJ, whereas the apical Par complex consists of Par-3, Par-6, and atypical protein kinase C. The lateral Scribble complex consists of Scribble, discs large, and lethal giant larvae. These complexes modulate kinase and small G protein activity such that the apical and basolateral complexes signal antagonistically, leading to the segregation of the apical and basolateral membranes. The polarity complexes also serve as scaffolds to direct and retain proteins at the apical membrane, the basolateral membrane, or the intervening tight junction. There is plasticity in apicobasal polarity, and this is best seen in the processes of epithelial-to-mesenchymal transition and the converse mesenchymal-to-epithelial transition. These transitions are important in kidney disease as well as kidney development, and modulation of the polarity complexes are critical for these transitions. Show less

Accumulated studies reported that the natruretic dopamine (DA) and the anti-natruretic angiotensin II (Ang II) represent an important mechanism to regulate renal Na(+) and water excretion through intracellular secondary messengers to inhibit or activate renal proximal tubule (PT) Na(+), K(+)-ATPase (NKA). The antagonistic actions were mediated by the phosphorylation of different position of NKA α₁-subunit and different Pals-associated tight junction protein (PATJ) PDZ domains, the different protein kinase C (PKC) isoforms (PKC-β, PKC-ζ), the common adenylyl cyclase (AC) pathway, and the crosstalk and balance between DA and Ang II to NKA regulation. Besides, Ang II-mediated NKA modulation has bi-phasic effects. Show less

Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis. Show less

Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV-infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients. Show less

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology. Show less

In Drosophila epithelial cells, specification and maintenance of the zonula adherens (za) is crucial to ensure epithelial tissue integrity. This depends on the intertwined function of Bazooka (Baz), Par6-DaPKC, and the Crumbs (Crb)-Stardust (Sdt)-PATJ complex. However, the detailed molecular basis for the interplay between these factors during this process is not fully understood. We demonstrate that during photoreceptor apicobasal polarity remodeling, Crb is required to exclude Baz from the subapical domain. This is achieved by recruiting Par6 and DaPKC to this membrane domain. This molecular sorting depends on Baz phosphorylation by DaPKC at the conserved serine 980 and on the activity of the small GTPase Cdc42 associated with Par6. Our data indicate that although Cdc42 binding to Par6 is not required for Baz phosphorylation by DaPKC, it is required for optimum recruitment of Crb at the subapical membrane, a process necessary for delineating the nascent za from this membrane domain. Binding of Cdc42 to the DaPKC regulatory subunit Par6 is required to promote Crb- and DaPKC-dependent apical exclusion of Baz. This molecular sorting mechanism results in setting up the boundary between the photoreceptor subapical membrane and the za. Show less

Cell polarity proteins regulate tight junction formation and directional migration in epithelial cells. To date, the mechanism by which these polarity proteins assemble at the leading edge of migrating epithelial cells remains unclear. We report that occludin, a transmembrane protein, is localized at the leading edge of migrating cells and regulates directional cell migration. During migration, occludin knockdown disrupted accumulation of aPKC-Par3 and PATJ at the leading edge, and led to a disorganized microtubule network and defective reorientation of the microtubule organization center (MTOC). Phosphorylation of occludin at tyrosine 473 residue allowed recruitment of p85 alpha to the leading edge via association with its C-terminal SH2 domain. Loss of occludin attenuated activation of PI3K, leading to disorganization of the actin cytoskeleton and reduced cell protrusions. Our data indicate that occludin is required for the leading-edge localization of polarity proteins aPKC-Par3 and PATJ and promotes cell protrusion by regulating membrane-localized activation of PI3K. Show less

Polarity proteins promote the asymmetric organization of cells by orienting intracellular sorting mechanisms, such as protein trafficking and cytoskeletal assembly. The localization of individual polarity proteins in turn is often determined by association with factors that mediate contact with other cells or the substratum. This arrangement for the Par and Crb apical polarity complexes at the tight junction is disrupted by the adaptor protein Amot. Amot directly binds the scaffolding proteins Patj and Mupp1 and redistributes them and their binding partners from the plasma membrane to endosomes. However, the mechanism by which Amot is targeted to endosomes is unknown. Here, a novel lipid binding domain within Amot is shown to selectively bind with high affinity to membranes containing monophosphorylated phosphatidylinositols and cholesterol. With similar lipid specificity, Amot inserts into and tubulates membranes in vitro and enlarges perinuclear endosomal compartments in cells. Based on the similar distribution of Amot with cholesterol, Rab11, and Arf6, such membrane interactions are identified at juxtanuclear endocytic recycling compartments. Taken together, these findings indicate that Amot is targeted along with associated apical polarity proteins to the endocytic recycling compartment via this novel membrane binding domain. Show less

The past two decades have seen an immense increase in our appreciation of the vast range of signalling processes and supporting machinery that occur in cells. Pivotal to this is the notion of signal compartmentalization (compartmentation). Targeting by protein domains is critical in allowing signalling complexes to be assembled at defined intracellular locales so as to confer correct function. This issue of the BJP contains two intriguing articles that address functional protein-protein interactions involving PDZ domains [Post-synaptic density protein-95 (PSD95), Drosophila disc large tumour suppressor (DlgA) and Zonula occludens-1 protein (zo-1)] and their implications for signalling. One involves targeting of neuronal nitric oxide synthase to the N-methyl D-aspartic acid (NMDA) receptor via the PDZ-containing signal scaffold, PSD95. The other involves controlling multiple receptor inputs into regulation of epithelial Na(+)K(+)-ATPase through the PDZ-containing signal scaffold Pals-associated tight junction. Highlighted is not only the use of dominant-negative strategies to identify the importance of targeting at specific types of PDZ domains but also the exciting notion that small molecule disruptors of interaction at specific PDZ domains can be generated for potential therapeutic application. Show less

Nephronophthisis (NPH) is an autosomal recessive disorder characterized by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end-stage renal failure. The disease is caused by mutations in NPHP1-9 genes, which encode the nephrocystins, proteins localized to cell-cell junctions and centrosome/primary cilia. Here, we show that nephrocystin mRNA expression is dramatically increased during cell polarization, and shRNA-mediated knockdown of either NPHP1 or NPHP4 in MDCK cells resulted in delayed tight junction (TJ) formation, abnormal cilia formation and disorganized multi-lumen structures when grown in a three-dimensional collagen matrix. Some of these phenotypes are similar to those reported for cells depleted of the TJ proteins PALS1 or Par3, and interestingly, we demonstrate a physical interaction between these nephrocystins and PALS1 as well as their partners PATJ and Par6 and show their partial co-localization in human renal tubules. Taken together, these results demonstrate that the nephrocystins play an essential role in epithelial cell organization, suggesting a plausible mechanism by which the in vivo histopathologic features of NPH might develop. Show less