Apical Proteins Research Articles

Crumbs stabilises epithelial polarity during tissue remodelling

The apicobasal polarity of epithelia depends on the integrated activity of apical and basolateral proteins, and is essential for tissue integrity and body homeostasis. Yet these tissues are frequently on the move as they are sculpted by active morphogenetic cell rearrangements. How does cell polarity survive these stresses? We analyse this question in the renal tubules of Drosophila, a tissue that undergoes dramatic morphogenetic change as it develops. Here we show that, whereas the Bazooka and Scribble protein groups are required for the establishment of tubule cell polarity, the key apical determinant, Crumbs, is required for cell polarity in the tubules only from the time when morphogenetic movements start. Strikingly, if these movements are stalled, polarity persists in the absence of Crumbs. Similar rescue of the ectodermal phenotype of the crumbs mutant when germ-band extension is reduced suggests that Crumbs has a specific, conserved function in stabilising cell polarity during tissue remodelling rather than in its initial stabilisation. We also identify a requirement for the exocyst component Exo84 during tissue morphogenesis, which suggests that Crumbs-dependent stability of epithelial polarity is correlated with a requirement for membrane recycling and targeted vesicle delivery.

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft-mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures-spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL-laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via xx intramembrane protein-protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms.

The Hippo pathway regulates apical-domain size independently of its growth-control function

The Hippo pathway, identified in Drosophila and conserved in vertebrates, regulates tissue growth by promoting cell cycle exit and apoptosis. In addition to their well-characterised overproliferation phenotype, adult Drosophila epithelial cells mutant for the kinases Hippo and Warts have hypertrophic apical domains. Here we examine the molecular basis of this apical hypertrophy and its impact on cell proliferation. In the wing imaginal disc epithelium, we observe increased staining for members of the apical polarity complexes aPKC and Crumbs as well as adherens junction components when Hippo activity is compromised, while basolateral markers are not affected. This increase in apical proteins is correlated with a hypertrophy of the apical domain and adherens junctions. The cell surface localisation of the Notch receptor is also increased in mutant clones, opening the possibility that aberrant receptor signalling may participate in overgrowth of hpo-deficient tissue. Interestingly, however, although the polarity determinant Crumbs is required for the accumulation of apical proteins, this does not appear to significantly contribute to the overproliferation defect elicited by loss of Hippo signalling. Therefore, Hippo signalling controls growth and apical domain size by distinct mechanisms.

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Our previous work in Drosophila indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. Here, we examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. We found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells.

Comparative proteomic analysis of bacterial wilt susceptible and resistant tomato cultivars

To investigate the molecular mechanisms of bacterial resistance in susceptible and resistant cultivars of tomato, a proteomic approach was adopted. Four cultivars of tomato were selected on the basis of their response to bacterial ( Pseudomonas solanacearum) inoculation wherein cultivar Roma and Riogarande, and cultivar Pusa Ruby and Pant Bahr were considered as resistant and susceptible cultivars, respectively. Proteins were extracted from leaves of 3-week-old seedlings of the four cultivars and separated by 2-DE. A total of nine proteins were found to be differentially expressed between the susceptible and resistant cultivars. Amino acid sequences of these proteins were determined with a protein sequencer. The identified proteins belongs to the categories of energy, protein destination and storage, and defense. Of these proteins, a 60 kDa chaperonin and an apical membrane antigen were significantly upregulated in resistant cultivars compared with susceptible cultivars. Application of jasmonic acid and salicylic acid resulted in significant changes in levels of apical membrane antigen and protein disulfide-isomerase. Taken together, these results suggest that apical membrane antigen might be involved in bacterial resistance process through salicylic acid induced defense mechanism signaling in tomato plants.

PP2A Antagonizes Phosphorylation of Bazooka by PAR-1 to Control Apical-Basal Polarity in Dividing Embryonic Neuroblasts

Bazooka/Par-3 (Baz) is a key regulator of cell polarity in epithelial cells and neuroblasts (NBs). Phosphorylation of Baz by PAR-1 and aPKC is required for its function in epithelia, but little is known about the dephosphorylation mechanisms that antagonize the activities of these kinases or about the relevance of Baz phosphorylation for NB polarity. We found that protein phosphatase 2A (PP2A) binds to Baz via its structural A subunit. By using phospho-specific antibodies, we show that PP2A dephosphorylates Baz at the conserved serine residue 1085 and thereby antagonizes the kinase activity of PAR-1. Loss of PP2A function leads to complete reversal of polarity in NBs, giving rise to an "upside-down" polarity phenotype. Overexpression of PAR-1 or Baz, or mutation of 14-3-3 proteins that bind phosphorylated Baz, causes essentially the same phenotype, indicating that the balance of PAR-1 and PP2A effects on Baz phosphorylation determines NB polarity.

Ethanol selectively impairs clathrin-mediated internalization in polarized hepatic cells

Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.

Apical polarity protein PrkCi is necessary for maintenance of spinal cord precursors in zebrafish

During development, neural precursors divide to produce new precursors and cells that differentiate as neurons and glia. In Drosophila, apicobasal polarity and orientation of the mitotic spindle play important roles in specifying the progeny of neural precursors for different fates. We examined orientation of zebrafish spinal cord precursors using time-lapse imaging and tested the function of protein kinase C, iota (PrkCi), a member of the Par complex of proteins necessary for apicobasal polarity in the nervous system. We found that nearly all precursors divide within the plane of the neuroepithelium of wild-type embryos even when they must produce cells that have different fates. In the absence of PrkCi function, neural precursor divisions become oblique during late embryogenesis and excess oligodendrocytes form concomitant with loss of dividing cells. We conclude that PrkCi function and planar divisions are necessary for asymmetric, self-renewing division of spinal cord precursors.

394 Intestine Specific Deletion of N-WASP Leads to Alteration of Gut Homeostasis in Mice

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.

396 The ZO-1 Actin Binding Region (ABR) Is Required for Cytoskeletal Tight Junction Regulation

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.

393 Apical Recycling Endosome-Associated Myosin Vb Is Mutated in Microvillus Inclusion Disease and Is Involved in Intestinal Brush Border Development

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.

Interaction between PAR-3 and the aPKC–PAR-6 complex is indispensable for apical domain development of epithelial cells

The evolutionarily conserved polarity proteins PAR-3, atypical protein kinase C (aPKC) and PAR-6 critically regulate the apical membrane development required for epithelial organ development. However, the molecular mechanisms underlying their roles remain to be clarified. We demonstrate that PAR-3 knockdown in MDCK cells retards apical protein delivery to the plasma membrane, and eventually leads to mislocalized apical domain formation at intercellular regions in both two-dimensional and three-dimensional culture systems. The defects in PAR-3 knockdown cells are efficiently rescued by wild-type PAR-3, but not by a point mutant (S827/829A) that lacks the ability to interact with aPKC, indicating that formation of the PAR-3-aPKC-PAR-6 complex is essential for apical membrane development. This is in sharp contrast with tight junction maturation, which does not necessarily depend on the aPKC-PAR-3 interaction, and indicates that the two fundamental processes essential for epithelial polarity are differentially regulated by these polarity proteins. Importantly, highly depolarized cells accumulate aPKC and PAR-6, but not PAR-3, on apical protein-containing vacuoles, which become targeted to PAR-3-positive primordial cell-cell contact sites during the initial stage of the repolarization process. Therefore, formation of the PAR-3-aPKC-PAR-6 complex might be required for targeting of not only the aPKC-PAR-6 complex but also of apical protein carrier vesicles to primordial junction structures.

Polarity and Morphogenesis‐‐‐ the Planar Cell Polarity Pathway in Mammalian Embryogenesis

Originally identified in flies, the planar cell polarity (PCP) pathway is crucial in establishing the cellular polarity in the plane of the epithelium, perpendicular to the apical‐basal axis of the cell. In frogs and zebrafish, homologous PCP genes regulate directional cell behavior to mediate polarized tissue morphogenesis. In the mouse, we and others have found that a highly conserved mammalian PCP pathway regulates both cell polarity and morphogenetic movement to assemble cells into tissues and organs with distinct anatomical forms and structures. For example, auditory sensory hair cells develop uniformly oriented stereocilia on the apical surface and PCP proteins localize asymmetrically on the apical membrane during this process. Mutations in PCP genes disrupt polarized membrane distribution of other PCP proteins and cause randomized orientation of stereocilia. We have also found that the PCP pathway is essential for coordinated lengthening and narrowing of the neural plate during neural tube closure. The PCP pathway is also required for outflow tract formation in the heart and our genetic studies pinpointed a specific role of this pathway in cardiomyocytes derived from the secondary heart field. While the detailed cellular mechanisms are yet to be defined, the mammalian PCP pathway is undoubtedly a critical force in shaping various tissues and organs and may underlie a number of birth defects in humans.

Death receptor signal transducers: nodes of coordination in immune signaling networks

Death receptors (DRs) are members of the tumor necrosis factor receptor superfamily that possess a cytoplasmic death domain (DD). DRs regulate important operational and homeostatic aspects of the immune system. They transmit signals through apical protein complexes, which are nucleated by the DD adaptors FADD and TRADD, to control cellular outcomes that range from apoptosis to gene activation. FADD and TRADD also nucleate several distal signaling complexes, which mediate cross-talk between distinct DR signaling pathways. Moreover, together with other DR signal transducers, FADD and TRADD participate in functional complexes assembled by certain non-DR immune cell receptors, such as pattern-recognition receptors. Thus, DR signal transducers may provide important nodes of coordination in immune signaling networks.

Transepithelial fluxes of adenosine and 2′-deoxyadenosine across human renal proximal tubule cells: roles of nucleoside transporters hENT1, hENT2, and hCNT3

This study examined the roles of human nucleoside transporters (hNTs) in mediating transepithelial fluxes of adenosine, 2'-deoxyadenosine, and three purine nucleoside anti-cancer drugs across polarized monolayers of human renal proximal tubule cells (hRPTCs), which were shown in previous studies to have human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) activities (11). Early passage hRPTCs were cultured on transwell inserts under conditions that induced formation of polarized monolayers with experimentally accessible apical and basolateral domains. Polarized hRPTC cultures were monitored for inhibitor sensitivities and sodium-dependence of the following: 1) transepithelial fluxes of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine (9-beta-d-arabinosyl-2-fluoroadenine), cladribine (2-chloro-2'-deoxyadenosine), and clofarabine (2-chloro-2'-fluoro-deoxy-9-beta-d-arabinofuranosyladenine); 2) mediated uptake of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine, cladribine, and clofarabine from either apical or basolateral surfaces; and 3) relative apical cell surface hCNT3 protein levels. Transepithelial fluxes of adenosine were mediated from apical-to-basolateral sides by apical hCNT3 and basolateral hENT2, whereas transepithelial fluxes of 2'-deoxyadenosine were mediated from basolateral-to-apical sides by apical hENT1 and basolateral human organic anion transporters (hOATs). The transepithelial fluxes of adenosine, hCNT3-mediated cellular uptake of adenosine, and relative apical cell surface hCNT3 protein levels correlated positively in polarized hRPTCs. The purine nucleoside anti-cancer drugs fludarabine, cladribine, and clofarabine, like adenosine exhibited apical-to-basolateral fluxes. Collectively, this evidence suggested that apical hCNT3 and basolateral hENT2 are involved in proximal tubular reabsorption of adenosine and some nucleoside drugs and that apical hENT1 and basolateral hOATs are involved in proximal tubular secretion of 2'-deoxyadenosine.

The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro

Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.

A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells.

Bicarbonate-dependent and bicarbonate-independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HCO(3)(-) was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part (K(m) = 54 mM) and a bicarbonate-independent component (K(m) = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 microM DIDS, 400 microM p-chloromercuribenzene sulfonic acid, 800 microM p-hydroxymercuribenzoic acid, or 100 microM phloretin had no effects on acetate uptake although the latter two inhibited l-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter.

Ezrin-Radixin-Moesin-Binding Phosphoprotein (EBP50), an Estrogen-Inducible Scaffold Protein, Contributes to Biliary Epithelial Cell Proliferation

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) anchors and regulates apical membrane proteins in epithelia. EBP50 is inducible by estrogen and may affect cell proliferation, although this latter function remains unclear. The goal of this study was to determine whether EBP50 was implicated in the ductular reaction that occurs in liver disease. EBP50 expression was examined in normal human liver, in human cholangiopathies (ie, cystic fibrosis, primary biliary cirrhosis, and primary sclerosing cholangitis), and in rats subjected to bile-duct ligation. The regulation of EBP50 by estrogens and its impact on proliferation were assessed in both bile duct-ligated rats and Mz-Cha-1 human biliary epithelial cells. Analyses of cell isolates and immunohistochemical studies showed that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and cystic fibrosis transmembrane conductance regulator, in the apical domains of cholangiocytes. In both human cholangiopathies and bile duct-ligated rats, EBP50 was redistributed to the cytoplasmic and nuclear compartments. EBP50 underwent a transient increase in rat cholangiocytes after bile-duct ligation, whereas such expression was down-regulated in ovariectomized rats. In addition, in Mz-Cha-1 cells, EBP50 underwent up-regulation and intracellular redistribution in response to 17beta-estradiol, whereas its proliferation was inhibited by siRNA-mediated EBP50 knockdown. These results indicate that both the expression and distribution of EBP50 are regulated by estrogens and contribute to the proliferative response in biliary epithelial cells.

Defects in the ATR-dependent DNA damage response pathway and human syndromes

A multitude of clinically distinct human disorders exist whose underlying cause is a defect in the response to or repair of DNA damage. The clinical spectrum of these conditions provides evidence for the role of the DNA damage response (DDR) in mediating diverse processes such as genomic stability, immune system function and normal human development. Cell lines from these disorders provide a valuable resource to help dissect the consequences of compromised DDR at the molecular level. Ataxia telangiectasia and Rad3-related (ATR) and Ataxia telangiectasia Mutated (ATM) are apical protein kinases that play central roles in coordinating the cells response to DNA damage. Whilst ATM is activated by DNA double strand breaks (DSB's), ATR is activated by single stranded regions of DNA (ssDNA) which can occur, for example, during DNA replication fork stalling. There is significant functional overlap between these two kinases. In fact, they phosphorylate many of the same substrates, including p53 and Brca1. Nevertheless, ATR appears to be essential for embryonic development, unlike ATM. Mutations in ATM result in Ataxia telangiectasia (A-T) a progressive neurological disorder. Interestingly, a hypomorphic mutation in ATR is associated with Seckel syndrome, a clinically distinct disorder to that of A-T. Seckel syndrome is characterised by profound proportionate growth retardation with severe microcephaly. Why defects in these two related kinases should result in such distinct human disorders is unclear. Recently, mutations in Pericentrin/Kendrin (PCNT) have also been demonstrated in Seckel syndrome. PCNT encodes a core structural centrosomal protein. Interestingly, defective PCNT results in impaired ATR-dependent, but not ATM-dependent G2-M cell cycle checkpoint arrest. Using evidence from murine knockout studies and human cell-based work I will discuss the biological impact of compromised ATR-pathway function with the aim of trying to understand the link between genotype-phenotype in this context. ACTA MEDICA NAGASAKIENSIA 53: 23－30, 2008