Apical Proteins Research Articles

Uptake of Extracellular Double-Stranded RNA by SID-2

Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates, including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA and that, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity, including three extracellular, positively charged histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi.

The Apical Polarity Protein Network in Drosophila Epithelial Cells: Regulation of Polarity, Junctions, Morphogenesis, Cell Growth, and Survival

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.

The Role of a Basolateral Transporter in Rosuvastatin Transport and Its Interplay with Apical Breast Cancer Resistance Protein in Polarized Cell Monolayer Systems

Membrane transporters can play a clinically important role in drug absorption and disposition; Caco-2 and Madin-Darby canine kidney (MDCK) cells are the most widely used in vitro models for studying the functions of these transporters and associated drug interactions. Transport studies using these cell models are mostly focused on apical transporters, whereas basolateral drug transport processes are largely ignored. However, for some hydrophilic drugs, a basolateral uptake transporter may be required for drugs to enter cells before they can interact with apical efflux transporters. The objective of this study was to evaluate potential differences in drug transport across Caco-2 and MDCK basolateral membrane that could cause discrepancy in the identification of efflux transporter substrates and to elucidate the underlying factors that may cause such differences, using rosuvastatin as a model substrate. Bidirectional transport results in Caco-2 and breast cancer resistance protein-MDCK cells demonstrated the necessity of an uptake transporter at the basolateral membrane for rosuvastatin. Kinetic study revealed saturable and nonsaturable processes for rosuvastatin uptake across the Caco-2 basolateral membrane, with the saturable process encompassing >75% of overall rosuvastatin basolateral uptake at concentrations below the K(m) (4.2 μM). Furthermore, rosuvastatin basolateral transport exhibited cis-inhibition and trans-stimulation phenomena, indicating a facilitated diffusion mechanism. This basolateral transporter appeared to be a prerequisite for rosuvastatin and perhaps for other hydrophilic substrates to interact with apical efflux transporters. Deficit of such a basolateral transporter in certain cell models may lead to false-negative results when screening drug interactions with apical efflux transporters.

Requirement for a Uroplakin 3a-Like Protein in the Development of Zebrafish Pronephric Tubule Epithelial Cell Function, Morphogenesis, and Polarity

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na+/K+–ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression.

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix

This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻⁶ and (3.2 ± 0.3) × 10⁻⁶ cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 μL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 μM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

Drosophila Patj plays a supporting role in apical-basal polarity but is essential for viability

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.

Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity

Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PARproteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.

Crb Apical Polarity Proteins Maintain Zebrafish Retinal Cone Mosaics via Intercellular Binding of Their Extracellular Domains

Cone photoreceptors are assembled by unknown mechanisms into geometrically regular mosaics in many vertebrate species. The formation and maintenance of photoreceptor mosaics are speculated to require differential cell-cell adhesion. However, the molecular basis for this theory has yet to be identified. The retina and many other tissues express Crumbs (Crb) polarity proteins. The functions of theextracellular domains of Crb proteins remain to be understood. Here we report cell-type-specific expression of the crb2a and crb2b genes at the cell membranes of photoreceptor inner segments and Müller cell apical processes in the zebrafish retina. We demonstrate that the extracellular domains of Crb2a and Crb2b mediate a cell-cell adhesion function, which plays an essential role in maintaining the integrity of photoreceptor layer and cone mosaics. Because Crb proteins are expressed in many types of epithelia, the Crb-based cell-cell adhesion may underlie cellular patterning in other epithelium-derived tissues as well.

Polarised apical-like intracellular sorting and trafficking regulates invadopodia formation and degradation of the extracellular matrix in cancer cells

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. A current challenge is to understand how proteolytic activity is so precisely localised at discrete sites of the plasma membrane to produce focalised ECM degradation at invadopodia. Indeed, a number of components including metalloproteases need to be directed to invadopodia to ensure proper segregation of proteolytic activities.We recently found invadopodia to feature the properties of cholesterol-rich membrane domains (a.k.a. lipid drafts) and that ECM degradation depends on the tight control of cholesterol homeostasis. Since apically directed polarised sorting and transport in epithelial cells relies on segregation of proteins into lipid rafts at the Golgi complex, we hypothesised that invadopodia-dependent ECM degradation might also rely on lipid raft-dependent polarised transport routes.To investigate this issue we undertook a three-pronged approach. First, we found that microtubule depolymerisation, which is known to disrupt polarised transport in polarised cells, strongly inhibited invadopodia formation, while not affecting overall protein transport. In the second approach we found that glycosylphosphatidylinositol-anchored green fluorescent protein (an apical model protein), but not vesicular stomatitis virus G-protein or influenza virus hemagglutinin (both model basolateral model cargoes), was transported to sites of ECM degradation. Finally, RNAi-mediated knock-down of proteins known to specifically regulate polarised apical or basolateral transport in epithelial cells, such as caveolin 1 and annexin XIIIB or clathrin, respectively, demonstrated that the selective inhibition of the apical, but not the basolateral, transport route impairs invadopodia formation and ECM degradation.Taken together, our findings suggest that invadopodia are apical-like membrane domains, where signal transduction and local membrane remodelling events might be temporally and spatially confined via selective raft-dependent apical transport routes.

Human PXR-mediated induction of intestinal CYP3A4 attenuates 1α,25-dihydroxyvitamin D3 function in human colon adenocarcinoma LS180 cells

Oxidative catabolism of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is mediated by either CYP24A1 or CYP3A4. In this paper, we tested whether induction of CYP3A4 in the LS180 intestinal cell model enhances clearance of 1α,25(OH)2D3 and blunts its hormonal effect on expression of the apical membrane calcium transport protein, TRPV6. Treatment with the hPXR agonist rifampin significantly increased CYP3A4 mRNA content and catalytic activity, but had no effect on CYP24A1 or TRPV6 mRNA content. Pre-treating cells with rifampin for 48h, prior to a 24h 1α,25(OH)2D3 treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)2D3 and a 35% reduction of peak TRPV6 mRNA. Introduction of the CYP3A4 inhibitor, 6′,7′-dihydroxybergamottin, an active inhibitor in grapefruit juice, reversed the effects of rifampin on 1α,25(OH)2D3 clearance and TRPV6 expression. Over-expression of hPXR in LS180 cells greatly enhanced the CYP3A4 responsiveness to rifampin pretreatment, and elicited a greater relative suppression of TRPV6 expression and an increase in 1α,25(OH)2D3 disappearance rate, compared to vector expressed cells, following hormone administration. Together, these results suggest that induction of CYP3A4 in the intestinal epithelium by hPXR agonists can result in a greater metabolic clearance of 1α,25(OH)2D3 and reduced effects of the hormone on the intestinal calcium absorption, which may contribute to an increased risk of drug-induced osteomalacia/osteoporosis in patients receiving chronic therapy with potent hPXR agonists. Moreover, ingestion of grapefruit juice in the at-risk patients could potentially prevent this adverse drug effect.

Rab5 is necessary for the biogenesis of the endolysosomal system in vivo

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

AP-1 is required for the maintenance of apico-basal polarity in theC. elegansintestine

Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium.

Mo1804 Contribution of Intestinal Bacteria and Epithelial Mesenchymal Transition to Intestinal Inflammation and Fibrosis in Enterocyte-Specific Pipk III Deficient Mice

BACKGROUND AND AIM: Genome-wide association studies have provided MTMR3 as a Crohn's-disease-associated gene. Pipk III, which cooperatively functions in phosphatidyl inositol 3,5-bisphosphates [PI(3,5)P2] metabolism with MTMR3, is a kinase that produces PI(3,5)P2 from phophatidyl inositol 3-bisphosphate. We generated enterocyte-specific Pipk III deficient (KO) mice which displayed diarrhea, body weight loss, and pathological findings of epithelial vacuoles, transmural inflammatory cell infiltration, increased fibrosis, and goblet cell depletion, sharing the clinical and histological findings with Crohn's diseae. In this context, we tried to elucidate the mechanisms of intestinal inflammation and fibrosis in Crohn's disease by analyzing those in our KOmice. METHODS:We examined the localization of proteins functioning at apical and basolateral membranes of enterocytes using immunofluorescence staining, and the expression of these membranous proteins in intestinal mucosa of both KO and the control mice using western blot analysis. We compared the expression of inflammationand fibrosis-related genes in intestinal mucosa using RT-PCR. In addition, we monitored the survival of Pipk III KO mice with or without antibiotics to study the relevance of intestinal bacteria in intestinal inflammation of KO mice. Furthermore, we injected fluorescent dextran per anus and observed its mucosal distribution using immunofluorescence staining to evaluate how easily intestinal microbiota can invade to mucosa of KO mice. RESULTS: Although western blot analysis did not show any differences in expression of apical and basolateral membranous proteins between KO and the control mice, immunofluorescence staining revealed mislocalization of these proteins in KO enterocytes, suggesting impaired intracellular vesicle transport and cellular polarity. We found the increased genes expression of proinflammatory cytokines including TNF-alpha, IL-1beta, and IL-6. Meanwhile, the expression of fibrosis-promoting genes such as TGF-beta1 and CTGF, and Snail1, which is a transcription factor that promotes epithelial mesenchymal transition (EMT), was increased in KO mice compared to the control mice. Consistent with this result, immunofluorescence staining revealed that KO enterocytes expressed Vimentin protein more intensely than the control. Furthermore, the survival of KO mice treated with antibiotics was prolonged and fluorescent dextran injected per anus was distributed more scattered in KO mice with antibiotics than those without antibiotics. CONCLUSION: Facilitating entry of intestinal bacteria to mucoca based on impaired intracellular vesicle transport and cellular polarity in enterocytes seems to bring about intestinal inflammation in KO mice. Meanwhile, there is a possibility that EMT in KO enterocytes induces intestinal fibrosis.

Mo1805 Deletion of the KrüPpel-Like Factor 4 Gene in Colonic Epithelium Protects Mice From Dextran Sulfate Sodium (DSS)-Induced Colitis

BACKGROUND AND AIM: Genome-wide association studies have provided MTMR3 as a Crohn's-disease-associated gene. Pipk III, which cooperatively functions in phosphatidyl inositol 3,5-bisphosphates [PI(3,5)P2] metabolism with MTMR3, is a kinase that produces PI(3,5)P2 from phophatidyl inositol 3-bisphosphate. We generated enterocyte-specific Pipk III deficient (KO) mice which displayed diarrhea, body weight loss, and pathological findings of epithelial vacuoles, transmural inflammatory cell infiltration, increased fibrosis, and goblet cell depletion, sharing the clinical and histological findings with Crohn's diseae. In this context, we tried to elucidate the mechanisms of intestinal inflammation and fibrosis in Crohn's disease by analyzing those in our KOmice. METHODS:We examined the localization of proteins functioning at apical and basolateral membranes of enterocytes using immunofluorescence staining, and the expression of these membranous proteins in intestinal mucosa of both KO and the control mice using western blot analysis. We compared the expression of inflammationand fibrosis-related genes in intestinal mucosa using RT-PCR. In addition, we monitored the survival of Pipk III KO mice with or without antibiotics to study the relevance of intestinal bacteria in intestinal inflammation of KO mice. Furthermore, we injected fluorescent dextran per anus and observed its mucosal distribution using immunofluorescence staining to evaluate how easily intestinal microbiota can invade to mucosa of KO mice. RESULTS: Although western blot analysis did not show any differences in expression of apical and basolateral membranous proteins between KO and the control mice, immunofluorescence staining revealed mislocalization of these proteins in KO enterocytes, suggesting impaired intracellular vesicle transport and cellular polarity. We found the increased genes expression of proinflammatory cytokines including TNF-alpha, IL-1beta, and IL-6. Meanwhile, the expression of fibrosis-promoting genes such as TGF-beta1 and CTGF, and Snail1, which is a transcription factor that promotes epithelial mesenchymal transition (EMT), was increased in KO mice compared to the control mice. Consistent with this result, immunofluorescence staining revealed that KO enterocytes expressed Vimentin protein more intensely than the control. Furthermore, the survival of KO mice treated with antibiotics was prolonged and fluorescent dextran injected per anus was distributed more scattered in KO mice with antibiotics than those without antibiotics. CONCLUSION: Facilitating entry of intestinal bacteria to mucoca based on impaired intracellular vesicle transport and cellular polarity in enterocytes seems to bring about intestinal inflammation in KO mice. Meanwhile, there is a possibility that EMT in KO enterocytes induces intestinal fibrosis.

Abstract 5263: The APC tumor suppressor controls epithelial polarization and morphogenesis

Abstract Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor that is frequently mutated or lost in most epithelial-derived tumor types, including colorectal, breast and renal cancer. We have previously demonstrated that germline heterozygous Apc mutation disrupts mammary epithelial integrity. In addition, Apc mutation advances polyoma middle T antigen (PyMT)-mediated mammary tumorigenesis. Interestingly, in both of these models, the effect of Apc mutation was independent of Wnt/β-catenin pathway activation. The objective of the present study was to extend these observations to in vitro models and identify the molecular mechanism by which APC exerts its tumor suppressive function through regulation of epithelial polarity and morphogenesis. Using lentiviral-mediated RNA interference, APC was stably suppressed in non-transformed HC11 mammary and MDCK kidney epithelial cells. Although the APC-knockdown cells did not show any significant morphological or growth differences compared to control vector-only or scrambled shRNA-infected cells on plastic, they showed a dramatic multi-layering phenotype when cultured on permeable supports. Moreover, the polarized localization of basolateral markers, such as Dlg and Scrib tumor suppressor proteins, and apical proteins, including MUC1, was severely disrupted. When the cells were cultured under three-dimensional conditions and compared to controls, the APC-knockdown epithelial cells formed enlarged, non-spherical acini and cysts with filled-in lumens, many of which were invasive into the surrounding matrix. Remarkably, in the MDCK APC-knockdown cells, inverted polarity was observed with the apical marker gp135 localized to the periphery of the cysts. Similar to our previous in vivo studies, these polarization defects associated with APC loss did not appear to require activation of the Wnt signaling pathway but likely involve dysregulation of APC-associated cytoskeletal and polarity complex components. These data indicate that APC is a potent regulator of epithelial polarization and morphogenesis and suggest that one mechanism by which APC loss promotes tumorigenesis is by disrupting epithelial architecture to subsequently drive tumor cell growth, survival and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5263. doi:1538-7445.AM2012-5263

Mitotic spindle orientation can direct cell fate and bias Notch activity in chick neural tube.

Inheritance of apical membrane is proposed to maintain vertebrate neural stem cell proliferation. However, evidence for this is contradictory. Using direct clonal analysis and live imaging in chick neural tube, we show that divisions that separate apical and basal components generate an apical daughter, which becomes a neuron, and a basal daughter, which rapidly re-establishes apico-basal polarity and divides again. Using a recently described real-time reporter of Notch activity, we confirm progenitor status and demonstrate that division orientation can influence Notch signalling. In addition, we reveal loss of apical complex proteins on neuronal differentiation onset, suggesting that removal of this inherited complex is part of the neuronal differentiation mechanism. These findings reconcile contradictory data, link asymmetric division to Notch signalling dynamics and identify apical complex loss as a new step towards neuronal differentiation.

Analysis of the Conformation and Function of the Plasmodium falciparum Merozoite Proteins MTRAP and PTRAMP

Thrombospondin repeat (TSR)-like domains are structures involved with cell adhesion. Plasmodium falciparum proteins containing TSR domains play crucial roles in parasite development. In particular, the preerythrocytic P. falciparum circumsporozoite protein is involved in hepatocyte invasion. The importance of these domains in two other malaria proteins, the merozoite-specific thrombospondin-related anonymous protein (MTRAP) and the thrombospondin-related apical membrane protein (PTRAMP), were assessed using near-full-length recombinant proteins composed of the extracellular domains produced in Escherichia coli. MTRAP is thought to be released from invasive organelles identified as micronemes during merozoite invasion to mediate motility and host cell invasion through an interaction with aldolase, an actin binding protein involved in the moving junction. PTRAMP function remains unknown. In this study, the conformation of recombinant MTRAP (rMTRAP) appeared to be a highly extended protein (2 nm by 33 nm, width by length, respectively), whereas rPTRAMP had a less extended structure. Using an erythrocyte binding assay, rMTRAP but not rPTRAMP bound human erythrocytes; rMTRAP binding was mediated through the TSR domain. MTRAP- and in general PTRAMP-specific antibodies failed to inhibit P. falciparum development in vitro. Altogether, MTRAP is a highly extended bifunctional protein that binds to an erythrocyte receptor and the merozoite motor.

Podocalyxin in podocyte injury of proteinuria associated nephropathy

Podocalyxin,a sialomucin most closely related to CD34,is expressed by kidney podocytes,hematopoietic progenitors,vascular endothelia and a subset of neurons.As one of the important apical marker protein,podocalyxin plays an important role in the integrity of the structure and function of nomal podocyte,and is closely related to the prevention of proteinuria.The mechanism of podocyte injury associated with proteinuria remains unkown.The investigation of podocalyxin,a major podocyte protein,is significant in the pathogenesis and process of the primarily glomerulopathies.This paper summarizes the progresses of podocalyxin in podocyte injury of proteinuria associated nephropathy. Key words: Podocalyxin; Proteinuia; Podocyte injury; Nephrotic syndrome

Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH(4)(+) and HCO(3)(-) ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMV(PCT)) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na(+)-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na(+)-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMV(PCT) isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.