scholarly journals Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells

2005 ◽  
Vol 169 (3) ◽  
pp. 491-501 ◽  
Author(s):  
Delphine Delacour ◽  
Valérie Gouyer ◽  
Jean-Pierre Zanetta ◽  
Hervé Drobecq ◽  
Emmanuelle Leteurtre ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Functional Role ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Affinity Ligands ◽  
Membrane Markers ◽  
Polarized Trafficking ◽  
Hydroxylated Fatty Acids ◽  
Ht 29

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAcα-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4–depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.

Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron

2001 ◽  
Vol 281 (6) ◽  
pp. F1117-F1122 ◽  
Author(s):  
Tong Wang ◽  
Max Hropot ◽  
Peter S. Aronson ◽  
Gerhard Giebisch
Keyword(s):  
Proximal Tubule ◽  
Functional Role ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Significant Inhibition ◽  
Distal Convoluted Tubule ◽  
Loop Of Henle ◽  
Nhe Isoforms

This study assessed the functional role of Na+/H+ exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid ( J v) and HCO[Formula: see text]( J HCO3) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced J v and J HCO3, but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, J HCO3 was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, J HCO3was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na+/H+ exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.

scholarly journals Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

2011 ◽  
Vol 301 (2) ◽  
pp. G306-G316 ◽  
Author(s):  
Michelle A. Falkowski ◽  
Diana D. H. Thomas ◽  
Scott W. Messenger ◽  
Thomas F. Martin ◽  
Guy E. Groblewski
Keyword(s):  
Membrane Trafficking ◽  
Functional Role ◽  
Apical Membrane ◽  
Pancreatic Acinar Cells ◽  
Zymogen Granule ◽  
Pancreatic Acini ◽  
Protein Receptor ◽  
Intracellular Ca ◽  
Synaptotagmin 1 ◽  
Associated Family

Secretagogue-induced changes in intracellular Ca2+ play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca2+ are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca2+-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca2+-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.

scholarly journals Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression

2012 ◽  
Vol 23 (16) ◽  
pp. 3229-3239 ◽  
Author(s):  
Takao Yasuda ◽  
Chika Saegusa ◽  
Sachiko Kamakura ◽  
Hideki Sumimoto ◽  
Mitsunori Fukuda
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Apical Surface ◽  
Dependent Manner ◽  
Signaling Molecule ◽  
Downstream Target ◽  
Junction Protein ◽  
Polarized Trafficking

Most cells in tissues are polarized and usually have two distinct plasma membrane domains—an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin–Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a–knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.

Chronic PKC-β activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

2006 ◽  
Vol 291 (2) ◽  
pp. G318-G330 ◽  
Author(s):  
James R. Broughman ◽  
Limin Sun ◽  
Shahid Umar ◽  
Jason Scott ◽  
Joseph H. Sellin ◽  
...  
Keyword(s):  
Phorbol Esters ◽  
Prolonged Exposure ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Membrane Association ◽  
High Dose ◽  
Camp Generation ◽  
Pkc Β ◽  
Ht 29

We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl−current ( ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISCgeneration. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-β-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC α-, β1-, γ-, and ε-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISCcorrelated with PKC-α membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-β1, decreased PKC-β2membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-β1and a significant reduction in PKC-ε mass occurred. Thus activated PKC-β1and/or PKC-ε prevented, whereas activated PKC-α facilitated, apical membrane ISC-cAMP. PKC-β-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC β-isoform.

PKC-ε regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS

1999 ◽  
Vol 277 (6) ◽  
pp. C1239-C1249 ◽  
Author(s):  
Jaekyung Cecilia Song ◽  
Bruce J. Hrnjez ◽  
Omid C. Farokhzad ◽  
Jeffrey B. Matthews
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Basolateral Membrane ◽  
Phosphorylation Site ◽  
Fluid Phase ◽  
Cytochalasin D ◽  
Pkc Inhibitor ◽  
Intestinal Epithelia ◽  
Stimulation Of

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-ε. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-ε, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

H(+)-K(+)-ATPase in rabbit cortical collecting duct B-type intercalated cell

1996 ◽  
Vol 270 (3) ◽  
pp. F518-F530 ◽  
Author(s):  
I. D. Weiner ◽  
A. E. Milton
Keyword(s):  
B Cell ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cortical Collecting Duct ◽  
Intercalated Cell ◽  
Acid Load ◽  
Phi Regulation

The role of H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) in the cortical collecting duct (CCD) B-type intercalated cell (B cell) is unclear. This study examined whether H(+)-K(+)-ATPase contributes to B cell intracellular pH (pHi) regulation and, if so, whether it is present at the apical or basolateral membrane. B cell Na(+)-independent pHi recovery from an acid load was only partially inhibited by peritubular N-ethylmaleimide (NEM). Complete inhibition required combining peritubular NEM either with luminal Sch-28080 or with luminal K+ removal. In contrast, neither peritubular Sch-28080 nor peritubular K+ removal altered pHi regulation. Tomato lectin, which binds to the gastric H(+)-K(+)-ATPase beta-subunit, labeled the B cell apical membrane. We conclude that the rabbit CCD B cell possesses an apical H(+)-K(+)-ATPase that plays an important role in pHi recovery from an in vitro acid load.

Contribution of the Na+-K+-2Cl−cotransporter NKCC1 to Cl− secretion in rat OMCD

2001 ◽  
Vol 280 (5) ◽  
pp. F913-F921 ◽  
Author(s):  
Susan M. Wall ◽  
Michael P. Fischer ◽  
Pramod Mehta ◽  
Kathryn A. Hassell ◽  
Stanley J. Park
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Fluid Secretion ◽  
Transepithelial Potential ◽  
In Series

In rat kidney the “secretory” isoform of the Na+-K+-2Cl− cotransporter (NKCC1) localizes to the basolateral membrane of the α-intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes Cl− and whether transepithelial Cl− transport occurs, in part, through Cl− uptake across the basolateral membrane mediated by NKCC1 in series with Cl− efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical HCO[Formula: see text]/CO2-buffered solutions. Cl− secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 μM), when added to the bath, reduced Cl− secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive Cl− secretion was dependent on extracellular Na+ and either K+ or NH[Formula: see text], consistent with the ion dependency of NKCC1-mediated Cl− transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete Cl−into the luminal fluid through NKCC1-mediated Cl− uptake across the basolateral membrane in series with Cl− efflux across the apical membrane. The physiological role of NKCC1-mediated Cl− uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated.

scholarly journals Role of NBCe1 and AE2 in Secretory Ameloblasts

2008 ◽  
Vol 87 (4) ◽  
pp. 391-395 ◽  
Author(s):  
M.L. Paine ◽  
M.L. Snead ◽  
H.J. Wang ◽  
N. Abuladze ◽  
A. Pushkin ◽  
...  
Keyword(s):  
Sodium Bicarbonate ◽  
Anion Exchanger ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Transport Processes ◽  
Bicarbonate Secretion ◽  
Enamel Formation

The H+/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

scholarly journals Caveolin-1 Is Not Essential for Biosynthetic Apical Membrane Transport

2005 ◽  
Vol 25 (22) ◽  
pp. 10087-10096 ◽  
Author(s):  
Aki Manninen ◽  
Paul Verkade ◽  
Soazig Le Lay ◽  
Juha Torkko ◽  
Michael Kasper ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Membrane Trafficking ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Embryonic Fibroblasts ◽  
Caveolin 1 ◽  
Influenza Virus Hemagglutinin ◽  
Kinetics Of ◽  
Darby Canine Kidney

ABSTRACT Caveolin-1 has been implicated in apical transport of glycosylphosphatidylinositol (GPI)-anchored proteins and influenza virus hemagglutinin (HA). Here we have studied the role of caveolin-1 in apical membrane transport by generating caveolin-1-deficient Madin-Darby canine kidney (MDCK) cells using retrovirus-mediated RNA interference. The caveolin-1 knockdown (cav1-KD) MDCK cells were devoid of caveolae. In addition, caveolin-2 was retained in the Golgi apparatus in cav1-KD MDCK cells. However, we found no significant alterations in the apical transport kinetics of GPI-anchored proteins or HA upon depletion of caveolin-1. Similar results were obtained using embryonic fibroblasts from caveolin-1-knockout mice. Thus, we conclude that caveolin-1 does not play a major role in lipid raft-mediated biosynthetic membrane trafficking.

scholarly journals Cdc42 negatively regulates endocytosis during apical membrane maintenance in live animals

2019 ◽  
Vol 30 (3) ◽  
pp. 324-332 ◽  
Author(s):  
Akiko Shitara ◽  
Lenka Malec ◽  
Seham Ebrahim ◽  
Desu Chen ◽  
Christopher Bleck ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Apical Membrane ◽  
Fluid Secretion ◽  
Small Gtpase ◽  
Model Systems ◽  
Adult Mice ◽  
Apical Domain ◽  
Endocytic Pathways

Lumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. Depletion of Cdc42 in adult mice induced a significant expansion of the apical canaliculi, whereas depletion at late embryonic stages resulted in a complete inhibition of their postnatal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and toward the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.

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