scholarly journals MUC1 traverses apical recycling endosomes along the biosynthetic pathway in polarized MDCK cells

2009 ◽  
Vol 390 (7) ◽  
Author(s):  
Polly E. Mattila ◽  
Carol L. Kinlough ◽  
Jennifer R. Bruns ◽  
Ora A. Weisz ◽  
Rebecca P. Hughey
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Metabolic Labeling ◽  
Apical Surface ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Myosin Vb ◽  
Targeting Signals ◽  
Golgi Network

AbstractMUC1 is a heavily glycosylated transmembrane protein localized at the apical surface of polarized epithelial cells. Here, we examined the biosynthetic route of newly synthesized MUC1 in polarized Madin-Darby canine kidney (MDCK) cells. Apically and basolaterally destined cargo are sorted at thetrans-Golgi network into distinct vesicles, and proteins with lipid raft-dependent apical targeting signals and glycan-dependent apical targeting signals appear to specifically transit apical early endosomes (AEEs) and apical recycling endosomes (AREs), respectively. Using metabolic labeling we found that MUC1 is efficiently targeted to the apical surface of polarized MDCK cells with at1/2of 45 min. Apical delivery was not altered by inactivation of AEEs by treatment with hydrogen peroxide and diaminobenzidine treatment after apical loading of endosomes with horseradish peroxidase-conjugated wheat germ agglutinin. However, expression of a GFP-tagged myosin Vb tail fragment (GFP-MyoVbT) that disrupts export from the ARE significantly reduced MUC1 apical expression. Moreover, MUC1 expressed for brief periods in MDCK cells co-localized with GFP-MyoVbT. We conclude that MUC1 traffics to the apical surface via AREs in polarized renal epithelial cells.

PKC induces internalization and retention of the EAAC1 glutamate transporter in recycling endosomes of MDCK cells

2009 ◽  
Vol 297 (4) ◽  
pp. C835-C844 ◽  
Author(s):  
Valeria Padovano ◽  
Silvia Massari ◽  
Silvia Mazzucchelli ◽  
Grazia Pietrini
Keyword(s):  
Excitatory Amino Acid ◽  
Mdck Cells ◽  
Glutamate Transporter ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Metabolic Labeling ◽  
Apical Surface ◽  
Recycling Endosomes ◽  
Intracellular Compartments

Here we show that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate transport activity due to relocalization of the excitatory amino acid carrier 1 (EAAC1) glutamate transporter from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cells to intracellular compartments. The PKC-induced internalization of EAAC1 is negatively regulated by the calcineurin inhibitor cyclosporine A and by the expression of a dominant-negative mutant of the endocytic protein dynamin 1, a well-known target of the phosphatase activity of calcineurin. Using 32P-metabolic labeling experiments, we found unchanged levels of phosphorylated EAAC1, indicating that EAAC1 relocalization does not depend on PKC and calcineurin modification of the transporter, while we found that a target of these modifications was the serine778 residue of dynamin, a calcineurin substrate that in its dephosphorylated form activates the endocytic functions of dynamin. These data suggest that PMA stimulates endogenous dynamin and that this activation is required to mediate internalization of EAAC1 in MDCK cells. By immunofluorescence experiments with endosomal markers we demonstrated that internalized EAAC1 accumulates in endosomes also containing the basolateral betaine-GABA transporter BGT1 and activated PKCα. The sustained activation of PKC was required to maintain the transporters in the endosomal compartment, while a posttreatment with a PKC-specific inhibitor induced the recycling of the transporters to their appropriate surfaces. Taken together, our data indicate that PKC activity regulates EAAC1 surface density in MDCK cells by inducing its internalization and retention in PKCα-labeled recycling endosomes common to apical and basolateral proteins.

scholarly journals Sorting of newly synthesized galactosphingolipids to the two surface domains of epithelial cells.

1996 ◽  
Vol 132 (5) ◽  
pp. 813-821 ◽  
Author(s):  
P van der Bijl ◽  
M Lopes-Cardozo ◽  
G van Meer
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
High Concentration ◽  
Selective Transport ◽  
Endogenous Lipid ◽  
Surface Domains ◽  
Golgi Network

The high concentration of glycosphingolipids on the apical surface of epithelial cells may be generated by selective transport from their site of synthesis to the cell surface. Previously, we showed that canine kidney MDCK and human intestinal Caco-2 cells converted a ceramide carrying the short fluorescent fatty acid C6-NBD to glucosylceramide (GlcCer) and sphingomyelin (SM), and that GlcCer was preferentially transported to the apical surface as compared to SM. Here, we address the point that not all glycosphingolipid classes are apically enriched in epithelia. We show that a ceramide containing the 2-hydroxy fatty acid C6OH was preferentially converted by MDCK and Caco-2 cells to galactosylceramide (GalCer) and its derivatives galabiosylceramide (Ga2Cer) and sulfatide (SGalCer) as compared to SM and GlcCer--all endogenous lipid classes of these cells. Transport to the apical and basolateral cell surface was monitored by a BSA-depletion assay. In MDCK cells, GalCer reached the cell surface with two- to sixfold lower apical/basolateral polarity than GlcCer. Remarkably, in Caco-2 cells GalCer and GlcCer displayed the same apical/basolateral polarity, but it was sixfold lower for lipids with a C6OH chain than for C6-NBD lipids. Therefore, the sorting of a sphingolipid appears to depend on lipid structure and cell type. We propose that the different ratios of gluco- and galactosphingolipid synthesis in the various epithelial tissues govern lipid sorting in the membrane of the trans Golgi network by dictating the composition of the domains from where vesicles bud to the apical and basolateral cell surface.

scholarly journals Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture

1980 ◽  
Vol 84 (3) ◽  
pp. 808-814 ◽  
Author(s):  
M Klagsbrun
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Calf Serum ◽  
Morphological Characteristics ◽  
Bovine Colostrum ◽  
Apical Surface ◽  
Early Passage ◽  
Rat Fibroblasts ◽  
Canine Kidney

Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco's modified eagle's medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow in DMEM supplemented with colostrum. Sparse cultures of early-passage human and rat fibroblasts that grow readily in DMEM supplemented with serum do not grow in DMEM supplemented with colostrum. Canine kidney epithelial cells (MDCK), when plated sparsely, grow exponentially in DMEM supplemented with day 1 bovine colostrum. The generation time is 26 h, the same growth rate as in DMEM supplemented with calf serum. The MDCK cells can be subcultured and regrown to confluence repeatedly in colostrum-supplemented DMEM. Growth in DMEM supplemented with colostrum does not alter the morphological characteristics of the MDCK cells, which are polygonal, contain microvilli at the apical surface, and are connected by tight junctions and desmosomes. MDCK cells do not proliferate in DMEM supplemented with milk obtained 1 wk after the birth of a calf.

scholarly journals GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

2006 ◽  
Vol 172 (7) ◽  
pp. 1023-1034 ◽  
Author(s):  
Simona Paladino ◽  
Thomas Pocard ◽  
Maria Agata Catino ◽  
Chiara Zurzolo
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Biochemical Assays ◽  
Intracellular Sorting ◽  
Apical Sorting ◽  
Golgi Network ◽  
Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

1995 ◽  
Vol 108 (1) ◽  
pp. 369-377 ◽  
Author(s):  
K.L. Soole ◽  
M.A. Jepson ◽  
G.P. Hazlewood ◽  
H.J. Gilbert ◽  
B.H. Hirst
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Gpi Anchor ◽  
Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

scholarly journals Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells

2007 ◽  
Vol 18 (10) ◽  
pp. 3978-3992 ◽  
Author(s):  
Asli Oztan ◽  
Mark Silvis ◽  
Ora A. Weisz ◽  
Neil A. Bradbury ◽  
Shu-Chan Hsu ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Small Gtpase ◽  
Junctional Complex ◽  
Recycling Endosomes ◽  
Permeabilized Cells ◽  
Exocyst Complex ◽  
C Terminus ◽  
Streptolysin O ◽  
Early Endosomes ◽  
Endocytic Traffic

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

scholarly journals Regulation of Membrane Trafficking by a Novel Cdc42-related Protein in Caenorhabditis elegans Epithelial Cells

2005 ◽  
Vol 16 (4) ◽  
pp. 1629-1639 ◽  
Author(s):  
S. Jenna ◽  
M.-E. Caruso ◽  
A. Emadali ◽  
D. T. Nguyên ◽  
M. Dominguez ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Rho Gtpases ◽  
Related Protein ◽  
Recycling Endosomes ◽  
C Elegans ◽  
Apical Trafficking ◽  
Golgi Network

Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C6-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.

scholarly journals Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

1992 ◽  
Vol 118 (1) ◽  
pp. 51-62 ◽  
Author(s):  
S H Low ◽  
B L Tang ◽  
S H Wong ◽  
W Hong
Keyword(s):  
Protein Targeting ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Selective Inhibition ◽  
Surface Expression ◽  
Apical Surface ◽  
Golgi Transport ◽  
Apical Domain ◽  
Golgi Network ◽  
Basolateral Targeting

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.

scholarly journals Pili Binding to Asialo-GM1 on Epithelial Cells Can Mediate Cytotoxicity or Bacterial Internalization byPseudomonas aeruginosa

1999 ◽  
Vol 67 (7) ◽  
pp. 3207-3214 ◽  
Author(s):  
James C. Comolli ◽  
Leslie L. Waite ◽  
Keith E. Mostov ◽  
Joanne N. Engel
Keyword(s):  
Epithelial Cells ◽  
Cell Injury ◽  
Mdck Cells ◽  
Type Iv Pili ◽  
Receptor Interaction ◽  
Apical Surface ◽  
Epithelial Monolayers ◽  
Type Iv ◽  
Mediate Cytotoxicity ◽  
Asialo Gm1

ABSTRACT The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to an untreated monolayer,P. aeruginosa PA103 displayed an eightfold increase in association with and fivefold more cytotoxicity toward MDCK cells pretreated with aGM1. Cytotoxicity of either carrier-treated or aGM1-treated monolayers required the type III secreted protein ExoU. Asialo-GM1 pretreatment of MDCK monolayers likewise augmented bacterial internalization of an isogenic invasive strain approximately fourfold. These increases were not seen in monolayers treated with GM1, the sialyated form of the glycolipid, and were inhibited by treatment with an antibody to aGM1. Also, the aGM1-mediated adhesion, cytotoxicity, and internalization required intact type IV pili since nonpiliated PA103 mutants were unaffected by aGM1 pretreatment of MDCK cells. These results demonstrate that epithelial cell injury and bacterial internalization can proceed from the same adhesin-receptor interaction, and they indicate that P. aeruginosa exoproducts solely determine the steps subsequent to adhesion.

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