Regulation of pathways within cultured epithelial cells for the transcytosis of a basal membrane-bound peroxidase-polylysine conjugate

1993 ◽  
Vol 106 (4) ◽  
pp. 1313-1321
Author(s):  
M.E. Taub ◽  
W.C. Shen
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Fold Increase ◽  
Basal Membrane ◽  
Size Exclusion ◽  
Apical Surface ◽  
Time Treatment ◽  
Apical Direction ◽  
Exclusion Chromatography

A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used as a non-specific adsorptive probe to study transcytosis in MDCK strain I and Caco-2 epithelial cells. As we have shown previously, HRP-PLL transcytosis proceeds via an intracellular, non-lysosomal proteolytic compartment in MDCK cells; yet, this compartment is utilized for transcytosis only in the basal-to-apical direction (Taub, M. E. and Shen, W.-C. J. Cell. Physiol., 150, 283–290, 1992). Using size exclusion chromatography, we demonstrate that the PLL moiety of the conjugate is effectively cleaved during transcytosis, thus releasing free HRP from the apical surface of the cells. Pulse-chase studies indicate that approximately 6% of basolateral surface-associated HRP-PLL conjugate in Transwell-grown cell monolayers enters the basal-to-apical transcytotic pathway. Brief (1 hour) treatment with 160 nM phorbol ester (PMA), a protein kinase C stimulator, elicits a 2-fold increase in the rate and amount of HRP-PLL transcytosis following a 1 hour lag time. Treatment with 1.6 micrograms/ml brefeldin A (BFA) inhibits HRP-PLL transcytosis by approximately 30%; additionally, BFA is able to abolish completely the PMA stimulatory effect. Removal of BFA causes a re-establishment of the normal rate of transcytosis within 2 hours, demonstrating the reversibility of BFA inhibition with respect to HRP-PLL transcytosis. Thus, PMA most likely elicits an increase in the amount of basally internalized conjugate delivered to BFA-sensitive transcytotic compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Epithelial sphingolipid sorting is insensitive to reorganization of the Golgi by nocodazole, but is abolished by monensin in MDCK cells and by brefeldin A in Caco-2 cells

1993 ◽  
Vol 104 (3) ◽  
pp. 833-842 ◽  
Author(s):  
G. van Meer ◽  
W. van't Hof
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Inhibitory Effect ◽  
Lipid Transport ◽  
Apical Surface ◽  
Transport Pathway ◽  
Apical Direction ◽  
Golgi Structure ◽  
Surface Domains

In epithelial MDCK and Caco-2 cells, short-chain analogs of glucosylceramide and sphingomyelin are delivered from the Golgi to the cell surface with different apical/basolateral polarities, which results in an apical enrichment of the glycolipid glucosylceramide over the phospholipid sphingomyelin. Here, we have interfered with the integrity of the Golgi complex in various ways and tested the effects on lipid transport and sorting. Nocodazole, which depolymerizes microtubules, dispersed the Golgi over the cytoplasm of MDCK cells and reduced transport of newly synthesized C6-NBD-(N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminocaproyl)-glucosy lceramide and C6-NBD-sphingomyelin to the apical surface by 40%. The lipids were not mistargeted to the basolateral surface and upon removal of nocodazole, apical transport recovered. Nocodazole did not affect the apical enrichment of glucosylceramide over sphingomyelin. The ionophore monensin led to swelling of the Golgi of MDCK cells and inhibited lipid transport to the cell surface by 30–50%. Whereas sphingomyelin transport to both surface domains was equally affected, monensin mainly inhibited apical transport of glucosylceramide. At 10–20 microM of monensin, the two lipids displayed the same polarity of delivery: sorting between the two lipids was abolished. Brefeldin A at 1 microgram/ml, which resulted in disruption of the Golgi in HepG2 cells and completely inhibited protein secretion, had no inhibitory effect on transport of the C6-NBD-lipids to the surface. The same was observed in Caco-2 cells. However, brefeldin A selectively shifted transport of sphingomyelin towards the apical direction which abolished the apical enrichment of glucosylceramide over sphingomyelin. Caco-2 cells were used because in MDCK cells brefeldin A did not change Golgi structure nor lipid transport and sorting. In summary, modification of the Golgi by monensin and brefeldin A, but not nocodazole, interfered with the sorting event by which glucosylceramide is enriched over sphingomyelin in the transport pathway from the Golgi to the apical 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.

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.

Metabolic inhibition-induced transient Ca2+ increase depends on mitochondria in a human proximal renal cell line

2007 ◽  
Vol 293 (2) ◽  
pp. F533-F540 ◽  
Author(s):  
Adrian Caplanusi ◽  
Andrew J. Fuller ◽  
Romer A. Gonzalez-Villalobos ◽  
Timothy G. Hammond ◽  
L. G. Navar
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Fold Increase ◽  
Recovery Phase ◽  
Metabolic Inhibition ◽  
Human Origin ◽  
Renal Epithelial Cells ◽  
Proximal Tubular ◽  
Renal Cell Line ◽  
Signaling Process

During ischemia or hypoxia an increase in intracellular cytosolic Ca2+ induces deleterious events but is also implicated in signaling processes triggered in such conditions. In MDCK cells (distal tubular origin), it was shown that mitochondria confer protection during metabolic inhibition (MI), by buffering the Ca2+ overload via mitochondrial Na+-Ca2+ exchanger (NCX). To further assess this process in cells of human origin, human cortical renal epithelial cells (proximal tubular origin) were subjected to MI and changes in cytosolic Ca2+ ([Ca2+]i), Na+, and ATP concentrations were monitored. MI was accomplished with both antimycin A and 2-deoxyglucose and induced a 3.5-fold increase in [Ca2+]i, reaching 136.5 ± 15.8 nM in the first 3.45 min. Subsequently [Ca2+]i dropped and stabilized to 62.7 ± 7.3 nM by 30 min. The first phase of the transient increase was La3+ sensitive, not influenced by diltiazem, and abolished when mitochondria were deenergized with the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The subsequent recovery phase was impaired in a Na+-free medium and weakened when the mitochondrial NCX was blocked with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Thus Ca2+ entry is likely mediated by store-operated Ca2+ channels and depends on energized mitochondria, whereas [Ca2+]i recovery relied partially on the activity of mitochondrial NCX. These results indicate a possible mitochondrial-mediated signaling process triggered by MI, support the hypothesis that mitochondrial NCX has an important role in the Ca2+ clearance, and overall suggest that mitochondria play a preponderant role in the regulation of responses to MI in human renal epithelial cells.

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.

Opposite sorting and transcytosis of the polymeric immunoglobulin receptor in transfected endothelial and epithelial cells

1998 ◽  
Vol 111 (9) ◽  
pp. 1197-1206
Author(s):  
T. Su ◽  
K.K. Stanley
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Epithelial Cells ◽  
Cell Line ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Secretory Component ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Immunoglobulin Receptor

We have transfected a polarised endothelial cell line, ECV 304, and an epithelial cell line, MDCK, with a well characterised epithelial protein, the rat polymeric immunoglobulin receptor (pIgR), in order to study the protein sorting and transcytosis in endothelial cells. The expressed protein was normally processed and the steady state distribution between apical and basolateral surfaces was similar in both cell types. MDCK cells, however, showed a marked polarity in the delivery of newly synthesised pIgR to the cell surface, and in the release of secretory component. 88% of newly synthesised pIgR in MDCK cells was first delivered to the basolateral surface and 99% of secretory component was released from the apical surface. In contrast the basolateral targeting signal of pIgR was only partially recognised in endothelial cells, with 63% of the newly synthesised pIgR being first delivered to the basolateral surface. At steady state only 43% of the pIgR was found on the basolateral membrane. The direction of dimeric IgA transcytosis in endothelial cells was from apical to basolateral surfaces, opposite to that in MDCK cells. These data suggest that endothelial cells poorly recognise the targeting signals of proteins from epithelial cells, and that the direction of transcytosis is linked to the biological role of the cells.

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 Entry of simian virus 40 is restricted to apical surfaces of polarized epithelial cells.

1988 ◽  
Vol 8 (8) ◽  
pp. 3391-3396 ◽  
Author(s):  
E T Clayson ◽  
R W Compans
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Apical Surface ◽  
Surface Binding ◽  
Virus Binding ◽  
Single Class ◽  
Infected Cells ◽  
Polarized Epithelial Cells

The uptake of simian virus 40 (SV40) by polarized epithelial cells was investigated by growth of cells on permeable supports and inoculation on either the apical or the basolateral surface. Binding of radiolabeled SV40 occurred on the apical but not the basolateral surfaces of permissive polarized Vero C1008 cells and nonpermissive polarized MDCK cells. When similar experiments were performed on nonpolarized Vero or CV-1 cells, virus binding occurred regardless of the direction of virus input. Electron micrographs of Vero C1008 cells infected at high multiplicities revealed virions lining the surfaces of apically infected cells, while the surfaces of basolaterally infected cells were devoid of virus particles. Analysis of the binding data revealed a single class of virus receptors (9 x 10(4) per cell) with a high affinity for SV40 (Kd = 3.76 pM) on the apical surfaces of Vero C 1008 cells. Indirect immunofluorescence studies revealed that synthesis of viral capsid proteins in Vero C1008 cells occurred only when input virions had access to the apical surface. Virus yields from apically infected Vero C1008 cells were 10(5) PFU per cell, while yields obtained from basolaterally infected cells were less than one PFU per cell. These results indicate that a specific receptor for SV40 is expressed exclusively on the apical surfaces of polarized Vero C1008 cells.

scholarly journals The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts.

1993 ◽  
Vol 121 (6) ◽  
pp. 1299-1310 ◽  
Author(s):  
S J Neame ◽  
C M Isacke
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Constitutive Expression ◽  
Cytoplasmic Tail ◽  
Apical Surface ◽  
Wild Type ◽  
Coated Pits ◽  
Triton X ◽  
Cortical Cytoskeleton ◽  
Human Specific

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.

Sign in / Sign up

Export Citation Format

Share Document