scholarly journals Role of Mitochondrial Na+ Concentration, Measured by CoroNa Red, in the Protection of Metabolically Inhibited MDCK Cells

2005 ◽  
Vol 16 (12) ◽  
pp. 3490-3497 ◽  
Author(s):  
Szilvia Baron ◽  
Adrian Caplanusi ◽  
Martin van de Ven ◽  
Mihai Radu ◽  
Sanda Despa ◽  
...  
Keyword(s):  
Mdck Cells

scholarly journals Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

2012 ◽  
Vol 23 (12) ◽  
pp. 2302-2318 ◽  
Author(s):  
Lynne A. Lapierre ◽  
Kenya M. Avant ◽  
Cathy M. Caldwell ◽  
Asli Oztan ◽  
Gerard Apodaca ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Cellular Polarity ◽  
Interacting Protein ◽  
P120 Catenin ◽  
Myosin Vb ◽  
Darby Canine Kidney

The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin–Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)–expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.

scholarly journals Pseudomonas aeruginosa-Mediated Damage Requires Distinct Receptors at the Apical and Basolateral Surfaces of the Polarized Epithelium

2009 ◽  
Vol 78 (3) ◽  
pp. 939-953 ◽  
Author(s):  
Iwona Bucior ◽  
Keith Mostov ◽  
Joanne N. Engel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mdck Cells ◽  
Three Dimensional ◽  
Opportunistic Pathogen ◽  
Necessary And Sufficient ◽  
Plastic Surfaces ◽  
Pharmacologic Inhibition ◽  
Increased Susceptibility ◽  
Polarized Epithelium

ABSTRACT Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.

The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells

2001 ◽  
Vol 114 (7) ◽  
pp. 1331-1341 ◽  
Author(s):  
A.K. Criss ◽  
D.M. Ahlgren ◽  
T.S. Jou ◽  
B.A. McCormick ◽  
J.E. Casanova
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Bacterial Pathogen ◽  
Small Gtpases ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Intestinal Epithelial ◽  
Actin Structure

The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Ρ family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two ‘Rgr; family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

Volume-activated K+ and Cl- pathways of dissociated epithelial cells (MDCK): role of Ca2+

1990 ◽  
Vol 258 (5) ◽  
pp. C827-C834 ◽  
Author(s):  
A. Rothstein ◽  
E. Mack
Keyword(s):  
Mdck Cells ◽  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
Ionophore A23187 ◽  
Madin Darby Canine Kidney ◽  
Osmotic Swelling ◽  
Volume Activation ◽  
Darby Canine Kidney ◽  
Volume Decrease

Osmotic swelling of dissociated Madin-Darby canine kidney (MDCK) cells in NaCl medium is followed by shrinking (regulatory volume decrease, or RVD) or in KCl medium by secondary swelling. The cation ionophore gramicidin has little effect on volumes of isotonic cells but accelerates volume-activated changes in either medium. Immediately after hypotonic exposure, the membrane becomes transiently hyperpolarized followed by depolarization. The depolarization phase is diminished by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Swelling is also associated with an almost immediate increase in Ca2+ influx and elevation of cytoplasmic Ca2+ ([Ca2+]i) preceding RVD. In Ca2(+)-free medium, [Ca2+]i rapidly declines to a low level. Osmotic swelling, under these circumstances, is associated with a small transient increase in [Ca2+]i, but RVD or secondary swelling (in KCl) are minimal. Under these conditions, addition of gramicidin or the Ca2(+)-ionophore A23187 induces significant volume changes, although not as large as those found in the presence of Ca2+. Quinine inhibits RVD in the absence of gramicidin, but not in its presence; oligomycin C, DIDS, and trifluoperazine, on the other hand, inhibit in the presence of the ionophore. These findings suggest that in MDCK cells RVD involves activation of distinct conductive K+ and Cl- pathways which allow escape of KCl and osmotically obligated water and that activation of both pathways is associated with elevated [Ca2+]i derived largely from volume activation of a Ca2(+)-influx pathway.

scholarly journals 150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells

2000 ◽  
Vol 278 (6) ◽  
pp. C1172-C1182 ◽  
Author(s):  
Yoshio Bando ◽  
Satoshi Ogawa ◽  
Atsushi Yamauchi ◽  
Keisuke Kuwabara ◽  
Kentaro Ozawa ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Protein Transport ◽  
Atp Hydrolysis ◽  
Mdck Cells ◽  
Secretory Protein ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney

To assess the participation of the 150-kDa oxygen-regulated protein (ORP150) in protein transport, its function in Madin-Darby canine kidney (MDCK) cells was studied. Exposure of MDCK cells to hypoxia resulted in an increase of ORP150 antigen and increased binding of ORP150 to GP80/clusterin (80-kDa glycoprotein), a natural secretory protein in this cell line. In ORP150 antisense transformant MDCK cells, GP80 was retained within the endoplasmic reticulum after exposure to hypoxia. Metabolic labeling showed the delay of GP80 maturation in antisense transformants in hypoxia, whereas its matured form was detected in wild-type cells, indicating a role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 suggested its ability to bind to ATP-agarose. Furthermore, the ATP hydrolysis analysis showed that ORP150 can release GP80 at a lower ATP concentration. These data indicate that ORP150 may function as a unique molecular chaperone in renal epithelial cells by facilitating protein transport/maturation in an environment where less ATP is accessible.

scholarly journals Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

1996 ◽  
Vol 135 (1) ◽  
pp. 153-167 ◽  
Author(s):  
J Peränen ◽  
P Auvinen ◽  
H Virta ◽  
R Wepf ◽  
K Simons
Keyword(s):  
Epithelial Cells ◽  
Membrane Transport ◽  
Vesicular Stomatitis Virus ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Wild Type ◽  
Expression Systems ◽  
Vesicular Stomatitis

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

scholarly journals Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton

2004 ◽  
Vol 164 (5) ◽  
pp. 717-727 ◽  
Author(s):  
David Cohen ◽  
Patrick J. Brennwald ◽  
Enrique Rodriguez-Boulan ◽  
Anne Müsch
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Cell Contact ◽  
Bile Canaliculi ◽  
Madin Darby Canine Kidney ◽  
Lumen Formation ◽  
Contact Sites ◽  
Darby Canine Kidney ◽  
Canine Kidney

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.

Human IgA inhibits adherence ofAcanthamoeba polyphagato epithelial cells and contact lenses

2004 ◽  
Vol 50 (9) ◽  
pp. 711-718 ◽  
Author(s):  
Rafael Campos-Rodríguez ◽  
Gabriela Oliver-Aguillón ◽  
Luz M Vega-Pérez ◽  
Adriana Jarillo-Luna ◽  
Dolores Hernández-Martínez ◽  
...  
Keyword(s):  
Contact Lenses ◽  
Mdck Cells ◽  
Secretory Iga ◽  
Acanthamoeba Polyphaga ◽  
Madin Darby Canine Kidney ◽  
Corneal Epithelial ◽  
Human Colostrum ◽  
Iga Antibodies ◽  
Darby Canine Kidney

Specific anti-Acanthamoeba IgA antibodies have been detected in the serum and tears of patients and healthy individuals. However, the role of human secretory IgA antibodies in inhibiting the adherence of Acanthamoeba had not been previously investigated. Therefore, the purpose of this study was to purify secretory IgA from human colostrum and analyze its effect on the adherence of Acanthamoeba trophozoites to contact lenses and Madin–Darby canine kidney (MDCK) cells. IgA antibodies to Acanthamoeba polyphaga in colostrum of healthy women as well as in saliva and serum of healthy subjects were analyzed by ELISA and Western blot analysis. In serum, saliva, and colostrum, we detected IgA antibodies that recognized several antigens of A. polyphaga. In addition, colostrum and IgA antibodies purified from it inhibited adherence of A. polyphaga trophozoites to contact lenses and MDCK cells. These results suggest that IgA antibodies may participate in the resistance to the amoebic infection, probably by inhibiting the adherence of the trophozoites to contact lenses and corneal epithelial cells.Key words: Acanthamoeba polyphaga, free-living amoebas, colostrum, IgA.

scholarly journals Inactivation of the Wnt/β-catenin signaling contributes to the epithelial barrier dysfunction induced by sodium oxalate in canine renal epithelial cells

2021 ◽  
Author(s):  
Yun Ji ◽  
Shuting Fang ◽  
Ying Yang ◽  
Zhenlong Wu
Keyword(s):  
Epithelial Cells ◽  
Barrier Function ◽  
Mdck Cells ◽  
Renal Stones ◽  
Sodium Oxalate ◽  
Epithelial Barrier ◽  
Barrier Dysfunction ◽  
Epithelial Barrier Function ◽  
Epithelial Barrier Dysfunction

Abstract Background Nephrolithiasis (also known as renal stones) is a common disease condition in companion animals, including dogs and cats. Dysfunction of renal tubular epithelial cells involves in the pathogenesis of renal stones. However, a functional role of Wnt/β-catenin signaling and its contribution to nephrolithiasis remains unknown. Results In the present study, we found that Mardin-Darby canine kidney (MDCK) cells treated with sodium oxalate resulted in reduced cell proliferation and migration, which was associated with the G0/G1 phase arrest of cell cycle progression. In addition, sodium oxalate exposure led to decreased transepithelial electrical resistance (TEER) and increased paracellular permeability. The deleterious effect of sodium oxalate on epithelial barrier function was related to decreased protein abundances of claudin-1, occludin, zonula occludens (ZO)-1, ZO-2 and ZO-3. Of note, protein levels of p-β-catenin (Ser552) in MDCK cells were repressed by sodium oxalate, indicating an inhibitory effect on the Wnt/β-catenin signaling. Intriguingly, SB216763, a GSK-3β inhibitor, enhanced the expression p-β-catenin (Ser552), and protected against epithelial barrier dysfunction in sodium oxalate-treated MDCK cells. Conclusion Taken together, our results revealed a critical role of Wnt/β-catenin signaling on the epithelial barrier function of MDCK cells. Activation of Wnt/β-catenin signaling might be an potentially therapeutic target for the treatment of renal stones in animals.

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