scholarly journals Regulated Synthesis and Functions of Laminin 5 in Polarized Madin-Darby Canine Kidney Epithelial Cells

2006 ◽  
Vol 17 (8) ◽  
pp. 3664-3677 ◽  
Author(s):  
Grace Z. Mak ◽  
Gina M. Kavanaugh ◽  
Mary M. Buschmann ◽  
Shaun M. Stickley ◽  
Manuel Koch ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Madin Darby Canine Kidney ◽  
Wound Edge ◽  
Laminin 5 ◽  
Renal Epithelium ◽  
Renal Tubular ◽  
And Function ◽  
Darby Canine Kidney ◽  
Canine Kidney

Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical–basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 α3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical–basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin α5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium.

scholarly journals Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

1990 ◽  
Vol 1 (12) ◽  
pp. 921-936 ◽  
Author(s):  
M J van Zeijl ◽  
K S Matlin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Sodium Dodecyl ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.

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.

scholarly journals Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells.

1987 ◽  
Vol 104 (6) ◽  
pp. 1527-1537 ◽  
Author(s):  
W J Nelson ◽  
P J Veshnock
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Membrane Skeleton ◽  
Cell Contact ◽  
Madin Darby Canine Kidney ◽  
The Stability ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

During growth of Madin-Darby canine kidney (MDCK) epithelial cells, there is a dramatic change in the stability, biophysical properties, and distribution of the membrane skeleton (fodrin) which coincides temporally and spatially with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral domain of the plasma membrane. These changes occur maximally upon the formation of a continuous monolayer of cells, indicating that extensive cell-cell contact may play an important role in the organization of polarized MDCK cells (Nelson, W. J., and P. J. Veshnock, 1986, J. Cell Biol., 103:1751-1766). To directly analyze the role of cell-cell contact in these events, we have used an assay in which the organization of fodrin and membrane proteins is analyzed in confluent monolayers of MDCK cells in the absence or presence of cell-cell contact by adjusting the concentration Ca++ in the growth medium. Our results on the stability and solubility properties of fodrin reported here show directly that there is a positive correlation between cell-cell contact and increased stability and insolubility of fodrin. Furthermore, we show that fodrin can be recruited from an unstable pool of protein to a stable pool during induction of cell-cell contact; significantly, the stabilization of fodrin is not affected by the addition of cyclohexamide, indicating that proteins normally synthesized during the induction of cell-cell contact are not required. Together these results indicate that cell-cell contact may play an important role in the development of polarity in MDCK cells by initiating the formation of a stable, insoluble matrix of fodrin with preexisting (membrane) proteins at the cell periphery. This matrix may function subsequently to trap proteins targeted to the membrane, resulting in the maintenance of membrane domains.

scholarly journals Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

1999 ◽  
Vol 10 (1) ◽  
pp. 179-195 ◽  
Author(s):  
Frederik Vilhardt ◽  
Morten Nielsen ◽  
Kirsten Sandvig ◽  
Bo van Deurs
Keyword(s):  
Epithelial Cells ◽  
Plasminogen Activator ◽  
Mdck Cells ◽  
Membrane Microdomains ◽  
Madin Darby Canine Kidney ◽  
Urokinase Plasminogen Activator Receptor ◽  
Coated Pits ◽  
Plasminogen Activator Receptor ◽  
Darby Canine Kidney ◽  
Canine Kidney

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.

Effect of triethyltin on Ca2+ movement in Madin–Darby canine kidney cells

2002 ◽  
Vol 21 (8) ◽  
pp. 457-462 ◽  
Author(s):  
B-P Jiann ◽  
K-J Chou ◽  
H-T Chang ◽  
W-C Chen ◽  
J-K Huang ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Independent Manner ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Renal Tubular ◽  
Darby Canine Kidney ◽  
Canine Kidney

The effects of the environmental toxicant, triethyltin, on Ca2 + mobilization in Madin–Darby canine kidney (MDCK) cells have been examined. Triethyltin induced an increase in cytosolic free Ca2 + levels ([Ca2 +]i) at concentrations larger than 2 mM in a concentrationdependent manner. Within 5 min, the [Ca2 +]i signal was composed of a gradual rise and a sustained phase. The [Ca2 +]i signal was partly reduced by removing extracellular Ca2 +. In Ca2 +-free medium, pretreatment with thapsigargin (1 mM), an endoplasmic reticulum Ca2 + pump inhibitor, reduced 50 mM triethyltin-induced [Ca2 +]i increase by 80%. Conversely, pretreatment with triethyltin abolished thapsigargin-induced Ca2 + release. Pretreatment with U73122 (2 mM) to inhibit phospholipase C-coupled inositol 1,4,5-trisphosphate formations failed to alter 50 mM triethyltin-induced Ca2 + release. Incubation with triethyltin at a concentration (1 mM) that did not increase basal [Ca2 +]i for 3 min did not alter ATP (10 mM)and bradykinin (1 mM)-induced [Ca2 +]i increases. Collectively, this study shows that triethyltin altered Ca2 + movement in renal tubular cells by releasing Ca2 + from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing Ca2 + influx.

scholarly journals Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules.

1987 ◽  
Vol 105 (3) ◽  
pp. 1283-1296 ◽  
Author(s):  
M H Bré ◽  
T E Kreis ◽  
E Karsenti
Keyword(s):  
Epithelial Cells ◽  
Dynamic Instability ◽  
Mdck Cells ◽  
Vero Cells ◽  
Madin Darby Canine Kidney ◽  
Microtubule Network ◽  
Alpha Tubulin ◽  
Immunofluorescence Labeling ◽  
Darby Canine Kidney ◽  
Canine Kidney

The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti-centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole-resistant microtubules in MDCK cells contained detyrosinated alpha-tubulin (glu tubulin). On the other hand, the first microtubules to regrow after nocodazole removal contained only tyrosinated tubulin. Glu-tubulin became detectable only after 30 min of microtubule regrowth. This strongly supports the hypothesis that alpha-tubulin detyrosination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount of glu-tubulin that we found in taxol-treated cells.

scholarly journals Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin.

1985 ◽  
Vol 101 (4) ◽  
pp. 1307-1315 ◽  
Author(s):  
J Behrens ◽  
W Birchmeier ◽  
S L Goodman ◽  
B A Imhof
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Apical Cell ◽  
Junctional Complex ◽  
Madin Darby Canine Kidney ◽  
Surface Antibody ◽  
Darby Canine Kidney ◽  
Canine Kidney

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.

scholarly journals PAR1b Promotes Cell–Cell Adhesion and Inhibits Dishevelled-mediated Transformation of Madin-Darby Canine Kidney Cells

2006 ◽  
Vol 17 (8) ◽  
pp. 3345-3355 ◽  
Author(s):  
Maya Elbert ◽  
David Cohen ◽  
Anne Müsch
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Wnt Signaling ◽  
Mdck Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin–dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell–cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin–dependent adhesion complexes.

scholarly journals Transformation of Madin-Darby Canine Kidney Epithelial Cells by Sheep Retrovirus Envelope Proteins

2005 ◽  
Vol 79 (2) ◽  
pp. 927-933 ◽  
Author(s):  
Shan-Lu Liu ◽  
A. Dusty Miller
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
Fibroblast Cell ◽  
Akt Pathway ◽  
Epithelial Cell Line ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) induce epithelial tumors in the airways of sheep and goats. In both of these simple retroviruses, the envelope (Env) protein is the active oncogene. Furthermore, JSRV Env can transform cultured cells by two distinct mechanisms. In rat and mouse fibroblasts, the cytoplasmic tail of JSRV Env is essential for transformation, which involves activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and the virus receptor hyaluronidase 2 (Hyal2) is not involved. In contrast, in the BEAS-2B human bronchial epithelial cell line, transformation is mediated by JSRV Env binding to Hyal2 followed by Hyal2 degradation and activation of the receptor tyrosine kinase RON, the activity of which is normally suppressed by Hyal2. Here we show that JSRV and ENTV Env proteins can also transform Madin-Darby canine kidney (MDCK) epithelial cells, but by a mechanism similar to that observed in fibroblast cell lines. In particular, the cytoplasmic tail of Env is required for transformation, the PI3K/Akt pathway is activated, expression of RON (which is not normally expressed in MDCK cells) does not affect transformation, and canine Hyal2 appears uninvolved. These results show that the JSRV and ENTV Env proteins can transform epithelial cells besides BEAS-2B cells and argue against a model for Env transformation involving different pathways that are uniquely active in fibroblasts or epithelial cells.

scholarly journals Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells.

1986 ◽  
Vol 103 (5) ◽  
pp. 1751-1765 ◽  
Author(s):  
W J Nelson ◽  
P J Veshnock
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Membrane Skeleton ◽  
Cell Contact ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Basolateral Plasma Membrane ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document