scholarly journals Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1172-1184 ◽  
Author(s):  
Takahiko Hara ◽  
Hiroshi Ishida ◽  
Razi Raziuddin ◽  
Stephan Dorkhom ◽  
Keiju Kamijo ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Contact ◽  
Kidney Cells ◽  
Actin Assembly ◽  
Madin Darby Canine Kidney ◽  
Contact Sites ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin–coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin–coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.

scholarly journals Myosin-1c regulates the dynamic stability of E-cadherin–based cell–cell contacts in polarized Madin–Darby canine kidney cells

2013 ◽  
Vol 24 (18) ◽  
pp. 2820-2833 ◽  
Author(s):  
Hiroshi Tokuo ◽  
Lynne M. Coluccio
Keyword(s):  
Dynamic Stability ◽  
Motor Function ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Cell Contacts ◽  
Cell Adhesions ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell–cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin–based cell–cell contacts. In Myo1c-depleted Madin–Darby canine kidney cells, E-cadherin localization was dis­organized and lateral membranes appeared less vertical with convoluted edges versus control cells. In polarized monolayers, Myo1c-knockdown (KD) cells were more sensitive to reduced calcium concentration. Myo1c separated in the same plasma membrane fractions as E-cadherin, and Myo1c KD caused a significant reduction in the amount of E-cadherin recovered in one peak fraction. Expression of green fluorescent protein (GFP)–Myo1c mutants revealed that the phosphatidylinositol-4,5-bisphosphate–binding site is necessary for its localization to cell–cell adhesions, and fluorescence recovery after photobleaching assays with GFP-Myo1c mutants revealed that motor function was important for Myo1c dynamics at these sites. At 18°C, which inhibits vesicle recycling, Myo1c-KD cells accumulated more E-cadherin–positive vesicles in their cytoplasm, suggesting that Myo1c affects E-cadherin endocytosis. Studies with photoactivatable GFP–E-cadherin showed that Myo1c KD reduced the stability of E-cadherin at cell–cell adhesions. We conclude that Myo1c stabilizes E-cadherin at adherens junctions in polarized epithelial cells and that the motor function and ability of Myo1c to bind membrane are critical.

scholarly journals Par1b Promotes Hepatic-type Lumen Polarity in Madin Darby Canine Kidney Cells via Myosin II- and E-Cadherin–dependent Signaling

2007 ◽  
Vol 18 (6) ◽  
pp. 2203-2215 ◽  
Author(s):  
David Cohen ◽  
Yuan Tian ◽  
Anne Müsch
Keyword(s):  
Cell Adhesion ◽  
Mdck Cells ◽  
Myosin Ii ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Luminal Compartment ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin ◽  
Cell Cell

Kidney-derived Madin Darby canine kidney (MDCK) cells form lumina at their apices, and target luminal proteins to an intracellular vacuolar apical compartment (VAC) when prevented from polarizing. Hepatocytes, by contrast, organize their luminal surfaces (the bile canaliculi; BC) between their lateral membranes, and, when nonpolarized, they display an intracellular luminal compartment that is distinct from the VACs of MDCK cells. Overexpression of the serine/threonine kinase Par1b/EMK1/MARK2 induces BC-like lateral lumina and a hepatic-type intracellular luminal compartment in MDCK cells, suggesting a role for Par1b in the branching decision between kidney- and hepatic-type epithelial phenotypes. Here, we report that Par1b promotes lateral lumen polarity in MDCK cells independently of Ca2+-mediated cell–cell adhesion by inhibiting myosin II in a rho kinase-dependent manner. Polarization was inhibited by E-cadherin depletion but promoted by an adhesion-defective E-cadherin mutant. By contrast, apical surface formation in control MDCK cells required Ca2+-dependent cell–cell adhesion, but it occurred in the absence of E-cadherin. We propose that E-cadherin, when in an adhesion-incompetent state at the lateral domain, serves as targeting patch for the establishment of lateral luminal surfaces. E-cadherin depletion also reverted the hepatic-type intracellular luminal compartment in Par1b-MDCK cells to VACs characteristic of control MDCK cells, indicating a novel link between E-cadherin and luminal protein targeting.

scholarly journals Involvement of nectin in the localization of IQGAP1 at the cell–cell adhesion sites through the actin cytoskeleton in Madin–Darby canine kidney cells

Oncogene ◽  
2003 ◽  
Vol 22 (14) ◽  
pp. 2097-2109 ◽  
Author(s):  
Tatsuo Katata ◽  
Kenji Irie ◽  
Atsunori Fukuhara ◽  
Tomomi Kawakatsu ◽  
Akio Yamada ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Adhesion Sites ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

Enhancement of cell-cell contact by claudin-4 in renal epithelial madin-darby canine kidney cells

2012 ◽  
Vol 113 (2) ◽  
pp. 499-507 ◽  
Author(s):  
Akira Ikari ◽  
Kosuke Atomi ◽  
Ayumi Takiguchi ◽  
Yasuhiro Yamazaki ◽  
Hisayoshi Hayashi ◽  
...  
Keyword(s):  
Cell Contact ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

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 Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells.

1988 ◽  
Vol 107 (6) ◽  
pp. 2389-2399 ◽  
Author(s):  
J D Siliciano ◽  
D A Goodenough
Keyword(s):  
Plasma Membrane ◽  
Extracellular Calcium ◽  
Cell Contact ◽  
Suspension Cells ◽  
Madin Darby Canine Kidney ◽  
Low Calcium ◽  
Normal Calcium ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1. In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex. If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm. After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis. However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized. ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium. These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane. An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.

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.

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