scholarly journals Junction Protein Shrew-1 Influences Cell Invasion and Interacts with Invasion-promoting Protein CD147

2007 ◽  
Vol 18 (4) ◽  
pp. 1272-1281 ◽  
Author(s):  
Alexander Schreiner ◽  
Mika Ruonala ◽  
Viktor Jakob ◽  
Jan Suthaus ◽  
Eckhard Boles ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Hela Cells ◽  
Adherens Junctions ◽  
Basolateral Membrane ◽  
Cellular Invasion ◽  
Junction Protein ◽  
Fibrosarcoma Cells ◽  
Interfering Rna ◽  
Darby Canine Kidney

Shrew-1 was previously isolated from an endometriotic cell line in our search for invasion-associated genes. It proved to be a membrane protein that targets to the basolateral membrane of polarized epithelial cells, interacting with E-cadherin–catenin complexes of adherens junctions. Paradoxically, the existence of adherens junctions is incompatible with invasion. To investigate whether shrew-1 can indeed influence cellular invasion, we overexpressed it in HT1080 fibrosarcoma cells. This resulted in enhanced invasiveness, accompanied by an increased matrix metalloprotease (MMP)-9 level in the supernatant, raising the question about the role of shrew-1 in this process. Logic suggested we looked for an interaction with CD147, a known promoter of invasiveness and MMP activity. Indeed, genetics-based, biochemical, and microscopy experiments revealed shrew-1– and CD147-containing complexes in invasive endometriotic cells and an interaction in epithelial cells, which was stronger in MCF7 tumor cells, but weaker in Madin-Darby canine kidney cells. In contrast to the effect mediated by overexpression, small interfering RNA-mediated down-regulation of either shrew-1 or CD147 in HeLa cells decreased invasiveness without affecting the proliferation behavior of HeLa cells, but the knockdown cells displayed decreased motility. Altogether, our results imply that shrew-1 has a function in the regulation of cellular invasion, which may involve its interaction with CD147.

scholarly journals Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

1997 ◽  
Vol 136 (4) ◽  
pp. 919-934 ◽  
Author(s):  
Jani E. Lewis ◽  
James K. Wahl ◽  
Kristin M. Sass ◽  
Pamela J. Jensen ◽  
Keith R. Johnson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Common Component ◽  
Classical Cadherins ◽  
E Cadherin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.

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.

scholarly journals Abelson kinase regulates epithelial morphogenesis in Drosophila

2001 ◽  
Vol 155 (7) ◽  
pp. 1185-1198 ◽  
Author(s):  
Elizabeth E. Grevengoed ◽  
Joseph J. Loureiro ◽  
Traci L. Jesse ◽  
Mark Peifer
Keyword(s):  
Epithelial Cells ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Epithelial Morphogenesis ◽  
Actin Dynamics ◽  
Nonreceptor Tyrosine Kinase ◽  
Abelson Kinase ◽  
Adherens Junction Protein ◽  
Junction Protein ◽  
Guidance Receptors

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and α-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.

scholarly journals The Interaction of JRAB/MICAL-L2 with Rab8 and Rab13 Coordinates the Assembly of Tight Junctions and Adherens Junctions

2008 ◽  
Vol 19 (3) ◽  
pp. 971-983 ◽  
Author(s):  
Rie Yamamura ◽  
Noriyuki Nishimura ◽  
Hiroyoshi Nakatsuji ◽  
Seiji Arase ◽  
Takuya Sasaki
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Tight Junctions ◽  
Binding Protein ◽  
Adherens Junctions ◽  
Binding Domain ◽  
Endocytic Recycling ◽  
E Cadherin

The assembly of tight junctions (TJs) and adherens junctions (AJs) is regulated by the transport of integral TJ and AJ proteins to and/or from the plasma membrane (PM) and it is tightly coordinated in epithelial cells. We previously reported that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) mediated the endocytic recycling of an integral TJ protein occludin and the formation of functional TJs. Here, we investigated the role of Rab13 and JRAB/MICAL-L2 in the transport of other integral TJ and AJ proteins claudin-1 and E-cadherin to the PM by using a Ca2+-switch model. Although knockdown of Rab13 specifically suppressed claudin-1 and occludin but not E-cadherin transport, knockdown of JRAB/MICAL-L2 and expression of its Rab13-binding domain (JRAB/MICAL-L2-C) inhibited claudin-1, occludin, and E-cadherin transport. We then identified Rab8 as another JRAB/MICAL-L2-C-binding protein. Knockdown of Rab8 inhibited the Rab13-independent transport of E-cadherin to the PM. Rab8 and Rab13 competed with each other for the binding to JRAB/MICAL-L2 and functionally associated with JRAB/MICAL-L2 at the perinuclear recycling/storage compartments and PM, respectively. These results suggest that the interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of AJs and TJs.

scholarly journals Molecular mechanisms underlying active desalination and low water permeability in the esophagus of eels acclimated to seawater

2017 ◽  
Vol 312 (2) ◽  
pp. R231-R244 ◽  
Author(s):  
Yoshio Takei ◽  
Marty K.-S. Wong ◽  
Supriya Pipil ◽  
Haruka Ozaki ◽  
Yutaka Suzuki ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Water Permeability ◽  
Molecular Mechanisms ◽  
Basolateral Membrane ◽  
Scaffolding Protein ◽  
Disulfonic Acid ◽  
Marine Teleost ◽  
Coupled Transport ◽  
Nacl Transport ◽  
Junction Protein

Marine teleosts can absorb imbibed seawater (SW) to maintain water balance, with esophageal desalination playing an essential role. NaCl absorption from luminal SW was enhanced 10-fold in the esophagus of SW-acclimated eels, and removal of Na+ or Cl− from luminal SW abolished the facilitated absorption, indicating coupled transport. Mucosal/serosal application of various blockers for Na+/Cl− transporters profoundly decreased the absorption. Among the transporter genes expressed in eel esophagus detected by RNA-seq, dimethyl amiloride-sensitive Na+/H+ exchanger (NHE3) and 4,4′-diisothiocyano-2,2′-disulfonic acid-sensitive Cl−/[Formula: see text] exchanger (AE) coupled by the scaffolding protein on the apical membrane of epithelial cells, and ouabain-sensitive Na+-K+-ATPases (NKA1α1c and NKA3α) and diphenylamine-2-carboxylic acid-sensitive Cl− channel (CLCN2) on the basolateral membrane, may be responsible for enhanced transcellular NaCl transport because of their profound upregulation after SW acclimation. Upregulated carbonic anhydrase 2a (CA2a) supplies H+ and [Formula: see text] for activation of the coupled NHE and AE. Apical hydrochlorothiazide-sensitive Na+-Cl− cotransporters and basolateral Na+-[Formula: see text] cotransporter (NBCe1) and AE1 are other possible candidates. Concerning the low water permeability that is typically seen in marine teleost esophagus, downregulated aquaporin genes ( aqp1a and aqp3) and upregulated claudin gene ( cldn15a) are candidates for transcellular/paracellular route. In situ hybridization showed that these upregulated transporters and tight-junction protein genes were expressed in the absorptive columnar epithelial cells of eel esophagus. These results allow us to provide a full picture of the molecular mechanism of active desalination and low water permeability that are characteristic to marine teleost esophagus and gain deeper insights into the role of gastrointestinal tracts in SW acclimation.

Abstract 1459: Connexin 43 Hemi Channels are Permeable to Exogenously Added NAD: Implications in Protection of Cardiomyocytes against PARP-mediated Cell Death

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Vinodkumar B Pillai ◽  
Jyothish B Pillai ◽  
Senthilkumar Rajamohan ◽  
Madhu Gupta ◽  
Jai Raman ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Gap Junction ◽  
Hela Cells ◽  
Connexin 43 ◽  
Neonatal Rat ◽  
Parp Activation ◽  
Junction Protein ◽  
Nad Depletion

Robust activation of poly (ADP ribose) polymerase-1 (PARP) following oxidative stress and DNA damage is a major cause of caspase independent cardiomyocyte cell death. PARP-mediated cell-death is associated with cellular NAD depletion. Connexin 43 (Cx43) is a major gap junction protein which forms hemi channels in cardiomyocytes. This study was designed to investigate whether exogenously added NAD can enter into cardiomyocytes via Cx43 hemi channels, and protect cells from PARP-mediated cell death. Primary cultures of neonatal rat heart myocytes were treated at various time points with a free-radical generating mixture of H 2 O 2 and FeSO4 (0.1mM each). To examine permeability of cells to NAD, myocytes were treated with FITC-tagged-NAD (NAD-FITC) and intracellular localization of NAD was determined by confocal microscopy. We found that following oxidative stress myocyte cell death was proportionally related to intensity of PARP activation and NAD depletion. In these cells with PARP activation, exogenously added NAD-FITC (green) readily entered into the cell interior. This NAD transport was abolished when myocytes were treated with gap junction blockers such as carbenoxolone or 18β-glycyrrhetinic acid. To further confirm a role of Cx43 in NAD transport, we utilized HeLa cells in which Cx43 was either epigenetically silenced or constitutively over expressed. We found that HeLa cells lacking Cx43 had no NAD transport activity, while cells over expressing Cx43 showed abundant entry of NAD into the cells, thus validating a role of Cx43 in cellular NAD transport. Addition of NAD into culture media prior to PARP activation also rescued cells from PARP-mediated cell death. Cellular NAD levels are known to control the activity of the longevity factor SIRT1 deacetylase. To test if SIRT1 could be involved in mediating the beneficial effects of NAD, we did an identical cell death experiment with myocytes in which SIRT1 levels were reduced 50% by siRNA silencing. In these cells we found no beneficial effect of NAD following oxidative stress, thus indicating that SIRT1 is taking part in cell survival effects of NAD. These results demonstrate that exogenously added NAD can enter into cardiomyocytes via Cx43 hemi channels and protect cells against PARP-mediated cell death.

Imaging of filter-grown epithelial cells: MDCK Na(+)-H+ exchanger is basolateral

1991 ◽  
Vol 260 (4) ◽  
pp. C868-C876 ◽  
Author(s):  
S. O. Rosenberg ◽  
P. A. Berkowitz ◽  
L. Li ◽  
V. L. Schuster
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Simple Method ◽  
Apical Side ◽  
Inverted Microscope ◽  
Permeable Supports ◽  
Darby Canine Kidney

We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto “inverted” filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pHi decreased. After an NH4Cl load, the H+ extrusion rate (JH+) in control saline was 2.42 +/- 0.62 mM/min. JH+ was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pHi in single cells and for the imaging of apical/subapical structures, and 2) the Na(+)-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.

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