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Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.
Koumangoye R, Omer S, Delpire E
(2018) Am J Physiol Cell Physiol 315: C258-C276
MeSH Terms: Animals, Cell Membrane, Cells, Cultured, Colon, Cytoplasm, Dogs, Epithelial Cells, Female, Madin Darby Canine Kidney Cells, Male, Mice, Oocytes, Salivary Glands, Sodium-Potassium-Chloride Symporters, Sodium-Potassium-Exchanging ATPase, Solute Carrier Family 12, Member 2, Xenopus laevis
Show Abstract · Added May 4, 2018
We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na-K-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.
1 Communities
1 Members
0 Resources
17 MeSH Terms
Microtubules regulate brush border formation.
Tonucci FM, Ferretti A, Almada E, Cribb P, Vena R, Hidalgo F, Favre C, Tyska MJ, Kaverina I, Larocca MC
(2018) J Cell Physiol 233: 1468-1480
MeSH Terms: Actin Cytoskeleton, Animals, Cell Polarity, Centromere, Colon, Dogs, Enterocytes, Epithelial Cells, Humans, Kidney, Madin Darby Canine Kidney Cells, Microtubule-Associated Proteins, Microtubules, Microvilli, Nocodazole, Time Factors, Tubulin Modulators
Show Abstract · Added April 10, 2018
Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.
© 2017 Wiley Periodicals, Inc.
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1 Members
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MeSH Terms
Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition.
Lapierre LA, Manning EH, Mitchell KM, Caldwell CM, Goldenring JR
(2017) Mol Biol Cell 28: 1088-1100
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Cadherins, Carrier Proteins, Cell Polarity, Dogs, Endosomes, Epithelial Cells, Gene Knockout Techniques, HEK293 Cells, Humans, Intercellular Junctions, Madin Darby Canine Kidney Cells, Membrane Proteins, Occludin, Phosphorylation, Protein Binding, Protein Transport, rab GTP-Binding Proteins
Show Abstract · Added April 18, 2017
MARK2 regulates the establishment of polarity in Madin-Darby canine kidney (MDCK) cells in part through phosphorylation of serine 227 of Rab11-FIP2. We identified Eps15 as an interacting partner of phospho-S227-Rab11-FIP2 (pS227-FIP2). During recovery from low calcium, Eps15 localized to the lateral membrane before pS227-FIP2 arrival. Later in recovery, Eps15 and pS227-FIP2 colocalized at the lateral membrane. In MDCK cells expressing the pseudophosphorylated FIP2 mutant FIP2(S227E), during recovery from low calcium, Eps15 was trapped and never localized to the lateral membrane. Mutation of any of the three NPF domains within GFP-FIP2(S227E) rescued Eps15 localization at the lateral membrane and reestablished single-lumen cyst formation in GFP-FIP2(S227E)-expressing cells in three-dimensional (3D) culture. Whereas expression of GFP-FIP2(S227E) induced the loss of E-cadherin and occludin, mutation of any of the NPF domains of GFP-FIP2(S227E) reestablished both proteins at the apical junctions. Knockdown of Eps15 altered the spatial and temporal localization of pS227-FIP2 and also elicited formation of multiple lumens in MDCK 3D cysts. Thus an interaction of Eps15 and pS227-FIP2 at the appropriate time and location in polarizing cells is necessary for proper establishment of epithelial polarity.
© 2017 Lapierre et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
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19 MeSH Terms
Contribution of Organic Anion-Transporting Polypeptides 1A/1B to Doxorubicin Uptake and Clearance.
Lee HH, Leake BF, Kim RB, Ho RH
(2017) Mol Pharmacol 91: 14-24
MeSH Terms: ATP Binding Cassette Transporter, Subfamily B, Member 1, Animals, Biological Transport, Cell Membrane, Dogs, Doxorubicin, HeLa Cells, Humans, Kinetics, Liver, Liver-Specific Organic Anion Transporter 1, Madin Darby Canine Kidney Cells, Male, Mice, Models, Biological, Mutant Proteins, Organic Anion Transporters, Organic Anion Transporters, Sodium-Independent, Organic Cation Transport Proteins, Rats, Transfection
Show Abstract · Added November 10, 2016
The organic anion-transporting polypeptides represent an important family of drug uptake transporters that mediate the cellular uptake of a broad range of substrates including numerous drugs. Doxorubicin is a highly efficacious and well-established anthracycline chemotherapeutic agent commonly used in the treatment of a wide range of cancers. Although doxorubicin is a known substrate for efflux transporters such as P-glycoprotein (P-gp; MDR1, ABCB1), significantly less is known regarding its interactions with drug uptake transporters. Here, we investigated the role of organic anion transporting polypeptide (OATP) transporters to the disposition of doxorubicin. A recombinant vaccinia-based method for expressing uptake transporters in HeLa cells revealed that OATP1A2, but not OATP1B1 or OATP1B3, and the rat ortholog Oatp1a4 were capable of significant doxorubicin uptake. Interestingly, transwell assays using Madin-Darby canine kidney II cell line cells stably expressing specific uptake and/or efflux transporters revealed that OATP1B1, OATP1B3, and OATP1A2, either alone or in combination with MDR1, significantly transported doxorubicin. An assessment of polymorphisms in SLCO1A2 revealed that four variants were associated with significantly impaired doxorubicin transport in vitro. In vivo doxorubicin disposition studies revealed that doxorubicin plasma area under the curve was significantly higher (1.7-fold) in Slco1a/1b versus wild-type mice. The liver-to-plasma ratio of doxorubicin was significantly decreased (2.3-fold) in Slco1a/1b2 mice and clearance was reduced by 40% compared with wild-type mice, suggesting Oatp1b transporters are important for doxorubicin hepatic uptake. In conclusion, we demonstrate important roles for OATP1A/1B in transporter-mediated uptake and disposition of doxorubicin.
Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
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1 Members
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21 MeSH Terms
Focal adhesions control cleavage furrow shape and spindle tilt during mitosis.
Taneja N, Fenix AM, Rathbun L, Millis BA, Tyska MJ, Hehnly H, Burnette DT
(2016) Sci Rep 6: 29846
MeSH Terms: Animals, Cell Differentiation, Cell Shape, Centrosome, Dogs, Focal Adhesion Protein-Tyrosine Kinases, Focal Adhesions, HeLa Cells, Humans, Madin Darby Canine Kidney Cells, Mitosis, Spindle Apparatus, Vinculin
Show Abstract · Added April 7, 2017
The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.
1 Communities
2 Members
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13 MeSH Terms
p120-catenin controls contractility along the vertical axis of epithelial lateral membranes.
Yu HH, Dohn MR, Markham NO, Coffey RJ, Reynolds AB
(2016) J Cell Sci 129: 80-94
MeSH Terms: Amino Acid Sequence, Animals, Cadherins, Catenins, Cell Membrane, Cell Polarity, Cell Shape, Dogs, Epithelial Cells, Madin Darby Canine Kidney Cells, Molecular Sequence Data, Nonmuscle Myosin Type IIA, Phenotype, Protein Binding, rho-Associated Kinases, rhoA GTP-Binding Protein
Show Abstract · Added May 2, 2016
In vertebrate epithelia, p120-catenin (hereafter referred to as p120; also known as CTNND1) mediates E-cadherin stability and suppression of RhoA. Genetic ablation of p120 in various epithelial tissues typically causes striking alterations in tissue function and morphology. Although these effects could very well involve p120's activity towards Rho, ascertaining the impact of this relationship has been complicated by the fact that p120 is also required for cell-cell adhesion. Here, we have molecularly uncoupled p120's cadherin-stabilizing and RhoA-suppressing activites. Unexpectedly, removing p120's Rho-suppressing activity dramatically disrupted the integrity of the apical surface, irrespective of E-cadherin stability. The physical defect was tracked to excessive actomyosin contractility along the vertical axis of lateral membranes. Thus, we suggest that p120's distinct activities towards E-cadherin and Rho are molecularly and functionally coupled and this, in turn, enables the maintenance of cell shape in the larger context of an epithelial monolayer. Importantly, local suppression of contractility by cadherin-bound p120 appears to go beyond regulating cell shape, as loss of this activity also leads to major defects in epithelial lumenogenesis.
© 2016. Published by The Company of Biologists Ltd.
1 Communities
2 Members
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16 MeSH Terms
Discovery of a Selective and CNS Penetrant Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 3 with Antidepressant and Anxiolytic Activity in Rodents.
Engers JL, Rodriguez AL, Konkol LC, Morrison RD, Thompson AD, Byers FW, Blobaum AL, Chang S, Venable DF, Loch MT, Niswender CM, Daniels JS, Jones CK, Conn PJ, Lindsley CW, Emmitte KA
(2015) J Med Chem 58: 7485-500
MeSH Terms: Allosteric Regulation, Animals, Anti-Anxiety Agents, Antidepressive Agents, Brain, Calcium, Dogs, Heterocyclic Compounds, 2-Ring, Humans, Madin Darby Canine Kidney Cells, Mice, Microsomes, Liver, Permeability, Pyridines, Rats, Receptors, Metabotropic Glutamate, Stereoisomerism, Structure-Activity Relationship
Show Abstract · Added February 18, 2016
Previous preclinical work has demonstrated the therapeutic potential of antagonists of the group II metabotropic glutamate receptors (mGlus). Still, compounds that are selective for the individual group II mGlus (mGlu2 and mGlu3) have been scarce. There remains a need for such compounds with the balance of properties suitable for convenient use in a wide array of rodent behavioral studies. We describe here the discovery of a selective mGlu3 NAM 106 (VU0650786) suitable for in vivo work. Compound 106 is a member of a series of 5-aryl-6,7-dihydropyrazolo[1,5-a]pyrazine-4(5H)-one compounds originally identified as a mGlu5 positive allosteric modulator (PAM) chemotype. Its suitability for use in rodent behavioral models has been established by extensive in vivo PK studies, and the behavioral experiments presented here with compound 106 represent the first examples in which an mGlu3 NAM has demonstrated efficacy in models where prior efficacy had previously been noted with nonselective group II antagonists.
0 Communities
1 Members
0 Resources
18 MeSH Terms
Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin.
Singh B, Bogatcheva G, Starchenko A, Sinnaeve J, Lapierre LA, Williams JA, Goldenring JR, Coffey RJ
(2015) J Cell Sci 128: 3444-55
MeSH Terms: Amino Acid Sequence, Animals, Betacellulin, Cell Polarity, Dogs, EGF Family of Proteins, ErbB Receptors, Fluorescent Antibody Technique, Madin Darby Canine Kidney Cells, Mutation, Protein Sorting Signals, Protein Structure, Tertiary
Show Abstract · Added September 28, 2015
Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically (156)EEMETL(161)). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization.
© 2015. Published by The Company of Biologists Ltd.
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4 Members
0 Resources
12 MeSH Terms
Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22.
Schlebach JP, Narayan M, Alford C, Mittendorf KF, Carter BD, Li J, Sanders CR
(2015) J Am Chem Soc 137: 8758-68
MeSH Terms: Amino Acid Sequence, Animals, Charcot-Marie-Tooth Disease, Dogs, Humans, Madin Darby Canine Kidney Cells, Metals, Models, Molecular, Molecular Sequence Data, Mutation, Missense, Myelin Proteins, Peripheral Nervous System Diseases, Protein Conformation, Protein Folding, Protein Stability, Protein Transport, Thermodynamics
Show Abstract · Added February 20, 2016
Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot-Marie-Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT.
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2 Members
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17 MeSH Terms
Intracellular and extracellular forces drive primary cilia movement.
Battle C, Ott CM, Burnette DT, Lippincott-Schwartz J, Schmidt CF
(2015) Proc Natl Acad Sci U S A 112: 1410-5
MeSH Terms: Animals, Centrosome, Cilia, Dogs, Madin Darby Canine Kidney Cells, Microscopy, Electron, Movement
Show Abstract · Added August 25, 2017
Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.
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7 MeSH Terms