Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells

2000 ◽  
Vol 279 (2) ◽  
pp. C375-C382 ◽  
Author(s):  
Marybeth Howard ◽  
Xiaosui Jiang ◽  
Donna Beer Stolz ◽  
Warren G. Hill ◽  
Jennifer A. Johnson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Surface Expression ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Madin Darby Canine Kidney ◽  
Cystic Fibrosis Transmembrane ◽  
Camp Stimulation ◽  
Darby Canine Kidney

Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2–901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565–C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080–C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2–901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2–901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2–901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2–901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2–901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.

scholarly journals Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells.

1987 ◽  
Vol 104 (2) ◽  
pp. 231-241 ◽  
Author(s):  
M J Rindler ◽  
I E Ivanov ◽  
D D Sabatini
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
G Protein ◽  
Mdck Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Vesicular Stomatitis ◽  
Ha Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

The synchronized directed transfer of the envelope glycoproteins of the influenza and vesicular stomatitis viruses from the Golgi apparatus to the apical and basolateral surfaces, respectively, of polarized Madin-Darby canine kidney (MDCK) cells can be achieved using temperature-sensitive mutant viruses and appropriate temperature shift protocols (Rindler, M. J., I. E. Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100:136-151). The microtubule-depolymerizing agents colchicine and nocodazole, as well as the microtubule assembly-promoting drug taxol, were found to interfere with the normal polarized delivery and exclusive segregation of hemagglutinin (HA) to the apical surface but not with the delivery and initial accumulation of G on the basolateral surface. Immunofluorescence analysis of permeabilized monolayers of influenza-infected MDCK cells treated with the microtubule-acting drugs demonstrated the presence of substantial amounts of HA protein on both the apical and basolateral surfaces. Moreover, in cells infected with the wild-type influenza virus, particles budded from both surfaces. Viral counts in electron micrographs showed that approximately 40% of the released viral particles accumulated in the intercellular spaces or were trapped between the cell and monolayer and the collagen support as compared to less than 1% on the basolateral surface of untreated infected cells. The effect of the microtubule inhibitors was not a result of a rapid redistribution of glycoprotein molecules initially delivered to the apical surface since a redistribution was not observed when the inhibitors were added to the cells after the HA was permitted to reach the apical surface at the permissive temperature and the synthesis of new HA was inhibited with cycloheximide. The altered segregation of the HA protein that occurs may result from the dispersal of the Golgi apparatus induced by the inhibitors or from the disruption of putative microtubules containing tracks that could direct vesicles from the trans Golgi apparatus to the cell surface. Since the vesicular stomatitis virus G protein is basolaterally segregated even when the Golgi elements are dispersed and hypothetical tracks disrupted, it appears that the two viral envelope glycoproteins are segregated by fundamentally different mechanisms and that the apical surface may be incapable of accepting vesicles carrying the G protein.

scholarly journals Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.

1989 ◽  
Vol 109 (5) ◽  
pp. 2117-2127 ◽  
Author(s):  
M P Lisanti ◽  
A Le Bivic ◽  
M Sargiacomo ◽  
E Rodriguez-Boulan
Keyword(s):  
Steady State ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Steady State Distribution ◽  
State Distribution ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.

Epitope tagging permits cell surface detection of functional CFTR

1995 ◽  
Vol 269 (6) ◽  
pp. C1565-C1576 ◽  
Author(s):  
M. Howard ◽  
M. D. DuVall ◽  
D. C. Devor ◽  
J. Y. Dong ◽  
K. Henze ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Surface Expression ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
External Loop ◽  
Glycosylation Sites ◽  
Apical Membranes ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.

scholarly journals Calreticulin Negatively Regulates the Cell Surface Expression of Cystic Fibrosis Transmembrane Conductance Regulator

2006 ◽  
Vol 281 (18) ◽  
pp. 12841-12848 ◽  
Author(s):  
Kazutsune Harada ◽  
Tsukasa Okiyoneda ◽  
Yasuaki Hashimoto ◽  
Keiko Ueno ◽  
Kimitoshi Nakamura ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

scholarly journals Sorting Nexin 27 (SNX27): A Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking

2018 ◽  
Author(s):  
Mark I. McDermott ◽  
William R. Thelin ◽  
Yun Chen ◽  
Patrick T. Lyons ◽  
Gabrielle Reilly ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Pdz Domain ◽  
Type I ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sorting Nexin ◽  
Cystic Fibrosis Transmembrane

AbstractThe underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.

scholarly journals Polarization of Specific Tropomyosin Isoforms in Gastrointestinal Epithelial Cells and Their Impact on CFTR at the Apical Surface

2003 ◽  
Vol 14 (11) ◽  
pp. 4365-4375 ◽  
Author(s):  
Jacqueline Rae Dalby-Payne ◽  
Edward Vincent O'Loughlin ◽  
Peter Gunning
Keyword(s):  
Epithelial Cells ◽  
Cell Types ◽  
Surface Expression ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Cell Monolayers ◽  
Polarized Delivery ◽  
Cystic Fibrosis Transmembrane

Microfilaments have been reported to be polarized in a number of cell types based both on function and isoform composition. There is evidence that microfilaments are involved in the movement of vesicles and the polarized delivery of proteins to specialized membrane domains. We have investigated the composition of actin microfilaments in gastrointestinal epithelial cells and their role in the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) into the apical membrane using cultured T84 cells as a model. We identified a specific population of microfilaments containing the tropomyosin (Tm) isoforms Tm5a and/or Tm5b, which are polarized in T84 cell monolayers. Polarization of this microfilament population occurs very rapidly in response to cell-cell and cell-substratum contact and is not inhibited by jasplakinolide, suggesting this involves the movement of intact filaments. Colocalization of Tm5a and/or Tm5b and CFTR was observed in long-term cultures. A reduction in Tm5a and Tm5b expression, induced using antisense oligonucleotides, resulted in an increase in both CFTR surface expression and chloride efflux in response to cAMP stimulation. We conclude that Tm isoforms Tm5a and/or Tm5b mark an apical population of microfilaments that can regulate the insertion and/or retention of CFTR into the plasma membrane.

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