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.

scholarly journals Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane

2003 ◽  
Vol 374 (3) ◽  
pp. 793-797 ◽  
Author(s):  
Mohabir RAMJEESINGH ◽  
Jackie F. KIDD ◽  
Ling Jun HUAN ◽  
Yanchun WANG ◽  
Christine E. BEAR
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Chinese Hamster Ovary Cells ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700–10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

scholarly journals Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

2009 ◽  
Vol 20 (8) ◽  
pp. 2337-2350 ◽  
Author(s):  
Mark R. Silvis ◽  
Carol A. Bertrand ◽  
Nadia Ameen ◽  
Franca Golin-Bisello ◽  
Michael B. Butterworth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Expression System ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

Targeting the regulation of CFTR channels

2011 ◽  
Vol 435 (2) ◽  
pp. e1-e4 ◽  
Author(s):  
Paul D.W. Eckford ◽  
Christine E. Bear
Keyword(s):  
Epithelial Cells ◽  
Protein Interaction ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Protein Protein Interaction ◽  
Biochemical Journal ◽  
New Strategy ◽  
Small Molecule Compound ◽  
Cystic Fibrosis Transmembrane

In this issue of the Biochemical Journal, Zhang et al. reveal a new strategy for modifying the regulated function of CFTR (cystic fibrosis transmembrane conductance regulator) on the apical surface of epithelial cells. Simply stated, these authors tested the idea that the cAMP-dependent channel activity of CFTR could be effectively enhanced by disruption of a protein–protein interaction which is normally inhibitory for the production of cAMP. This particular protein–protein interaction [between the PDZ motif of LPA2 (type 2 lysophosphatidic acid receptor) and the scaffold protein Nherf2 (Na+/H+ exchanger regulatory factor 2)] is localized in the CFTR interactome on the apical membrane of epithelial cells. Hence disruption of the LPA2–Nherf2 interaction should lead to a localized elevation in cAMP and, consequently, increased cAMP-dependent CFTR activity on the surface of epithelial cells. Zhang et al. confirmed these expectations for a small-molecule compound targeting the LPA2–Nherf2 interaction using relevant cultures and tissues thought to model the human respiratory epithelium. The success of this strategy depended on previous knowledge regarding the role for multiple PDZ-motif-mediated interactions in signalling (directly or indirectly) to CFTR. Given the number and diversity of such PDZ-mediated interactions, future structural and computational studies will be essential for guiding the design of specific pharmacological interventions.

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 Chenodeoxycholic acid stimulates Cl− secretion via cAMP signaling and increases cystic fibrosis transmembrane conductance regulator phosphorylation in T84 cells

2013 ◽  
Vol 305 (4) ◽  
pp. C447-C456 ◽  
Author(s):  
Mei Ao ◽  
Jayashree Sarathy ◽  
Jada Domingue ◽  
Waddah A. Alrefai ◽  
Mrinalini C. Rao
Keyword(s):  
Cystic Fibrosis ◽  
Chenodeoxycholic Acid ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Intracellular Camp ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Cystic Fibrosis Transmembrane

High levels of chenodeoxycholic acid (CDCA) and deoxycholic acid stimulate Cl− secretion in mammalian colonic epithelia. While different second messengers have been implicated in this action, the specific signaling pathway has not been fully delineated. Using human colon carcinoma T84 cells, we elucidated this cascade assessing Cl− transport by measuring I− efflux and short-circuit current ( Isc). CDCA (500 μM) rapidly increases I− efflux, and we confirmed by Isc that it elicits a larger response when added to the basolateral vs. apical surface. However, preincubation with cytokines increases the monolayer responsiveness to apical addition by 55%. Nystatin permeabilization studies demonstrate that CDCA stimulates an eletrogenic apical Cl− but not a basolateral K+ current. Furthermore, CDCA-induced Isc was inhibited (≥67%) by bumetanide, BaCl2, and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172. CDCA-stimulated Isc was decreased 43% by the adenylate cyclase inhibitor MDL12330A and CDCA increases intracellular cAMP concentration. The protein kinase A inhibitor H89 and the microtubule disrupting agent nocodazole, respectively, cause 94 and 47% reductions in CDCA-stimulated Isc. Immunoprecipitation with CFTR antibodies, followed by sequential immunoblotting with Pan-phospho and CFTR antibodies, shows that CDCA increases CFTR phosphorylation by approximately twofold. The rapidity and side specificity of the response to CDCA imply a membrane-mediated process. While CDCA effects are not blocked by the muscarinic receptor antagonist atropine, T84 cells possess transcript and protein for the bile acid G protein-coupled receptor TGR5. These results demonstrate for the first time that CDCA activates CFTR via a cAMP-PKA pathway involving microtubules and imply that this occurs via a basolateral membrane receptor.

The cystic fibrosis transmembrane conductance regulator is not a base transporter in isolated duodenal epithelial cells

2002 ◽  
Vol 174 (4) ◽  
pp. 327-336 ◽  
Author(s):  
J. PRAETORIUS ◽  
U. G. FRIIS ◽  
M. A. AINSWORTH ◽  
O. B. SCHAFFALITZKY DE MUCKADELL ◽  
T. JOHANSEN
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane
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