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• W2063315216 abstract "The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments. The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments. The cystic fibrosis transmembrane conductance regulator (CFTR) 1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosisENaCepithelial sodium channelGFPgreen fluorescent proteinGPIglycosylphosphatidylinositol4-PBA4-phenylbutyric acidDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminHRPhorseradish peroxidaseTGNtrans-Golgi networkfunctions as an apical membrane chloride channel (1.Welsh M.J. Tsui L.-C. Boat T.F. Beaudet A.L. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Cystic Fibrosis. III. McGraw-Hill Inc., New York1995: 3799-3876Google Scholar). Different CFTRmutations causing cystic fibrosis (CF) affect the processing, intracellular localization, and function of the corresponding protein (2.Schwiebert E.M. Benos D.J. Egan M.E. Stutts M.J. Guggino W.B. Physiol. Rev. 1999; 79: S145-S166Crossref PubMed Scopus (379) Google Scholar, 3.Bradbury N.A. Physiol. Rev. 1999; 79: S175-S191Crossref PubMed Scopus (141) Google Scholar). The most common mutant form of CFTR in CF, ΔF508 CFTR, does not enter the organelles of the secretory pathway and is not delivered to the plasma membrane as it is not properly folded and remains trapped in the endoplasmic reticulum. Mutations in CFTR result in reduced apical chloride transport but also have pleiotropic effects on the function of other ion transporters including the amiloride-sensitive epithelial sodium channel (ENaC) (4.Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (957) Google Scholar, 5.Schreiber R. Hopf A. Mall M. Greger R. Kunzelmann K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5310-5315Crossref PubMed Scopus (83) Google Scholar), outwardly rectifying chloride channels (6.Schwiebert E.M. Egan M.E. Hwang T.H. Fulmer S.B. Allen S.S. Cutting G.R. Guggino W.B. Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (595) Google Scholar, 7.Schwiebert E.M. Cid-Soto L.P. Stafford D. Carter M. Blaisdell C.J. Zeitlin P.L. Guggino W.B. Cutting G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3879-3884Crossref PubMed Scopus (128) Google Scholar), the Na+/H+ exchanger via EBP50 (ezrin-binding protein), Na+/H+exchanger regulatory factor (8.Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (250) Google Scholar), bicarbonate conductance (9.Illek B. Yankaskas J.R. Machen T.E. Am. J. Physiol. 1997; 272: L752-L761PubMed Google Scholar, 10.Lee M.G. Choi J.Y. Luo X. Strickland E. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 14670-14677Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and aquaporin 3 (5.Schreiber R. Hopf A. Mall M. Greger R. Kunzelmann K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5310-5315Crossref PubMed Scopus (83) Google Scholar). cystic fibrosis transmembrane conductance regulator cystic fibrosis epithelial sodium channel green fluorescent protein glycosylphosphatidylinositol 4-phenylbutyric acid Dulbecco's modified Eagle's medium bovine serum albumin horseradish peroxidase trans-Golgi network It has been proposed that CFTR also plays a role in facilitating acidification of intracellular compartments, such as endosomes, by providing anions (Cl−) and maintaining charge neutrality as protons are pumped into the lumen of these organelles (11.Barasch J. Kiss B. Prince A. Saiman L. Gruenert D. al-Awqati Q. Nature. 1991; 352: 70-73Crossref PubMed Scopus (425) Google Scholar). According to this proposal, a loss of CFTR and chloride conductance would result in increased pH (11.Barasch J. Kiss B. Prince A. Saiman L. Gruenert D. al-Awqati Q. Nature. 1991; 352: 70-73Crossref PubMed Scopus (425) Google Scholar, 12.Barasch J. al-Awqati Q. J. Cell Sci. 1993; 17: 229-233Crossref Google Scholar). However, repeated studies have failed to detect alkalinization of intracellular compartments in CF (13.Lukacs G.L. Chang X.B. Kartner N. Rotstein O.D. Riordan J.R. Grinstein S. J. Biol. Chem. 1992; 267: 14568-14572Abstract Full Text PDF PubMed Google Scholar, 14.Seksek O. Biwersi J. Verkman A.S. J. Biol. Chem. 1996; 271: 15542-15548Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15.Biwersi J. Verkman A.S. Am. J. Physiol. 1994; 266: C149-C156Crossref PubMed Google Scholar, 16.Gibson G.A. Hill W.G. Weisz O.A. Am. J. Physiol. 2000; 279: C1088-C1099Crossref Google Scholar, 17.Dunn K.W. Park J. Semrad C.E. Gelman D.L. Shevell T. McGraw T.E. J. Biol. Chem. 1994; 269: 5336-5345Abstract Full Text PDF PubMed Google Scholar). It has been shown that CFTR is present in endosomes of stably transfected Swiss 3T3 and T84 cells, which normally express CFTR (15.Biwersi J. Verkman A.S. Am. J. Physiol. 1994; 266: C149-C156Crossref PubMed Google Scholar). The absence of CFTR on the plasma membrane and organelles of the secretory pathway, which communicate with the endocytic pathway, prompted us to re-examine potential consequences in CF on the pH of endocytic organelles by specific targeting of pH-sensitive GFP (18.Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1966) Google Scholar) to a defined endocytic compartment. Here we show that cellubrevin-labeled endosomes are hyperacidified in CF lung epithelial cells and that the pH of the recycling endosome depends on CFTR and its effects on sodium transport. In addition, we show physiological defects in the function of the endocytic pathway in CF, as recycling of receptor-mediated endocytic tracers (transferrin) is affected in CF lung epithelial cells. CFT1 (19.Olsen J.C. Johnson L.G. Stutts M.J. Sarkadi B. Yankaskas J.R. Swanstrom R. Boucher R.C. Hum. Gene Ther. 1992; 3: 253-266Crossref PubMed Scopus (84) Google Scholar, 20.Lee A. Chow D. Haus B. Tseng W. Evans D. Fleiszig S. Chandy G. Machen T. Am. J. Physiol. 1999; 277: L204-L217PubMed Google Scholar) is a cell line derived from the tracheal epithelium of a CF patient homozygous for the most common CFTR ΔF508 mutation. Stably transfected derivatives of CFT1were the following: CFT1-LCFSN, expressing the wild-type CFTR gene; CFT1-Δ508, transfected with ΔF508 mutant CFTR gene; and CFT1-LC3, the vector-transfected control cells. CFT1 and derivative cells were grown in F12 media (Invitrogen) supplemented with 10 μg/ml insulin, 1 μmhydrocortisone, 1 nm triiodothyronine, 10 ng/ml cholera toxin (Sigma), 3.75 μg/ml endothelial cell growth supplement, 25 ng/ml epidermal growth factor, and 5 μg/ml transferrin (Collaborative Research Inc., Bedford, MA) (19.Olsen J.C. Johnson L.G. Stutts M.J. Sarkadi B. Yankaskas J.R. Swanstrom R. Boucher R.C. Hum. Gene Ther. 1992; 3: 253-266Crossref PubMed Scopus (84) Google Scholar). IB3-1 is a human bronchial epithelial cell line derived from a CF patient with a ΔF508/W1282X CFTR mutant genotype (21.Zeitlin P.L. Lu L. Rhim J. Cutting G. Stetten G. Kieffer K.A. Craig R. Guggino W.B. Am. J. Respir. Cell Mol. Biol. 1991; 4: 313-319Crossref PubMed Scopus (273) Google Scholar). C38 and S9 are derivatives of IB3-1 cells and are stably transfected with a functionalCFTR corrected for chloride conductance (22.Egan M. Flotte T. Afione S. Solow R. Zeitlin P.L. Carter B.J. Guggino W.B. Nature. 1992; 358: 581-584Crossref PubMed Scopus (377) Google Scholar). The physiological levels of expression of CFTR and its functionality have been established previously for C38 cells (22.Egan M. Flotte T. Afione S. Solow R. Zeitlin P.L. Carter B.J. Guggino W.B. Nature. 1992; 358: 581-584Crossref PubMed Scopus (377) Google Scholar). The cells were maintained in LHC-8 media (BIOSOURCE Int., Rockville, MD), 10% fetal bovine serum, and 50 units/ml penicillin-streptomycin (Invitrogen). All cells were grown in a humidified incubator at 37 °C under 5% CO2. Cellubrevin-pHluorin GFP and glycosylphosphatidylinositol (GPI)-pHluorin GFP DNA constructs were from J. Rothman (18.Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1966) Google Scholar). IB3-1 cells and its derivatives were seeded at 105 cells/ml on 25-mm coverslips in 6-well plates. Cells were transfected with 1 μg/ml DNA using Lipofectin (Invitrogen) for 6 h at 37 °C, 5% CO2. CFT1 cells and their derivatives were seeded at 105 cells/ml on 25-mm coverslips in 6-well plates and grown in the medium without cholera toxin. Cells were transfected with GenePorter (Gene Therapy Systems, San Diego, CA) with 2.5 μg/ml DNA for 4 h at 37 °C, 5% CO2. Transfected cells were mounted in a perfusion chamber after 48 h of expression (Harvard Instruments, Holliston, MA) set at 37 °C for live microscopy or otherwise processed for colocalization studies. Fluorescence microscopy was carried out using an Olympus IX-70 microscope and Olympix KAF1400 CCD camera (LSR, Olympus, Melville, NY). The ratio of emission at 508 nm upon excitation at 410 versus 470 nm was obtained using the previously described (18.Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1966) Google Scholar) filter sets (Chroma Technology Corp., Brattleboro, VT) mounted in a Sutter filter wheel (Sutter Instruments, Novato, CA) and controlled by the Merlin program (version 1.89, LSR, Olympus, Melville, NY). For the pH standard curve, two types of calibrations were carried out. (i) Cells transfected with GPI-pHluorin GFP were mounted in a perfusion chamber and incubated in buffer A (25 mm HEPES (pH changing from 7.4 to 5.5), 119 mm NaCl, 2.5 mm KCl, 2 mmCaCl2, 2 mm MgCl2, 30 mm glucose) at 37 °C. Fluorescence images were taken upon excitation at 410 and 470 nm (six consecutive exposures). Three regions of interest were selected, and the standard curve was plotted as averaged 410/470 ratio values for a given buffer pH. (ii) At the end of experiments, the pH gradient was collapsed by incubating cells in 10 μm monensin and 10 μm nigericin for 30 min at 37 °C in buffer A at pH 7.4 or 5.5, and ratios were recorded for internal standards. Sample pH was determined the same way as for the external standard curve. IB3-1 and derivative cells were seeded onto glass coverslips in 6-well plates at the density described above. After 72 h cells were washed and incubated with the water-soluble, membrane-impermeant, pH-sensitive ratiometric probe 8-hydroxypyrene-1,3,6-trisulfonic acid 5 mm (HPTS, Molecular Probes, Eugene, OR) at 37 °C. Cells were washed after 10 and 60 min, and the ratio of fluorescence emission at 508 nm was determined upon altered excitation at 410 and 470 nm. For H+-ATPase inhibition, cells were incubated with 100 nm bafilomycin A (Sigma) in buffer A at pH 7.4 for 2.5 h at 37 °C. For inhibition of sodium channels, 100 μm amiloride (Sigma) was added in buffer A at pH 7.4 for 60 or 120 min at 37 °C. For Na+/K+-ATPase inhibition, cells were incubated with 10 μm acetylstrophanthidin in buffer A for 60 min, and pH was measured as above. In experiments where CFTR folding and trafficking were rescued (23.Egan M.E. Schwiebert E.M. Guggino W.B. Am. J. Physiol. 1995; 268: C243-C251Crossref PubMed Google Scholar) by low temperature, mutant IB3-1 cells were grown at 26 °C, 5% CO2 for 40 h on glass coverslips. Organellar pH was determined using ratiometric GFP-pHluorin as described in sections above. 4-Phenylbutyric acid (4-PBA; gift from Triple Crown America Inc., Perkasie, PA) was used as an agent that promotes CFTR trafficking and rescue of its function (24.Rubenstein R.C. Zeitlin P.L. Am. J. Physiol. Cell Physiol. 2000; 278: C259-C267Crossref PubMed Google Scholar,25.Zeitlin P.L. J. Clin. Invest. 1999; 103: 447-452Crossref PubMed Scopus (79) Google Scholar). IB3-1 cells were grown in the presence of 2.5 mm 4-PBA at 37 °C, 5% CO2 for 40 h. Cells were then subjected to ratiometric determination of organellar pH as described above. Cells were grown for 48 h in complete LHC-8 media in the presence of 0.1–1.0 mmNH4Cl (from Sigma) at 37 °C in 5% CO2, and pH measurements were carried out as describe above. For localization studies with fluorescently labeled transferrin, IB3-1 cells and derivatives grown on glass slides were transfected with cellubrevin-pHluorin GFP as described above. After 48 h of expression, cells were incubated for 30 min in DMEM (BioWhittaker, Walkersville, MD), 0.2% BSA (Sigma) at 37 °C followed by a change of medium and incubation at 4 °C for 30 min. 20 μg/ml human transferrin conjugated to Texas Red (Molecular Probes) in DMEM, 0.2% BSA was added for 30 min at 4 °C followed by three washes and incubation with DMEM, 0.2% BSA at 37 °C for 15 and 120 min. When indicated, cells were treated with 20 μg/ml nocodazole (Sigma) in DMEM, 0.2% BSA for 60 min following a 120-min treatment with transferrin. Samples were fixed with 3.7% paraformaldehyde, 5% sucrose for 10 min at room temperature, mounted with PermaFluor (Shandon, Pittsburgh, PA), and examined by fluorescence microscopy using a 570/20 excitation filter and a dichroic mirror/emitter cube set 8300 (Chroma Technology Corp.). For localization studies with CFTR-GFP and transferrin, IB3-1 cells and derivatives were transfected with CFTR-GFP. Transfection and transferrin incubation were as described above. For localization studies with α2,6-sialyltransferase, cells were co-transfected with 0.5 μg of cellubrevin-pHluorin GFP and Myc-tagged α2,6-sialyltransferase DNA using 10 μl of Lipofectin. After 48 h of expression, cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.2% saponin for 5 min. Mouse monoclonal antibody (9E10) against c-myc (Santa Cruz Biotechnology, Santa Cruz, CA) was followed by goat anti-mouse secondary antibody conjugated to Alexa 568 (Molecular Probes). Glass slides were mounted using PermaFluor and analyzed by fluorescence microscopy using a 570/20 excitation filter and a dichroic mirror/emitter cube set 8300. For localization studies with dextran-Texas Red, cellubrevin-pHluorin-transfected IB3-1 cells and IB3-1 derivatives were incubated with 10 μg/ml dextran-Texas Red followed by three washes. Cells were either fixed or live sequences were recorded immediately after removal of dextran-Texas Red every 30 s for 30 min using a monochromator excitation light source and emission filter sets on a microscope and camera controlled by TILLvisTRAC, version 3.3 (T.I.L.L. Vision Photonics, GMBH). For localization studies of cellubrevin-pHluorin GFP and EEA-1, IB3-1 and derivative cells were transfected with cellubrevin-pHluorin GFP. EEA1 was visualized using primary human anti-EEA1 antibody (Transduction Laboratories, Lexington, KY) and secondary Alexa 568-conjugated antibody. Transferrin recycling was carried out as described previously (16.Gibson G.A. Hill W.G. Weisz O.A. Am. J. Physiol. 2000; 279: C1088-C1099Crossref Google Scholar). IB3-1 cells and their derivatives were incubated with 125I-labeled transferrin for 45 min in DMEM, 0.2% BSA at 37 °C. Cells were then washed three times with ice-cold DMEM, 0.2% BSA. The last wash was taken as 0 time point. Cells were then incubated for 15 min at 37 °C, and medium was collected and replaced with fresh DMEM, 0.2% BSA for a further 45 min. Medium was collected, and cells were lysed to establish 100% of counts. Samples were counted in a γ-counter (Beckman, Brae, CA) and expressed as % transferrin recycled at a given time point. The assay was carried out according to Li and co-workers (26.Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar, 27.Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (246) Google Scholar). Cells were seeded in 6-well plates at 5 × 105/well at 24 h prior to assay. After being washed in serum-free DMEM, cells were incubated for 15 min at 37 °C with either DMEM or 100 ng/ml wortmannin in DMEM. After washing, cells were incubated with 5 mg/ml HRP in DMEM, 0.2% BSA for 60 min at 37 °C. Uptake was stopped by washing with 4 °C phosphate-buffered saline, 0.2% BSA. Cells were lysed in phosphate-buffered saline, 0.1% Triton X-100. Lysate was added to O-phenylenediamine solution (HRP substrate) in a 96-well plate and incubated at room temperature for 5 min. Reaction was stopped by adding 1 mH2SO4, and A490was measured using a spectrophotometer (Shimadzu UV-1601, Shimadzu, Columbia, MD). Protein concentration of the lysate was determined by BCA reaction (Pierce), and uptake was expressed asA490/mg protein. All statistical analyses were carried out using Fisher's Protected LSD post hoc test (analysis of variance) (SuperANOVA v1.11, Abacus Concepts, Inc., Berkeley, CA). In this study, we employed the recently developed pH-sensitive GFP (pHluorin GFP) system for ratiometric determination of the lumenal pH in intracellular organelles (18.Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1966) Google Scholar). Two pHluorin GFP fusion constructs were used (Fig. 1, a–h), one with GPI-pHluorin GFP and another with (endosomal v-SNARE) cellubrevin (18.Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1966) Google Scholar). GPI-pHluorin GFP is expected to result in the exposure of pHluorin GFP on the plasma membrane to the extracellular fluid. The cellubrevin-pHluorin GFP fusion has GFP exposed luminally in the intracellular compartments containing cellubrevin. The endosomal (cellubrevin) and plasma membrane (GPI)-targeted pHluorin GFP probes were transfected into well characterized human bronchial epithelial cells (21.Zeitlin P.L. Lu L. Rhim J. Cutting G. Stetten G. Kieffer K.A. Craig R. Guggino W.B. Am. J. Respir. Cell Mol. Biol. 1991; 4: 313-319Crossref PubMed Scopus (273) Google Scholar, 22.Egan M. Flotte T. Afione S. Solow R. Zeitlin P.L. Carter B.J. Guggino W.B. Nature. 1992; 358: 581-584Crossref PubMed Scopus (377) Google Scholar): IB3-1 (from a compound heterozygote CFTR ΔF508/W1282X CF patient), C38 (IB3-1 cells corrected with a functional CFTR lacking the first ecto-loop), and S9 (IB3-1 cells corrected with a full size functional CFTR cDNA). These cells have been used as standard cell lines to model the effects of CFTR (6.Schwiebert E.M. Egan M.E. Hwang T.H. Fulmer S.B. Allen S.S. Cutting G.R. Guggino W.B. Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (595) Google Scholar, 21.Zeitlin P.L. Lu L. Rhim J. Cutting G. Stetten G. Kieffer K.A. Craig R. Guggino W.B. Am. J. Respir. Cell Mol. Biol. 1991; 4: 313-319Crossref PubMed Scopus (273) Google Scholar, 22.Egan M. Flotte T. Afione S. Solow R. Zeitlin P.L. Carter B.J. Guggino W.B. Nature. 1992; 358: 581-584Crossref PubMed Scopus (377) Google Scholar, 24.Rubenstein R.C. Zeitlin P.L. Am. J. Physiol. Cell Physiol. 2000; 278: C259-C267Crossref PubMed Google Scholar, 28.DiMango E. Ratner A.J. Bryan R. Tabibi S. Prince A. J. Clin. Invest. 1998; 101: 2598-2605Crossref PubMed Google Scholar). The plasma membrane localization of GPI-pHluorin GFP was demonstrated by responsiveness of GFP fluorescence to pH changes of the external buffer. Fig. 1, a–d, displays the fluorescence appearance of GPI-pHluorin GFP at pH 7.4 and 5.5. The cells expressing GPI-pHluorin GFP were used to generate a standard curve (Fig. 1i). All cells showed identical dependence of the GPI-pHluorin GFP fluorescence on pH of the external buffer. In addition to the plasma membrane labeling, as evidenced in Fig. 1,a–d, all cells transfected with GPI-pHluorin GFP showed a perinuclear fluorescence corresponding to a lipid raft recycling compartment, recently described by Lippincott-Schwartz and colleagues (29.Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-541Crossref PubMed Scopus (457) Google Scholar). Based on our observations, this compartment responds to external buffer pH (Fig. 1, a–d), most likely because of the previously described rapid cycling of these membranes in constant communication with plasma membrane (29.Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-541Crossref PubMed Scopus (457) Google Scholar). GPI-pHluorin GFP fluorescence was not dependent on changes in concentration of other ions in the medium (e.g. sodium; data not shown). There were no differences in fluorescence ratios obtained with GPI-pHluorin GFP in IB3-1, C38, and S9 cells. Localization of cellubrevin-pHluorin GFP was examined in both CF and CFTR-corrected cells by fluorescence microscopy using EEA1 antibodies, Texas Red-conjugated endocytic tracers. First, the cells were allowed to endocytose fluorescent transferrin, which was followed by chasing this marker of receptor-mediated endocytosis into the pericentriolar/paranuclear recycling compartment. This resulted in a significant colocalization of transferrin with cellubrevin-pHluorin GFP fluorescence in the transfected cells as evidenced by a similar overall organellar distribution (Fig. 2,a–c, IB3-1 cells; d–f, CFTR-corrected S-9 cells). Both the CF and CFTR-corrected cells showed similar overall organellar distribution. The colocalization of cellubrevin-pHluorin GFP and transferrin was not absolute in either cell line, as some of the cellubrevin- and transferrin-labeled profiles did not fully overlap, consistent with previous observations of strong but incomplete colocalization between transferrin and cellubrevin labeled vesicles (30.Teter K. Chandy G. Quinones B. Pereyra K. Machen T. Moore H.P. J. Biol. Chem. 1998; 273: 19625-19633Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The most complete overlap was seen in the pericentriolar recycling endosomal compartment, strongly labeled by fluorescent transferrin, which was also the site of the majority of cellubrevin-pHluorin GFP labeled intracellular organelles. In further support of the overlap between cellubrevin and the recycling endosomal compartment, the treatment of cells with nocodazole, which causes depolymerization of microtubules and dispersion of the recycling endosome, resulted in redistribution of both transferrin and cellubrevin-pHluorin GFP fluorescence with a preservation of the significant overlap between the two markers (Fig. 2, g–i). These observations suggest that cellubrevin-pHluorin GFP is localized in human bronchial epithelial cells with similar distribution in both CF and CFTR-corrected cells in the endosomal recycling compartment equivalent to what has been observed in several model cell lines (30.Teter K. Chandy G. Quinones B. Pereyra K. Machen T. Moore H.P. J. Biol. Chem. 1998; 273: 19625-19633Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 31.Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Abstract Full Text PDF PubMed Scopus (417) Google Scholar, 32.Hopkins C.R. Gibson A. Shipman M. Strickland D.K. Trowbridge I.S. J. Cell Biol. 1994; 125: 1265-1274Crossref PubMed Scopus (217) Google Scholar, 33.Marsh E.W. Leopold P.L. Jones N.L. Maxfield F.R. J. Cell Biol. 1995; 129: 1509-1522Crossref PubMed Scopus (131) Google Scholar). Importantly, CFTR partially overlapped with the recycling endosome in bronchial epithelial cells (Fig. 2, j–l). The colocalization of CFTR-GFP and transferrin was similar to the one observed with cellubrevin-pHluorin GFP and transferrin (Fig. 2,a–f). The cellubrevin-pHluorin GFP probe did not colocalize with the early endosomal marker EEA1, although the large EEA1-positive profiles and the cellubrevin recycling endosome appeared to be closely apposed (Fig. 3, a–c). Treatment of cells with nocodazole confirmed that cellubrevin-pHluorin GFP and EEA1 were in distinct compartments (Fig. 3, d–f). The organellar distribution of EEA1 and cellubrevin compartments was similar in CFTR-corrected (Fig. 3, a–c) and CF cells (Fig. 3,insets in a–c). Cellubrevin-pHluorin GFP did not colocalize with peripheral endocytic organelles labeled with the fluid phase tracer dextran-Texas Red in fixed cells (data not shown) and in live cells monitored by time lapse microscopy (Fig. 3,g–l). Cellubrevin-pHluorin GFP was also tightly apposed to the α2,6-sialyltransferase, as revealed by immunofluorescence (Fig. 4), but remained localized distinctly from the TGN marker. There were no differences in localization of cellubrevin-pHluorin GFP in the CFTR mutant cells and CFTR-corrected cells (Fig. 4, a–d, CFTR-corrected cells; insetsin b–d, CF cells). Collectively, these observations indicate that cellubrevin-pHluorin GFP probe was in the identical compartments in CFTR-corrected and CF cells and that the pH probe was in the recycling endosome.Figure 4Close apposition of cellubrevin-pHluorin GFP recycling endosomes and TGN in human bronchial epithelial cells.TGN was revealed by Myc-tagged α2,6-sialyltransferase Sttyr. IB3-1 and S9 cells were co-transfected with cellubrevin-pHluorin GFP and myc tagged α2,6-sialyltransferase Sttyr expressing constructs. a–d, main panels, S9 (CFTR-corrected cell). b–d,insets, IB3-1 (mutant CFTR cell): a, phase contrast; b, GFP fluorescence; c, immunofluorescent visualization of Myc-tagged α2,6-sialyltransferase, Sttyr, using anti-Myc antibody and secondary Alexa 568-conjugated antibody (redfluorescence); d, merged images b andc.View Large Image Figure ViewerDownload Hi-res image Download (PPT) IB3-1 (CF), C38 (CFTR-corrected IB3-1), and S9 cells (full size CFTR-corrected IB3-1), transfected with cellubrevin-pHluorin GFP (18.Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1966) Google Scholar) were used to determine the pH of cellubrevin-containing endosomal compartments. Fig. 1, e–h, illustrates the difference in fluorescence between cellubrevin-pHluorin GFP-transfected IB3-1 and C38 cells upon illumination at 410versus 470 nm. The apparent pH of cellubrevin-containing endosomes was 6.7 ± 0.1 (mean ± S.E., n = 15) for the CFTR-corrected C38 and 6.7 ± 0.1 (mean ± S.E.,n = 32) for S9 cells compared with the apparent pH of IB3-1 CFTR mutant cells, which was 6.2 ± 0.1 (mean ± S.E.,n = 19) (Table I). Thus, cellubrevin-labeled compartments in CF mutant cells show hyperacidification of 0.5 pH unit (p = 0.0001). The pH of the cellubrevin-labeled compartments remained unaltered regardless of whether the cells were subconfluent or confluent, retaining the difference in pH between CF and CFTR-corrected cells (n= 66).Table IHyperacidification of TGN38 and cellubrevin-labeled compartments in CF respiratory epithelial cellsCell line1-aIB3–1 is a human bronchial cell line derived from a CF patient with a ΔF508/W1282XCFTR mutant genotype (21). C38 cells express a functionalCFTR with a fortuitous N-terminal in frame deletion. S9 cells express a functional full size CFTR (22). CFT1 (19) is a tracheal cell line derived from a CF patient homozygous for mutant ΔF508/ΔF508 CFTR. CFT1-LCFSN (wild-typeCFTR), CFT1-Δ508 (Δ508 CFTR), and CFT1-LC3 (vector control) are stably transfected derivatives of CFT1.Apparent pH1-bP < 0.0001 for IB3–1versus C38; IB3–1 versus S9; CFT1-LCFSNversus CFT1-LC3; CFT1-LCFSN versus CFT1-Δ508; CFT1-LCFSN versus CFT1" @default.
• W2063315216 created "2016-06-24" @default.
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• W2063315216 date "2002-04-01" @default.
• W2063315216 modified "2023-10-11" @default.
• W2063315216 title "Hyperacidification of Cellubrevin Endocytic Compartments and Defective Endosomal Recycling in Cystic Fibrosis Respiratory Epithelial Cells" @default.
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• W2063315216 doi "https://doi.org/10.1074/jbc.m105441200" @default.
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