PMC

Wt and G551D CFTR expression, function, and postendocytic localization in CF respiratory epithelia and HeLa cells. (A) CFTR expression was probed by immunoblot analysis in mock, wt, and G551D CFTR-3HA expressing CFBE and IB3 respiratory epithelia, as well as in HeLa cells. Equal amounts of cell lysates were immunoblotted with anti-HA Ab. (B) Wt and G551D CFTR activity was measured by the iodide efflux assay as described in C. Data are means of triplicate determinations from a representative experiment. (C) Localization of internalized wt and G551D CFTR in CFBE and IB3 cells. Internalized CFTR was labeled as described in D. Endosomes were visualized by 15 μg/ml FITC-Tf loading or by indirect immunostaining of EEA1 and rab5. Lysosomes were identified by 50 μg/ml FITC-dextran loading as described in Materials and Methods or by indirect Lamp2 immunostaining. Single optical sections were obtained by laser confocal fluorescence microscopy. Bar, 10 μm.

Determination of the passive proton permeability of endocytic organelles. (A) Measurement of the buffer capacity of endocytic organelles. The lysosomal compartment of BHK cells were loaded with dextran as described in Materials and Methods, and the organellar pH was monitored by FRIA. The extent of lysosome alkalinization was measured after the addition of small amounts of NH₄Cl (e.g., 0.1–2 mM) in the presence of Baf to prevent the compensatory activation of the v-ATPase. The number indicates the final concentration of NH₄Cl in mM. The buffer capacity was calculated from the alkalinization, induced by the 0.5–1 mM NH₄Cl as described in and . (B) The buffer capacity of recycling endosomes, lysosomes, and phagosomes was measured on FITC-Tf– and FITC-dextran–loaded organelles as described in A. The early and mature phagosomal buffer capacities were measured after 5 and 30 min phagocytosis of FITC-conjugated P. aeruginosa. (C) The passive proton permeability of recycling endosomes, lysosomes, and phagosomes. The passive proton efflux rate from the indicated organelles was measured in the presence of 400 nM Baf by FRIA after their selective labeling as described in Materials and Methods. The passive proton permeability was calculated as described in Materials and Methods. For comparison, the passive proton permeability of the Golgi compartment and the ER was indicated obtained from previous publications (; ; ; ).

Expression, activity, and membrane trafficking of CFTR-3HA in RAW macrophages. (A) CFTR plasma membrane channel activity was measured by the iodide efflux assay in transiently transfected RAW cells as described in C. Data are means of triplicate determinations. (B) CFTR expression in transiently transfected RAW macrophages was visualized by immunoblotting using M3A7 and L12B4 anti-CFTR Ab. Equal amounts of cell lysates were immunoblotted. (C) Subcellular localization of internalized wt CFTR-3HA in transiently transfected RAW cells and the maturation of phagosomes. Top panels, internalized CFTR was colocalized with recycling endosomes and excluded from lysosomes. FITC-Tf and FITC-dextran labeling of recycling endosomes and lysosomes, respectively, was performed as described in D. Single optical sections were obtained by fluorescence laser confocal microscopy (FLCM). Lower panels, phagosomal maturation was monitored by the colocalization of synchronously ingested, FITC-conjugated P. aeruginosa (PAO1) with TRITC-Tf– and TRITC-dextran–labeled lysosomes as a function of incubation time at 37°C. The labeling of recycling endosomes and lysosomes are described in Materials and Methods. Single optical sections were obtained by FLCM. Bar, 10 μm. (D) Colocalization of FITC-conjugated P. aeruginosa with Ab-labeled CFTR in RAW macrophages. Transiently expressed CFTR-3HA was labeled in vivo by Ab capture for 90 min without chase with anti-HA Ab and TRITC-conjugated secondary Fab at 37°C. Synchronously initiated phagocytosis of FITC-conjugated P. aeruginosa was performed for the indicated time before fixing the cells with paraformaldehyde. Single optical sections were obtained by FLCM. Bar, 10 μm.

CFTR complementation of CF respiratory epithelia and CFTR overexpression in HeLa and MDCK cells has no effect on the endosomal pH. (A and B) Measurement of endosomal pH dissipation rates in wt and G551D CFTR-3HA complemented CFBE and IB3 CF respiratory epithelia and overexpressing HeLa and MDCK cells. CFTR labeling was performed as described in B. The endosomal pH dissipation rates were determined as described in , E and F. (C) Vesicular pH of CFTR-containing vesicle was measured after 3 min of PKA stimulation as in E in the indicated cell lines. Data were analyzed by two-tailed unpaired t tests and indicated as follows: * p = 0.0347 for CFTR G551D control versus forskolin in HeLa. (D) Vesicular pH of FITC-Tf containing vesicle before and after 3 min of PKA stimulation with the agonist cocktail. Means ± SEM; n = 3–5. FITC-Tf loading was described in Materials and Methods. (E) Initial acidification rates of endosomes were measured in wt and G551D CFTR-3HA–expressing HeLa and IB3 cells. CFTR labeling was performed on ice as described in A and monitored by FRIA after increasing the temperature to 37°C for the indicated time in the presence or absence of PKA activators. Means ± SEM from three independent experiments. The pH of ∼10,000 and 8000 vesicles was measured in studies depicted on the top and bottom panels, respectively. (F) Initial acidification rates were measured after 4 min CFTR internalization as in E in the indicated cell lines. Means ± SEM; n = 3.

Selective labeling of CFTR-3HA–containing endosomes with pH-sensitive probes. (A) Schematic comparison of the subcellular distribution of CFTR and various endosomal pH probes. It is predicted that CFTR has partially overlapping localization with the fluid-phase marker dextran-FITC (left panel) and cellubrevin or synaptobrevin coupled with GFP-Phluorin (middle panel). Completely overlapping distribution of the pH-probe and endosomal CFTR is anticipated by labeling the exofacial 3HA-tag of CFTR with anti-HA Ab and FITC-Fab. (B) Immunoblot analysis of wt and G551D CFTR-3HA expression in stably transfected BHK cells. Equal amounts of cell lysates were immunoblotted with anti-HA Ab. Core- and complex-glycosylated CFTR are labeled, indicated by empty and filled arrowhead, respectively. (C) Plasma membrane halide conductance was measured by the iodide efflux assay in BHK monolayers expressing wt or G551D CFTR. PKA was stimulated with 20 μM forskolin, 0.2 mM IBMX, and 0.5 mM CPT-cAMP cocktail (+FK) at the indicated time at 22°C. Data are means of triplicate determinations from a single representative experiment. (D) Subcellular localization of internalized CFTR in BHK cells. Internalized wt and G551D CFTR-3HA was labeled with anti-HA Ab and visualized by TRITC-conjugated secondary Fab. Lysosomes and recycling endosomes were labeled with 50 μg/ml FITC-dextran (loaded overnight and chased for 3 h) and 15 μg/ml FITC-Tf (loaded for 45 min in serum-free medium), respectively. Single optical sections were obtained by laser confocal fluorescence microscopy. Bar, 10 μm.

The phagosomal acidification is CFTR-independent in RAW macrophages. (A) The pH distribution profile of wt CFTR-containing endosomes in transiently transfect RAW macrophages. FRIA of internalized CFTR-3HA was performed as described in B. (B) The pH dissipation rate of FITC-Tf– or FITC-CFTR–containing endosomes was measured in mock or wt CFTR-3HA–transfected RAW cells as indicated. The pH dissipation was initiated by Baf+CCCP addition as described in E and also shown in C. MalH2, 20 μM, was added to inhibit CFTR during labeling and the measurement. (C) The CFTR-dependent PKA-stimulated endosomal pH dissipation was inhibited by MalH2. The pH dissipation of FITC-Tf–loaded endosomes was measured in the absence or presence of PKA stimulation (+FK) as in B. MalH2 (20 μM) was used during the labeling and the measurement to inhibit CFTR activity. (D) Vesicular pH of FITC-Tf– or FITC-CFTR–containing vesicle in the absence or presence of PKA activation (+FK) for 3 min. Vesicular pH measurements were performed as in C. When indicated, 20 μM MalH2 was included. (E) Phagosomal acidification kinetics of RAW macrophages. Phagosomal pH was measured after synchronous ingestion of FITC-conjugated P. aeruginosa (PAO1) by FRIA in control and transiently transfected RAW cells with CFTR-3HA (+CFTR). CFTR-expressing cells were identified by labeling the channel with anti-HA Ab and TRITC-conjugated Fab capture before phagocytosis. MalH2 was added to the medium as in B. Means ± SEM; n = 4. (F) Effect of phagocytosis on the pH of CFTR-containing vesicles in RAW cells. The postendocytic trafficking of synchronously internalized CFTR was measured by FRIA as in A. CFTR was labeled with anti-HA Ab and FITC-conjugated Fab on ice. TRITC-conjugated P. aeruginosa was adsorbed to the cell surface at 4°C. pH measurements were performed on double-stained cells after raising the temperature to 37°C for the indicated time. Means ± SEM; n = 4. (G) Vesicular pH and pH dissipation rates of CFTR-containing vesicles after 5- or 30-min chase in RAW cells transiently expressing wt CFTR-3HA. CFTR was labeled as described in F. (H) The steady-state pH of CFTR-containing endosomes and PAO1 ingested phagosomes are insensitive to PKA stimulation. The vesicular pH was determined before and after 3 min of stimulation with the PKA agonist cocktail (+FK) by FRIA. Means ± SEM; n = 4.

Acidification of phagosomes in CFTR-deficient primary alveolar (AM) and peritoneal (PM) macrophages is preserved. (A and B) CFTR is active in early endosomes, but not in lysosomes, of primary alveolar and peritoneal mouse macrophages from cftr^+/+ mice. pH dissipation rates were measured in FITC-Tf– or FITC/Oregon488-dextran–loaded endosomes and lysosomes, respectively in macrophages isolated from cftr^+/+ or cftr^−/− mice. Tf and dextran labeling was described in Materials and Methods. MalH2, 20 μM, was added to inhibit CFTR during labeling, and the measurement. pH dissipation rates were measured after Baf and CCCP addition as described in E. (C and D) CFTR can be activated in immature phagosomes of cftr^+/+ macrophages. pH dissipation rates of phagosomes ingesting FITC-labeled PAO1 was measured after 5 and 30 min of infection. MalH2, 20 μM, was added during phagocytosis. (E) Phagosomal acidification kinetics of cftr^+/+ and cftr^−/− alveolar macrophages are similar. Acidification of phagosomes, synchronously ingesting FITC-PAO1, was monitored by FRIA. Inset, the variation of immature and mature phagosomal pH after 3 min PKA stimulation is reported as the function of the initial pH. (F) Comparison of phagosomal pH of cftr^+/+ and cftr^−/− peritoneal macrophages by FRIA of FITC or FITC- and TRITC-labeled PAO1. Phagocytosis was initiated synchronously and the duration (5–45 min) is indicated. Means ± SEM; n = 4 mice. (G) Protonophore-induced increase of the pH dissipation rate of endosomes and lysosomes relative to the passive proton efflux rate as an indicator of counterion permeability. The pH dissipation rates of FITC-Tf– and FITC-dextran–loaded recycling endosomes and lysosomes, respectively, were measured by FRIA in the presence of Baf alone and Baf+ CCCP as in , D and E. CFTR-expressing HeLa, RAW and respiratory epithelia (CFBE and IB3) were used. Baf+CCCP simultaneous addition yielded results similar to those obtained by consecutive addition with 1-min delay. Results expressed as fold increase of the CCCP+Baf–induced proton efflux rate over that of the passive proton release. (H) Protonophore-induced increase of the pH dissipation rate relative to the passive proton efflux rate of endocytic organelles in primary macrophages. Baf- and Baf+CCCP–induced pH dissipation rates of recycling endosomes, lysosomes as well as immature and mature phagosomes were measured by the FRIA using the indicated pH-sensitive probe as described in Materials and Methods and in G. Means ± SEM; n = 4 mice.

Monitoring wt and G551D CFTR-3HA endocytic sorting and pH regulation by FRIA in BHK cells. (A) Determining the sorting pathway of internalized CFTR by vesicular pH (pH_v) measurement. Anti-HA antibody and FITC-conjugated secondary Fab was bound to CFTR-expressing BHK cells for 1 h at 0°C. Then the temperature was raised to 37°C for 0–1 h, and the pH_v was measured by FRIA. Data are expressed as frequency of pH_v and means (± SEM) pH_v of the major endosomal population. The number of vesicles analyzed in a single experiment is indicated. (B) Wt CFTR was labeled with anti-HA primary Ab and FITC-conjugated secondary Fab for 1 h at 37°C and was chased for 30 min before FRIA. (C) G551D CFTR was labeled as described in B. (D) Determination of the passive proton and relative counterion permeability of wt CFTR-containing endosomes. Right panel, to unravel the CFTR dependent counterion permeability, protonophere (CCCP) was used to rapidly dissipate the pH gradient in the presence of Baf. H⁺ egress generated a negative inside membrane potential that was dissipated by Cl⁻ efflux via CFTR and uptake of cations. Left panel, the endosomal pH was monitored as a function of time as described in B. For each experiment, 30–50 vesicles were tracked simultaneously. The vacuolar H⁺-ATPase was inhibited with 0.4 μM Baf to unmask the passive proton permeability. CCCP (20 μM), a protonophore, induced a rapid dissipation of the endosomal pH gradient, indicating the presence of constitutively active counterion conductance in endosomes. Addition of NH₄Cl (20 mM) dissipated the pH gradient. Wt CFTR was labeled as in B. (E) CFTR activation enhances the counterion permeability of wt but not G551D CFTR-expressing endosomes in BHK cells. Endosomes were labeled as described in B and the pH dissipation rate was measured after addition of 0.4 μM Baf and 20 μM CCCP by FRIA. CFTR was activated with the PKA agonist cocktail (20 μM forskolin, 0.2 mM IBMX, and 0.5 mM CPT-cAMP cocktail [FK]) for 2–3 min before the pH dissipation was induced. Traces obtained in the presence of activated PKA are labeled by +FK. (F) Quantification of pH dissipation rate (left panel) and vesicular pH (right panel) after PKA activation. The pH dissipation rate was measured in experiments described in E. (G) No significant variation in late and early endosomal pH of wt CFTR-containing vesicles was detectable. The PKA sensitivity of a selected subpopulation of CFTR-containing vesicles with distinct initial pH was determined as in B. Means ± SEM; n = 3–5.