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First published online 9 January 2007
doi: 10.1242/jcs.03350

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Misassembled mutant F508 CFTR in the distal secretory pathway alters cellular lipid trafficking

¹ Department of Cell and Developmental Biology and Cystic Fibrosis Research Center, University of North Carolina, Chapel Hill, NC 27599, USA
² Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
³ Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Scottsdale, AZ 85259, USA
⁴ Department of Biochemistry and Biophysics and Cystic Fibrosis Research Center, University of North Carolina, Chapel Hill, NC 27599, USA

View larger version (76K): [in this window] [in a new window]   Fig. 1. F508 CFTR perturbs intracellular cholesterol trafficking. (A) Cholesterol redistribution in cells expressing F508 CFTR is enhanced at reduced temperature. BHK-21 and CHO-K1 cells expressing CFTR and F508 CFTR were grown at 37°C and 27°C and stained with filipin. Bar, 10 µm. The histogram indicates the percentage of cells showing perinuclear Golgi-like staining versus punctate late endosomal/lysosomal staining. For quantification, at least 90 cells were counted from three different experiments and standard deviations are indicated. (B) F508 CFTR escapes ER quality control at reduced temperature. Western blot of lysates from BHK-21 cells expressing CFTR and F508 CFTR grown at 37°C and 27°C shows that F508 CFTR matures when cells are grown at low temperature. Cell lysates were separated by 6% SDS-PAGE and proteins were detected by immunoblotting after transfer to nitrocellulose using anti-CFTR antibody 596. Immunofluorescence microscopy on nonpermeabilized cells using mouse mAb HA11 to detect an external HA epitope in an expanded second extracytoplasmic loop of CFTR, followed by Alexa Fluor 488 goat anti-mouse IgG, confirms cell-surface localization of F508 CFTR in cells that were incubated at 27°C. BHK-21 clones stably expressing CFTR and F508 CFTR with HA epitope have been described earlier (Gentzsch et al., 2004).

View larger version (68K): [in this window] [in a new window]   Fig. 2. Most cholesterol colocalizes with Golgi markers GM130 and giantin in BHK cells expressing wild-type or no CFTR, but not in cells expressing F508 CFTR. GM130 was detected with mouse anti-GM130 mAb followed by goat anti-mouse Alexa Fluor 568 IgG conjugate; giantin was detected by rabbit anti-giantin antibody followed by goat anti-rabbit Alexa Fluor 568 IgG conjugate; and cholesterol was stained with filipin. In the overlay panels, GM130 and giantin are shown in red and filipin staining is shown in green.

View larger version (74K): [in this window] [in a new window]   Fig. 3. Misassembled rather than dysfunctional CFTR mutants cause cholesterol redistribution. (A) Influence of reduced temperature on maturation of different CFTR variants. Western blots showing maturation of CFTR and CFTR variants F508, 1410X, D572N and G551D grown at 37°C and 27°C. Maturation of CFTR mutants F508 and 1410X CFTR is rescued by low temperature. D572N CFTR is retained at the ER at high or low temperature and the severe-disease-causing mutation G551D, which prevents CFTR channel activation, is processed normally at 37°C and 27°C. Cell lysates were separated by 6% SDS-PAGE, transferred to nitrocellulose and proteins were detected by immunoblotting using anti-CFTR antibody 596. (B) Free cholesterol (Chol.) is elevated in CFTR-processing mutants. Free cholesterol content of cells expressing CFTR variants at 27°C was quantified after cellular lipid extraction from equal numbers of cells and thin-layer chromatographic analysis to compare amounts of cholesterol in cells expressing mutant CFTR with the amount of cholesterol detected in cells expressing wild-type CFTR. Values are representative of three independent experiments. Cells expressing F508 and 1410X CFTR show a significant increase in cholesterol content (*P<0.05) in comparison with cells expressing wild-type CFTR. (C) Cholesterol distribution in these variants grown at 27°C. Microscopy showing filipin staining of CFTR and CFTR variants F508, 1410X, D572N and G551D grown at 27°C. The percentage of cells showing perinuclear Golgi staining is shown in a histogram. At least 75 cells were counted and values are representative of three independent experiments. (D) CFTR inhibitor I-172 is without effect on cholesterol distribution. Filipin staining of BHK-21 cells expressing CFTR untreated (control) or treated with 10 µM CFTR inhibitor I-172 for 1, 8 or 24 hours. Neither the percentage of cells showing Golgi staining by filipin nor the relative filipin intensity changes in the presence of the inhibitor. The histogram shows the percentage of cells with perinuclear Golgi staining. At least 75 cells were counted and values are representative of three independent experiments.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Misprocessed ABC protein F728 MRP1 also affects cellular cholesterol distribution. (A) F728 MRP1 is a temperature-sensitive misfolding mutant. F728 MRP1 matures when cells are grown at low temperature. Western blot of lysates from BHK-21 cells expressing MRP1 or F728 MRP1. BHK-21 cells stably expressing MRP1 or F728 MRP1 were grown either at 37°C or shifted to 27°C. Lysates were separated by SDS-PAGE, transferred to nitrocellulose and detected using mAb 897.2. (B) F728 MRP1 escapes ER quality control at low temperature and proceeds to the cell surface. Immunofluorescence microscopy showing localization of MRP1 and F728 MRP1 at 37°C and 27°C. Immunostaining was performed on cells permeabilized with 0.1% saponin using mouse mAb 897.2 followed by Alexa Fluor 488 goat anti-mouse IgG. (C) Cells expressing F728 MRP1 show redistribution of cholesterol similar to cells expressing F508 CFTR. BHK-21 cells expressing MRP1 or F728 MRP1 were grown at 37°C or 27°C and stained with filipin. The percentage of cells showing Golgi staining is shown in a histogram. At least 75 cells were counted and values are representative of three independent experiments.

View larger version (78K): [in this window] [in a new window]   Fig. 5. Cells expressing F508 CFTR show accumulation of glycosphingolipids as well as cholesterol, which is blocked in transfer to the Golgi and ER for esterification. (A) Transfer of NBD-cholesterol to the Golgi and ER is blocked in cells expressing F508 CFTR. BHK-21 cells were grown at 27°C for 48 hours, labeled with NBD-cholesterol and chased for 4 hours with growth medium at 37°C. The histogram shows that the percentage of cells with perinuclear Golgi staining is drastically reduced in cells expressing F508. At least 75 cells were counted and values are representative of three independent experiments. (B) Nile Red staining is decreased in cells expressing F508 CFTR. Nile Red staining of BHK-21 cells grown at 27°C was reduced in cells expressing F508 CFTR. The average Nile Red intensity per cell is shown in a histogram. At least 90 cells were counted from three different experiments and standard deviations are indicated. (C) Distribution of fluorescent sphingolipid analog is altered in cells expressing F508 CFTR. BHK-21 cells expressing CFTR or F508 at 27°C for 48 hours were labeled with BODIPY-LacCer for 30 minutes at 4°C and subsequently chased for 1 hour at 37°C. The histogram shows that the percentage of cells with Golgi staining is reduced in cells expressing F508 CFTR.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Rab9 overexpression overcomes intracellular cholesterol accumulation. BHK-21 cells expressing CFTR or F508 CFTR were transfected with a plasmid encoding Rab9-EGFP (Choudhury et al., 2002). After 24 hours, the cells were shifted to 27°C and incubated for another 24 hours, before staining for intracellular cholesterol with filipin. Three representative panels of cells expressing wild-type or F508 CFTR are shown. Cells that overexpress Rab9-EGFP are indicated by an arrow in the filipin panel. The punctate filipin staining is cleared from cells expressing Rab9-EGFP and F508 CFTR, but remains unchanged in a perinuclear Golgi-like location in cells expressing Rab9-EGFP and CFTR. Approximately 90 cells of three different experiments were quantified and data are shown in a histogram.