Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Trikafta correctors restore F508del-CFTR clustering at the PM. HBE cells transduced with adenoviruses containing wt- or F508del-CFTR. (A,B) PM distribution of wt-CFTR shows clusters (white arrow, N exp =10; n cell =380) and platforms after Thaps (blue arrow, N exp =8; n cell =156). CFTR is also enriched near cell–cell junctions (rim, yellow arrow). (C) F508del-CFTR lacks cluster and does not accumulate near junctions ( N exp =14; n cell =315). (D) There is no recruitment of F508del-CFTR into platforms after Thaps treatment ( N exp =5; n cell =147). (E,F) Lumacaftor (VX-809, 24 h) does not restore F508del-CFTR clustering or entry into platforms ( N exp =6; n cell =213). (G,H) VX-445 plus VX-661 (24 h) restores F508del-CFTR membrane expression, clustering (white arrow) and rim formation (yellow arrow, N exp =19; n cell =842, see inset). Thaps treatment triggers formation of F508del-CFTR-enriched platforms (blue arrow, N exp =6; n cell =264, see inset), further evidence that corrected F508del-CFTR partitions inside lipid rafts. Magnified views of the indicated area are shown in G′,H′.

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Transduction, Membrane, Expressing

Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Trikafta correctors restore F508del-CFTR clustering and confinement inside microdomains. HBE cells transduced with wt- or F508del-CFTR adenoviruses. (A) Degree of aggregation (DA ratio) showing that wt-CFTR clusters are five times larger than F508del-CFTR. Thaps increases the wt-CFTR DA ratio due to incorporation in platforms whereas F508del-CFTR DA is unchanged. VX-445 plus VX-661 correction restores normal F508del-CFTR DA ratio and clustering. Thaps treatment increased the DA ratio, indicating entry of the mutant into platforms. (mean±s.e.m.; N exp =2; n cell , n wt =40, n wt+Thaps =37, n ΔF =108, n ΔF+Thaps =72, n ΔF(VX) =98, n ΔF(VX)+Thaps =95). **** P <0.0005; ns, not significant. (B) D micro from k-space image correction spectroscopy analysis indicates confined mobility of CFTR inside microdomains and is higher for F508del-CFTR than wt-CFTR, indicating weaker confinement. Thaps reduces mobility and increases confinement due to the presence of wt-CFTR inside ceramide-rich platforms but does not affect F508del-CFTR, which is excluded from ceramide-rich platforms. VX-445 plus VX-661 correction partially restores F508del-CFTR mobility and confinement under Ctr and Thaps conditions (mean±s.e.m.; N exp =2; n cell , n wt =40, n wt+Thaps =37, n ΔF =91, n ΔF+Thaps =51, n ΔF(VX) =85, n ΔF(VX)+Thaps =80). **** P <0.0005; ** P <0.025. (C,D). Differences in F508del-CFTR degree of aggregation (DA ratio) and confined mobility ( D micro ) in the PM and the ER indicate the presence of some weakly confined F508del-CFTR channels at the PM (mean±s.e.m.; N exp =2; n cell , n ΔF(PM) =39, n ΔF(ER) =30). **** P <0.0005. Unpaired one-tailed t -tests were used throughout the analysis, and each cell is an independent biological sample.

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Transduction, Mutagenesis, Spectroscopy, One-tailed Test

Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Trikafta correctors restore S13F-CFTR folding. HBE cells transduced with adenovirus containing S13F-CFTR. (A) Like F508del-CFTR, S13F-CFTR does not form clusters or a rim near junctions ( N exp =11; n cell =341). (B) Mid-section through cell reveals that most S13F-CFTR is intracellular (green arrow). (C,D) VX-445 plus VX-661 correction reduces S13F-CFTR intracellular retention and restores membrane expression, clustering (white arrow) and rim formation (yellow arrow, N exp =16; n cell =582). The green arrow in D highlights the decrease in intracellular S13F-CFTR level after correction therapy. (E,F) S13F-CFTR fails to form platforms after Thaps treatment ( N exp =3; n cell =90) (E), and Trikafta correctors ameliorate this defect ( N exp =7; n cell =306) (F). The blue arrow in F highlights the restoration of S13F-CFTR incorporation in ceramide-rich platforms after correction therapy. (G) As shown by co-immunoprecipitation, VX-445 plus VX-661 (and low temperature, 29°C for 24 h) restores interaction of S13F-CFTR and F508del-CFTR with FLNA suggesting both mutants are misfolded and rescued by these correctors ( N exp =3). 20 μg of protein were used for the lysate blot and 750 μg for the IP blot (2.7% of the IP).

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Transduction, Membrane, Expressing, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs

doi: 10.1242/jcs.259002

Figure Lengend Snippet: Reciprocal effects of CFTR on membrane lipids. Cells were exposed to CTXB conjugated to the fluorophore Alexa Fluor 594 (CTXB–594), which binds to the ganglioside GM1 and labels GM1-positive lipid rafts, at 0.5 μg/ml for 30 min before imaging. (A,B) CTXB–594 distribution at the PM of non-CF (A) and CF (B) cells showing GM1 clustering. (C–E) ICS analysis comparing distribution of GM1 clusters on HBE cells that overexpress wt- or F508del-CFTR (ΔF). (C) Total fluorescence due to CTXB–594 binding is similar on cells expressing wt- and F508del-CFTR. (D,E) GM1 aggregation (DA ratio) is 3-fold higher and cluster density (CD ratio) (# cluster per μm 2 ) is 3-fold lower for wt-CFTR than F508del-CFTR, suggesting it promotes lipid raft formation. I wt (×10 3 )=2.73±0.04 arbitrary units (AU), I ΔF (×10 3 )=2.49±0.04 AU, DA wt ratio=1.00±0.05, DA ΔF ratio=0.35±0.02, CD wt ratio=1.00±0.06, CD ΔF ratio=2.9±0.2 (mean±s.e.m.; N exp =3, n cell , n wt =52, n ΔF =89). **** P <0.0005. (F–H) Similar results were obtained using untransduced cells expressing endogenous wt- (nonCF) or F508del-CFTR (CF). Overall CTXB fluorescence was similar (F); however, CTXB clusters were 5-fold larger (H) and their number per unit area was ∼5-fold lower on non-CF cells (G). I nonCF (×10 3 )=0.77±0.02 AU, I CF (×10 3 )=0.81±0.01 AU, DA nonCF ratio=1.00±0.07, DA CF ratio=0.19±0.01, CD nonCF ratio=1.00±0.05, CD CF ratio=5.0±0.2 (mean±s.e.m.; N exp =3; n cell , n nonCF =100, n CF =62). **** P <0.0005; ns, not significant. (I,J) Trikafta correctors (VX) increase aggregation state of GM1-positive rafts (CTXB clusters) by ∼2.5-fold when HBE cells overexpress F508del-CFTR or S13F-CFTR [mean±s.e.m. N exp =2, n cell : n S13F =30, n S13F(VX) =30, n ΔF =30, n ΔF(VX) =25]. **** P <0.0005; ns, not significant. Each point on the histogram represents a cell, and each cell is an independent biological sample. Unpaired one-tailed t -tests were used throughout the analysis.

Article Snippet: CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM).

Techniques: Membrane, Imaging, Fluorescence, Binding Assay, Expressing, One-tailed Test

Journal: F1000Research

Article Title: An unexpected effect of TNF-α on F508del-CFTR maturation and function

doi: 10.12688/f1000research.6683.2

Figure Lengend Snippet: Differentiated primary HBE cell cultures grown at an air-liquid interface were incubated with 50ng/ml TNFα for 10–30min, 3–6h and 24h. CFTR immunodetection was performed with 24.1 anti-CFTR antibody and analyzed with confocal microscopy. Green staining represents CFTR (Alexa Fluor 488), red color staining represents ZO-1 protein of tight junctions (Alexa Fluor 594) and blue DAPI staining visualizes nuclei. Independent TNFα treatments and CFTR immunodetection were performed on HBE cells from three different F508del/F508del CF patients. Representative images of one experiment are demonstrated (Scale bars = 20µm).

Article Snippet: Anti-CFTR antibodies (abs): MM13-4 mouse monoclonal ab against N-terminus of CFTR, (Millipore, France, 05-581); 24-1 mouse monoclonal ab against C-terminus of CFTR (R&D Systems, MAB, 25031).

Techniques: Incubation, Immunodetection, Confocal Microscopy, Staining