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: PLOS One

Article Title: Alternative splicing and residual function potentially expand the therapeutic landscape of the CFTRdele2ins182 variant

doi: 10.1371/journal.pone.0330974

Figure Lengend Snippet: A A. Representative traces on nasal epithelia derived from two non-CF donors with the short-circuit current technique. During the experiment, the epithelia were treated with amiloride on the apical side (10 µM), CPT-cAMP (100 µM) both apical and basolateral side, ivacaftor (1 µM) and the inhibitor inh-172 (20 µM) on apical side. The dotted line marks the zero current level. B. Representative traces and scatter dot plot summarizing the results of the effect of vehicle alone (DMSO; gray trace) and ELX/TEZ (3 µM/10 µM; blue trace) obtained on nasal epithelia derived from an individual compound heterozygous for the F508del and 2183AA>G variants, with the short-circuit current technique. During the experiment, the epithelia were sequentially treated as indicated in A. Data reported in the scatter dot plot are the amplitude of the current blocked by 20 µM inh-172 (ΔIsc inh-172 ). For each experimental condition the number of biological replicates was n = 4. C. Representative traces of the effect of the vehicle alone (DMSO), or Tezacaftor (TEZ, 10 µM), or Elexacaftor/Tezacaftor (ELX/TEZ 3µM/10µM) on nasal epithelia derived from patient FI077 with the short-circuit current technique. Epithelia were acutely stimulated under the same conditions described in A. The scatter dot plot shows the summary of results. Data reported are the amplitude of the current blocked by 20 µM inh-172 (ΔIsc inh-172 ). For each experimental condition the number of biological replicates was n = 6-8. D. Representative traces and scatter dot plot summarizing the results of the effect of short-circuit current measurements performed as in B,C on nasal epithelia derived from MI253 epithelia. For each experimental condition the number of biological replicates was n = 4. E. Representative traces and scatter dot plot summarizing the results of the effect of short-circuit current measurements performed as in B,C on nasal epithelia from FI066 patient. In each experimental condition the number of biological replicates was n = 8. Asterisks indicate statistical significance of treatments: ***, p < 0.001.

Article Snippet: The CFTR modulators Ivacaftor and Tezacaftor were purchased from TargetMol (catalog ID:T2588 and T2263, respectively; Wellesley Hills, MA, USA), while Elexacaftor from MedChemExpress (catalog ID: HY-111772; Monmouth Junction, NJ, USA).

Techniques: Derivative Assay