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

Abstract

Membrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol-dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor+tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane.

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters

“…While structurally unrelated, it is plausible that these large transmembrane transporters require specific protein complexes for their proper folding and expression at the cell surface. Finally, both CFTR [ 51 , 52 ] and some SLCs [ 53 , 54 ] have been identified within and outside sphingolipid- and cholesterol-rich lipid nanodomains (or lipid rafts), raising the possibility of defining specific regions where these transporters are in close proximity. Taken together, the close proximity between CFTR and SLC transporters should be further explored.…”

βIII-Tubulin is a Brake on Extrinsic Cell-Death in Pancreatic Cancer

“…kICS CF was calculated as previously described [59][60][61] and azimuthally averaged at every temporal lag. The resulting kICS Cf was fitted with two dynamic components as detailed previously [59][60][61] and temporally (tau lag) varying amplitudes and decays of dynamic components assessed to extract the diffusion coefficients at micro and macro spatial scales.…”

“…To analyse the dynamics and diffusion of DR5-GFP, cells were imaged with 50 ms exposure on PCO edge sCMOS (pixel size 0.097 µm), using the same TIRF settings, and 1000 frames were acquired per field of view. DR5-GFP diffusion was quantified using k-space image correlation spectroscopy (kICS), as previously described [59][60][61]. Briefly, image time series were loaded in a custom-built script in MATLAB (MathWorks, Natick, MA).…”

“…Prior to application of kICS correlation function (CF) calculation, the padded image series were spatially Hann windowed, to remove the high spatial frequencies 'leakage' in kICS CF, coming from the image cell edges. kICS CF was calculated as previously described [59][60][61] and azimuthally averaged at every temporal lag. The resulting kICS Cf was fitted with two dynamic components as detailed previously [59][60][61] and temporally (tau lag) varying amplitudes and decays of dynamic components assessed to extract the diffusion coefficients at micro and macro spatial scales.…”

Changes in cystic fibrosis transmembrane conductance regulator protein expression prior to and during elexacaftor-tezacaftor-ivacaftor therapy

“…The effect of targeted CFTR modulator therapy on CFTR protein expression has been observed during short-term exposure to transfected cell lines where constraints of ER processing and Golgiassociated post-translational glycosylation may differ compared to the effects achieved in patients' epithelial tissues. Such in vitro results suggest that incubation with ELX/TEZ/IVA leads to an increase of mature CFTR (Capurro et al, 2021;Becq et al, 2022) and facilitates clustering of CFTR into lipid rafts and ceramide-rich platforms (Abu-Arish et al, 2022). Closer to the in vivo situation are primary epithelia derived from healthy controls or p.Phe508del-CFTR homozygous CF patients in which CFTR function has been assessed with electrophysiology.…”