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Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Ferru-Clément R, Fresquet F, Norez C, Métayé T, Becq F, Kitzis A, Thoreau V - PLoS ONE (2015)

Bottom Line: When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis.Total and PM CFTR amounts were increased, resulting in greater activation of CFTR.In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus

Cdc42 depletion reduces the long-term CFTR internalized fraction.The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, PM proteins were labelled and the cultures were submitted to additional 24 h incubation at 37°C. Purification occurred either directly (-), from 200 μg clarified lysates, or alternatively, after a MESNA-mediated stripping step (+), from 600 μg clarified lysates. After densitometric quantification of Western blot images (representative example in the bottom left panel), (+) to 3×(-) ratios were calculated. The Ctrl RNAi value was used to define 100% of long-term internalized fraction. In the bottom right panel, histograms express the relative CFTR internal fractions normalized to the Ctrl RNAi condition. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.
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pone.0118943.g011: Cdc42 depletion reduces the long-term CFTR internalized fraction.The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, PM proteins were labelled and the cultures were submitted to additional 24 h incubation at 37°C. Purification occurred either directly (-), from 200 μg clarified lysates, or alternatively, after a MESNA-mediated stripping step (+), from 600 μg clarified lysates. After densitometric quantification of Western blot images (representative example in the bottom left panel), (+) to 3×(-) ratios were calculated. The Ctrl RNAi value was used to define 100% of long-term internalized fraction. In the bottom right panel, histograms express the relative CFTR internal fractions normalized to the Ctrl RNAi condition. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.

Mentions: In addition, we evaluated CFTR internalization with an endocytic assay: surface proteins were labelled 48h after siRNA transfection and 5 min incubation at 37°C triggered internalization. Several studies [25,26] have shown that reactivating intracellular trafficking for 5 min allows for CFTR endocytosis measurement before recycling or degradation may interfere. After stripping of the labelled protein remaining at the cell surface, intracellular CFTR was streptavidin-bound and quantified. Internalization rate was then reduced by 20% upon Cdc42 depletions (Fig. 8D). We then used another protocol to assess longer term effect upon internalized CFTR amount; labelling was performed 48 h after siRNA transfection and cells were further cultured for 24 h; either whole labelled CFTR (at PM and intracellular) or labelled CFTR remaining inside cellular compartments (protected from stripping) were then captured and quantified. The ratio of intracellular to total biotinylated CFTR was calculated. The relative fraction of internal labelled CFTR appeared to have been lowered upon Cdc42 depletion (Fig. 11). This suggests that the decrease of CFTR endocytosis was sustained during 24 h or was perhaps balanced by increased recycling.


Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Ferru-Clément R, Fresquet F, Norez C, Métayé T, Becq F, Kitzis A, Thoreau V - PLoS ONE (2015)

Cdc42 depletion reduces the long-term CFTR internalized fraction.The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, PM proteins were labelled and the cultures were submitted to additional 24 h incubation at 37°C. Purification occurred either directly (-), from 200 μg clarified lysates, or alternatively, after a MESNA-mediated stripping step (+), from 600 μg clarified lysates. After densitometric quantification of Western blot images (representative example in the bottom left panel), (+) to 3×(-) ratios were calculated. The Ctrl RNAi value was used to define 100% of long-term internalized fraction. In the bottom right panel, histograms express the relative CFTR internal fractions normalized to the Ctrl RNAi condition. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359135&req=5

pone.0118943.g011: Cdc42 depletion reduces the long-term CFTR internalized fraction.The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, PM proteins were labelled and the cultures were submitted to additional 24 h incubation at 37°C. Purification occurred either directly (-), from 200 μg clarified lysates, or alternatively, after a MESNA-mediated stripping step (+), from 600 μg clarified lysates. After densitometric quantification of Western blot images (representative example in the bottom left panel), (+) to 3×(-) ratios were calculated. The Ctrl RNAi value was used to define 100% of long-term internalized fraction. In the bottom right panel, histograms express the relative CFTR internal fractions normalized to the Ctrl RNAi condition. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.
Mentions: In addition, we evaluated CFTR internalization with an endocytic assay: surface proteins were labelled 48h after siRNA transfection and 5 min incubation at 37°C triggered internalization. Several studies [25,26] have shown that reactivating intracellular trafficking for 5 min allows for CFTR endocytosis measurement before recycling or degradation may interfere. After stripping of the labelled protein remaining at the cell surface, intracellular CFTR was streptavidin-bound and quantified. Internalization rate was then reduced by 20% upon Cdc42 depletions (Fig. 8D). We then used another protocol to assess longer term effect upon internalized CFTR amount; labelling was performed 48 h after siRNA transfection and cells were further cultured for 24 h; either whole labelled CFTR (at PM and intracellular) or labelled CFTR remaining inside cellular compartments (protected from stripping) were then captured and quantified. The ratio of intracellular to total biotinylated CFTR was calculated. The relative fraction of internal labelled CFTR appeared to have been lowered upon Cdc42 depletion (Fig. 11). This suggests that the decrease of CFTR endocytosis was sustained during 24 h or was perhaps balanced by increased recycling.

Bottom Line: When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis.Total and PM CFTR amounts were increased, resulting in greater activation of CFTR.In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus