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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 increases CFTR stability at plasma membrane.Two approaches were used to assess CFTR turnover downstream of its targeting to plasma membrane. (A) Cdc42 depletion increases stability of the PM-targeted CFTR. The upper diagrams summarize the procedures followed. 48 h after cell transfection by the mentioned siRNA, surface proteins were labelled. Streptavidin capture occurred either subsequently (0), or after a 24 h incubation of cell cultures at 37°C (24). Labelled CFTR protein amounts were assessed in the resulting samples. After densitometric quantification of Western blot images (representative examples in the bottom left panel), (24) to (0) ratios were calculated. The Ctrl RNAi value was used to define 100% of labelled CFTR stability. In the bottom right panel, the histogram shows that the relative PM-CFTR stabilities increase when Cdc42 is depleted. (B) Cdc42 depletion increases remaining PM-CFTR after cycloheximide chase. The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, the PM proteins were labelled and purified (-). Alternatively, the cell cultures were submitted to additional 24 h incubation with 100 μg/mL cycloheximide (+), before biotinylation and capture were performed. The labelled CFTR protein amounts were assessed by densitometric quantification of Western blot bands (representative images in the bottom left panel). In both RNAi conditions, labelled CFTR amounts extracted from the same amount of whole cell lysates appeared higher after 24h CHX chase: this could be explained by stability differences between the various cellular proteins. To overcome this bias, we compared (+) to (-) ratios and the Ctrl RNAi value was used to define 100% of CFTR apparent PM stability. In the bottom right panel, histogram shows that CFTR stability at PM appears higher when Cdc42 is depleted. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.
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pone.0118943.g010: Cdc42 depletion increases CFTR stability at plasma membrane.Two approaches were used to assess CFTR turnover downstream of its targeting to plasma membrane. (A) Cdc42 depletion increases stability of the PM-targeted CFTR. The upper diagrams summarize the procedures followed. 48 h after cell transfection by the mentioned siRNA, surface proteins were labelled. Streptavidin capture occurred either subsequently (0), or after a 24 h incubation of cell cultures at 37°C (24). Labelled CFTR protein amounts were assessed in the resulting samples. After densitometric quantification of Western blot images (representative examples in the bottom left panel), (24) to (0) ratios were calculated. The Ctrl RNAi value was used to define 100% of labelled CFTR stability. In the bottom right panel, the histogram shows that the relative PM-CFTR stabilities increase when Cdc42 is depleted. (B) Cdc42 depletion increases remaining PM-CFTR after cycloheximide chase. The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, the PM proteins were labelled and purified (-). Alternatively, the cell cultures were submitted to additional 24 h incubation with 100 μg/mL cycloheximide (+), before biotinylation and capture were performed. The labelled CFTR protein amounts were assessed by densitometric quantification of Western blot bands (representative images in the bottom left panel). In both RNAi conditions, labelled CFTR amounts extracted from the same amount of whole cell lysates appeared higher after 24h CHX chase: this could be explained by stability differences between the various cellular proteins. To overcome this bias, we compared (+) to (-) ratios and the Ctrl RNAi value was used to define 100% of CFTR apparent PM stability. In the bottom right panel, histogram shows that CFTR stability at PM appears higher when Cdc42 is depleted. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.

Mentions: Independently of Cdc42 pathway implication in the early steps of CFTR processing, we tried to determine whether it might be involved in distal trafficking steps of CFTR i.e. downstream of its exit from the Golgi apparatus. To evaluate variations in CFTR stability upon depletion of Cdc42, we used two approaches. On the one hand, we estimated the degradation rate of the CFTR initially expressed at the plasma membrane: PM-CFTR was biotinylated, and the labelled CFTR amount was quantified at the beginning of the experiment and after 24 h of further cell incubation in culture medium at 37°C. The percentage of remaining labelled CFTR after the 24 h chase is an indicator of the stability of initial PM-CFTR; it increased when Cdc42 was depleted compared to control (Fig. 10A). On the other hand, we determined the variations of PM-CFTR amount between a condition in which we labelled surface proteins and extracted them 48 h after siRNA transfection, and a condition in which labelling and extraction were performed after 24 h of supplementary CHX chase. These CHX treatment conditions do not preclude the effects of Cdc42 depletion upon early steps of CFTR processing. Consequently, the difference should result only from an equilibrium between endocytosis, recycling and degradation. Cdc42 depletion then elicited an increase of remaining PM-CFTR, in comparison to control (Fig. 10B). Both results are consistent with the hypothesis of improved CFTR stability in PM when the Cdc42 pathway is impaired.


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 increases CFTR stability at plasma membrane.Two approaches were used to assess CFTR turnover downstream of its targeting to plasma membrane. (A) Cdc42 depletion increases stability of the PM-targeted CFTR. The upper diagrams summarize the procedures followed. 48 h after cell transfection by the mentioned siRNA, surface proteins were labelled. Streptavidin capture occurred either subsequently (0), or after a 24 h incubation of cell cultures at 37°C (24). Labelled CFTR protein amounts were assessed in the resulting samples. After densitometric quantification of Western blot images (representative examples in the bottom left panel), (24) to (0) ratios were calculated. The Ctrl RNAi value was used to define 100% of labelled CFTR stability. In the bottom right panel, the histogram shows that the relative PM-CFTR stabilities increase when Cdc42 is depleted. (B) Cdc42 depletion increases remaining PM-CFTR after cycloheximide chase. The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, the PM proteins were labelled and purified (-). Alternatively, the cell cultures were submitted to additional 24 h incubation with 100 μg/mL cycloheximide (+), before biotinylation and capture were performed. The labelled CFTR protein amounts were assessed by densitometric quantification of Western blot bands (representative images in the bottom left panel). In both RNAi conditions, labelled CFTR amounts extracted from the same amount of whole cell lysates appeared higher after 24h CHX chase: this could be explained by stability differences between the various cellular proteins. To overcome this bias, we compared (+) to (-) ratios and the Ctrl RNAi value was used to define 100% of CFTR apparent PM stability. In the bottom right panel, histogram shows that CFTR stability at PM appears higher when Cdc42 is depleted. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.
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pone.0118943.g010: Cdc42 depletion increases CFTR stability at plasma membrane.Two approaches were used to assess CFTR turnover downstream of its targeting to plasma membrane. (A) Cdc42 depletion increases stability of the PM-targeted CFTR. The upper diagrams summarize the procedures followed. 48 h after cell transfection by the mentioned siRNA, surface proteins were labelled. Streptavidin capture occurred either subsequently (0), or after a 24 h incubation of cell cultures at 37°C (24). Labelled CFTR protein amounts were assessed in the resulting samples. After densitometric quantification of Western blot images (representative examples in the bottom left panel), (24) to (0) ratios were calculated. The Ctrl RNAi value was used to define 100% of labelled CFTR stability. In the bottom right panel, the histogram shows that the relative PM-CFTR stabilities increase when Cdc42 is depleted. (B) Cdc42 depletion increases remaining PM-CFTR after cycloheximide chase. The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, the PM proteins were labelled and purified (-). Alternatively, the cell cultures were submitted to additional 24 h incubation with 100 μg/mL cycloheximide (+), before biotinylation and capture were performed. The labelled CFTR protein amounts were assessed by densitometric quantification of Western blot bands (representative images in the bottom left panel). In both RNAi conditions, labelled CFTR amounts extracted from the same amount of whole cell lysates appeared higher after 24h CHX chase: this could be explained by stability differences between the various cellular proteins. To overcome this bias, we compared (+) to (-) ratios and the Ctrl RNAi value was used to define 100% of CFTR apparent PM stability. In the bottom right panel, histogram shows that CFTR stability at PM appears higher when Cdc42 is depleted. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.
Mentions: Independently of Cdc42 pathway implication in the early steps of CFTR processing, we tried to determine whether it might be involved in distal trafficking steps of CFTR i.e. downstream of its exit from the Golgi apparatus. To evaluate variations in CFTR stability upon depletion of Cdc42, we used two approaches. On the one hand, we estimated the degradation rate of the CFTR initially expressed at the plasma membrane: PM-CFTR was biotinylated, and the labelled CFTR amount was quantified at the beginning of the experiment and after 24 h of further cell incubation in culture medium at 37°C. The percentage of remaining labelled CFTR after the 24 h chase is an indicator of the stability of initial PM-CFTR; it increased when Cdc42 was depleted compared to control (Fig. 10A). On the other hand, we determined the variations of PM-CFTR amount between a condition in which we labelled surface proteins and extracted them 48 h after siRNA transfection, and a condition in which labelling and extraction were performed after 24 h of supplementary CHX chase. These CHX treatment conditions do not preclude the effects of Cdc42 depletion upon early steps of CFTR processing. Consequently, the difference should result only from an equilibrium between endocytosis, recycling and degradation. Cdc42 depletion then elicited an increase of remaining PM-CFTR, in comparison to control (Fig. 10B). Both results are consistent with the hypothesis of improved CFTR stability in PM when the Cdc42 pathway is impaired.

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