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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

Impact of Cdc42 or N-WASP depletions upon CFTR amount and internalization.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h. We then used biotinylation experiments to evaluate CFTR amount at the cell surface and CFTR internalization, as described in Fig. 2 legend. (A) Representative Western blots and (B-D) histograms summarizing the data are presented. Following Cdc42 depletion, (B) total CFTR amount in whole cell lysate, (C) as well as CFTR amount at the plasma membrane, had increased compared to negative control RNAi conditions. (D) Internalized CFTR amount had decreased following Cdc42 depletion. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01.
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pone.0118943.g008: Impact of Cdc42 or N-WASP depletions upon CFTR amount and internalization.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h. We then used biotinylation experiments to evaluate CFTR amount at the cell surface and CFTR internalization, as described in Fig. 2 legend. (A) Representative Western blots and (B-D) histograms summarizing the data are presented. Following Cdc42 depletion, (B) total CFTR amount in whole cell lysate, (C) as well as CFTR amount at the plasma membrane, had increased compared to negative control RNAi conditions. (D) Internalized CFTR amount had decreased following Cdc42 depletion. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01.

Mentions: We then confronted these functional data with the quantification of CFTR protein amount: N-WASP depletion resulted in only a small decrease, while Cdc42 indeed elicited a 37% increase of total CFTR amount (Fig. 8A and 8B). Nevertheless, when protein synthesis was inhibited by cycloheximide for the last 24 h of the Cdc42 depletion, we observed no difference of total CFTR amount, compared to control (S4 Fig.). This suggests that Cdc42 may be involved in the stability of newly synthesized CFTR protein in early steps of its processing. We checked by real-time RT-PCR to make sure that neither of these depletions had an impact upon CFTR mRNA level (not shown), so the consequences may lie in post-transcriptional events such as translation, folding control, maturation or degradation of the protein. However, Western blots exhibited no apparent alteration of maturation from band B to band C, a process that terminates within the Golgi apparatus and appears unaffected.


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)

Impact of Cdc42 or N-WASP depletions upon CFTR amount and internalization.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h. We then used biotinylation experiments to evaluate CFTR amount at the cell surface and CFTR internalization, as described in Fig. 2 legend. (A) Representative Western blots and (B-D) histograms summarizing the data are presented. Following Cdc42 depletion, (B) total CFTR amount in whole cell lysate, (C) as well as CFTR amount at the plasma membrane, had increased compared to negative control RNAi conditions. (D) Internalized CFTR amount had decreased following Cdc42 depletion. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118943.g008: Impact of Cdc42 or N-WASP depletions upon CFTR amount and internalization.RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h. We then used biotinylation experiments to evaluate CFTR amount at the cell surface and CFTR internalization, as described in Fig. 2 legend. (A) Representative Western blots and (B-D) histograms summarizing the data are presented. Following Cdc42 depletion, (B) total CFTR amount in whole cell lysate, (C) as well as CFTR amount at the plasma membrane, had increased compared to negative control RNAi conditions. (D) Internalized CFTR amount had decreased following Cdc42 depletion. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01.
Mentions: We then confronted these functional data with the quantification of CFTR protein amount: N-WASP depletion resulted in only a small decrease, while Cdc42 indeed elicited a 37% increase of total CFTR amount (Fig. 8A and 8B). Nevertheless, when protein synthesis was inhibited by cycloheximide for the last 24 h of the Cdc42 depletion, we observed no difference of total CFTR amount, compared to control (S4 Fig.). This suggests that Cdc42 may be involved in the stability of newly synthesized CFTR protein in early steps of its processing. We checked by real-time RT-PCR to make sure that neither of these depletions had an impact upon CFTR mRNA level (not shown), so the consequences may lie in post-transcriptional events such as translation, folding control, maturation or degradation of the protein. However, Western blots exhibited no apparent alteration of maturation from band B to band C, a process that terminates within the Golgi apparatus and appears unaffected.

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