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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

Analysis of CFTR maturation and turnover by metabolic labelling.CFBE-wtCFTR cells were transfected with negative control or Cdc42 siRNA and cultured 48 h prior to pulse-chase experiments. Cells were pulse-labelled for 15 min with 100 μCi/mL of [35S]methionine and [35S]cysteine mix and then chased for 0, 0.5, 1, 2, and 4 h. CFTR was then immunoprecipited and subjected to SDS-PAGE. Bands corresponding to core-glycosylated (band B) and fully-glycosylated (band C) CFTR were quantified by densitometry for analysis. (A) Representative gels are shown. (B) Maturation of CFTR is evaluated as the ratio of band C detected at a given time relative to total CFTR (bands B+C). (C) CFTR turnover is displayed as the relative total CFTR amount (bands B+C) along the chase. Total CFTR amount is assigned a value of 100 in arbitrary units at the beginning of the chase (0 h), when the cells are transfected with negative control siRNA. (D) The rate of CFTR disappearance is estimated as the natural logarithm of the amount of CFTR (bands B+C) at a given time of chase relative to its amount at the beginning of the experiment (B0+C0). Displayed lines are the linear regressions to the data. Symbol and error bars are means ± SEM of the values at each point. The numbers of independent experiments used to build the graphs are indicated on the figures.
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pone.0118943.g009: Analysis of CFTR maturation and turnover by metabolic labelling.CFBE-wtCFTR cells were transfected with negative control or Cdc42 siRNA and cultured 48 h prior to pulse-chase experiments. Cells were pulse-labelled for 15 min with 100 μCi/mL of [35S]methionine and [35S]cysteine mix and then chased for 0, 0.5, 1, 2, and 4 h. CFTR was then immunoprecipited and subjected to SDS-PAGE. Bands corresponding to core-glycosylated (band B) and fully-glycosylated (band C) CFTR were quantified by densitometry for analysis. (A) Representative gels are shown. (B) Maturation of CFTR is evaluated as the ratio of band C detected at a given time relative to total CFTR (bands B+C). (C) CFTR turnover is displayed as the relative total CFTR amount (bands B+C) along the chase. Total CFTR amount is assigned a value of 100 in arbitrary units at the beginning of the chase (0 h), when the cells are transfected with negative control siRNA. (D) The rate of CFTR disappearance is estimated as the natural logarithm of the amount of CFTR (bands B+C) at a given time of chase relative to its amount at the beginning of the experiment (B0+C0). Displayed lines are the linear regressions to the data. Symbol and error bars are means ± SEM of the values at each point. The numbers of independent experiments used to build the graphs are indicated on the figures.

Mentions: To investigate the consequences of Cdc42 depletion upon CFTR processing and turnover, we performed pulse-chase experiments followed by CFTR immunoprecipitation (Fig. 9A). The time-course of CFTR glycosylation was estimated as the percentage of total CFTR that is fully-glycosylated (band C) along the chase (Fig. 9B). When comparing the curves corresponding to data obtained with negative control or Cdc42 RNAi, similar patterns were observed. The maturation statuses of CFTR at the end of the chase were found to be non-significantly different: C/(B+C) = 94.2±2.1% with Cdc42 depletion versus 89.4±1.5% in control conditions. In fact, Cdc42 did not appear to be involved in CFTR glycosylation steps occurring along the Golgi apparatus and should not be implicated in CFTR trafficking throughout this compartment.


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)

Analysis of CFTR maturation and turnover by metabolic labelling.CFBE-wtCFTR cells were transfected with negative control or Cdc42 siRNA and cultured 48 h prior to pulse-chase experiments. Cells were pulse-labelled for 15 min with 100 μCi/mL of [35S]methionine and [35S]cysteine mix and then chased for 0, 0.5, 1, 2, and 4 h. CFTR was then immunoprecipited and subjected to SDS-PAGE. Bands corresponding to core-glycosylated (band B) and fully-glycosylated (band C) CFTR were quantified by densitometry for analysis. (A) Representative gels are shown. (B) Maturation of CFTR is evaluated as the ratio of band C detected at a given time relative to total CFTR (bands B+C). (C) CFTR turnover is displayed as the relative total CFTR amount (bands B+C) along the chase. Total CFTR amount is assigned a value of 100 in arbitrary units at the beginning of the chase (0 h), when the cells are transfected with negative control siRNA. (D) The rate of CFTR disappearance is estimated as the natural logarithm of the amount of CFTR (bands B+C) at a given time of chase relative to its amount at the beginning of the experiment (B0+C0). Displayed lines are the linear regressions to the data. Symbol and error bars are means ± SEM of the values at each point. The numbers of independent experiments used to build the graphs are indicated on the figures.
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Related In: Results  -  Collection

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pone.0118943.g009: Analysis of CFTR maturation and turnover by metabolic labelling.CFBE-wtCFTR cells were transfected with negative control or Cdc42 siRNA and cultured 48 h prior to pulse-chase experiments. Cells were pulse-labelled for 15 min with 100 μCi/mL of [35S]methionine and [35S]cysteine mix and then chased for 0, 0.5, 1, 2, and 4 h. CFTR was then immunoprecipited and subjected to SDS-PAGE. Bands corresponding to core-glycosylated (band B) and fully-glycosylated (band C) CFTR were quantified by densitometry for analysis. (A) Representative gels are shown. (B) Maturation of CFTR is evaluated as the ratio of band C detected at a given time relative to total CFTR (bands B+C). (C) CFTR turnover is displayed as the relative total CFTR amount (bands B+C) along the chase. Total CFTR amount is assigned a value of 100 in arbitrary units at the beginning of the chase (0 h), when the cells are transfected with negative control siRNA. (D) The rate of CFTR disappearance is estimated as the natural logarithm of the amount of CFTR (bands B+C) at a given time of chase relative to its amount at the beginning of the experiment (B0+C0). Displayed lines are the linear regressions to the data. Symbol and error bars are means ± SEM of the values at each point. The numbers of independent experiments used to build the graphs are indicated on the figures.
Mentions: To investigate the consequences of Cdc42 depletion upon CFTR processing and turnover, we performed pulse-chase experiments followed by CFTR immunoprecipitation (Fig. 9A). The time-course of CFTR glycosylation was estimated as the percentage of total CFTR that is fully-glycosylated (band C) along the chase (Fig. 9B). When comparing the curves corresponding to data obtained with negative control or Cdc42 RNAi, similar patterns were observed. The maturation statuses of CFTR at the end of the chase were found to be non-significantly different: C/(B+C) = 94.2±2.1% with Cdc42 depletion versus 89.4±1.5% in control conditions. In fact, Cdc42 did not appear to be involved in CFTR glycosylation steps occurring along the Golgi apparatus and should not be implicated in CFTR trafficking throughout this compartment.

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