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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

Pharmacological treatments decrease actin polymerization.(A) Cell shape, polarity and peripheral actin cytoskeleton pattern appear preserved upon 10 μM wiskostatin or 10 μM L141 treatments compared to vehicle treatment, whereas actin is scattered throughout the cytoplasm with 100 μM wiskostatin. Cells were grown on Transwell permeable support until a monolayer was established. After ML141 or wiskostatin treatments at the indicated concentrations, ZO-1 proteins were immunostained (green) and actin was tracked using phalloidin-TRITC (red). TO-PRO-3 was used as a cell nucleus marker (blue). Transversal section images were acquired with a confocal microscope. Scale bars represent 20 μm. (B) Pharmacological inhibitions of Cdc42 pathway reduce fibrillar (F-) actin content. Cells were incubated with 10 μM ML141 or 10 μM wiskostatin, and 10 μM cytochalasin B was used as an F-actin polymerization inhibition positive control treatment, whereas 1% DMSO (v/v) was used as negative control treatment. Actin-bound phalloidin-FITC was methanol-extracted and fluorescence measurements were performed. Results were normalized to protein amount and the relative F-actin contents are expressed as the percentage of DMSO control condition in histograms. Data represent means ± SEM of 3 independent experiments, each performed in triplicate. **: p<0.01, *: p<0.05.
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pone.0118943.g005: Pharmacological treatments decrease actin polymerization.(A) Cell shape, polarity and peripheral actin cytoskeleton pattern appear preserved upon 10 μM wiskostatin or 10 μM L141 treatments compared to vehicle treatment, whereas actin is scattered throughout the cytoplasm with 100 μM wiskostatin. Cells were grown on Transwell permeable support until a monolayer was established. After ML141 or wiskostatin treatments at the indicated concentrations, ZO-1 proteins were immunostained (green) and actin was tracked using phalloidin-TRITC (red). TO-PRO-3 was used as a cell nucleus marker (blue). Transversal section images were acquired with a confocal microscope. Scale bars represent 20 μm. (B) Pharmacological inhibitions of Cdc42 pathway reduce fibrillar (F-) actin content. Cells were incubated with 10 μM ML141 or 10 μM wiskostatin, and 10 μM cytochalasin B was used as an F-actin polymerization inhibition positive control treatment, whereas 1% DMSO (v/v) was used as negative control treatment. Actin-bound phalloidin-FITC was methanol-extracted and fluorescence measurements were performed. Results were normalized to protein amount and the relative F-actin contents are expressed as the percentage of DMSO control condition in histograms. Data represent means ± SEM of 3 independent experiments, each performed in triplicate. **: p<0.01, *: p<0.05.

Mentions: As Cdc42 pathway is one of the regulation mechanisms involved in actin cytoskeleton remodelling, we checked the impact of previously applied treatments upon actin cytoskeleton integrity in CFBE-wtCFTR cells. First, we visualized the consequences of ML141 and wiskostatin treatments upon actin cytoskeleton organization by means of fluorescent confocal imagery, on a monolayer of polarized CFBE-wtCFTR cells grown on a permeable support (Fig. 5A). As depicted in transversal sections, ZO-1 immunostaining of tight junctions was not modified by the treatments. This further suggests that the polarity of the cells was not disrupted. In control condition (i.e. vehicle treatment), phalloidin staining highlighted cortical actin cytoskeleton above the apical and basolateral membranes. Upon 100 μM wiskostatin treatment, actin staining appeared blurry and scattered throughout the cell cytosol when compared to control. This severe disturbance of cytoskeleton organization is in accordance with the high level of cell mortality that we observed with the above-mentioned wiskostatin concentration. Conversely, in the presence of 10 μM wiskostatin or 10 μM ML141, the actin patterns within the cells appeared similar to the ones produced under control conditions.


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)

Pharmacological treatments decrease actin polymerization.(A) Cell shape, polarity and peripheral actin cytoskeleton pattern appear preserved upon 10 μM wiskostatin or 10 μM L141 treatments compared to vehicle treatment, whereas actin is scattered throughout the cytoplasm with 100 μM wiskostatin. Cells were grown on Transwell permeable support until a monolayer was established. After ML141 or wiskostatin treatments at the indicated concentrations, ZO-1 proteins were immunostained (green) and actin was tracked using phalloidin-TRITC (red). TO-PRO-3 was used as a cell nucleus marker (blue). Transversal section images were acquired with a confocal microscope. Scale bars represent 20 μm. (B) Pharmacological inhibitions of Cdc42 pathway reduce fibrillar (F-) actin content. Cells were incubated with 10 μM ML141 or 10 μM wiskostatin, and 10 μM cytochalasin B was used as an F-actin polymerization inhibition positive control treatment, whereas 1% DMSO (v/v) was used as negative control treatment. Actin-bound phalloidin-FITC was methanol-extracted and fluorescence measurements were performed. Results were normalized to protein amount and the relative F-actin contents are expressed as the percentage of DMSO control condition in histograms. Data represent means ± SEM of 3 independent experiments, each performed in triplicate. **: p<0.01, *: p<0.05.
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Related In: Results  -  Collection

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pone.0118943.g005: Pharmacological treatments decrease actin polymerization.(A) Cell shape, polarity and peripheral actin cytoskeleton pattern appear preserved upon 10 μM wiskostatin or 10 μM L141 treatments compared to vehicle treatment, whereas actin is scattered throughout the cytoplasm with 100 μM wiskostatin. Cells were grown on Transwell permeable support until a monolayer was established. After ML141 or wiskostatin treatments at the indicated concentrations, ZO-1 proteins were immunostained (green) and actin was tracked using phalloidin-TRITC (red). TO-PRO-3 was used as a cell nucleus marker (blue). Transversal section images were acquired with a confocal microscope. Scale bars represent 20 μm. (B) Pharmacological inhibitions of Cdc42 pathway reduce fibrillar (F-) actin content. Cells were incubated with 10 μM ML141 or 10 μM wiskostatin, and 10 μM cytochalasin B was used as an F-actin polymerization inhibition positive control treatment, whereas 1% DMSO (v/v) was used as negative control treatment. Actin-bound phalloidin-FITC was methanol-extracted and fluorescence measurements were performed. Results were normalized to protein amount and the relative F-actin contents are expressed as the percentage of DMSO control condition in histograms. Data represent means ± SEM of 3 independent experiments, each performed in triplicate. **: p<0.01, *: p<0.05.
Mentions: As Cdc42 pathway is one of the regulation mechanisms involved in actin cytoskeleton remodelling, we checked the impact of previously applied treatments upon actin cytoskeleton integrity in CFBE-wtCFTR cells. First, we visualized the consequences of ML141 and wiskostatin treatments upon actin cytoskeleton organization by means of fluorescent confocal imagery, on a monolayer of polarized CFBE-wtCFTR cells grown on a permeable support (Fig. 5A). As depicted in transversal sections, ZO-1 immunostaining of tight junctions was not modified by the treatments. This further suggests that the polarity of the cells was not disrupted. In control condition (i.e. vehicle treatment), phalloidin staining highlighted cortical actin cytoskeleton above the apical and basolateral membranes. Upon 100 μM wiskostatin treatment, actin staining appeared blurry and scattered throughout the cell cytosol when compared to control. This severe disturbance of cytoskeleton organization is in accordance with the high level of cell mortality that we observed with the above-mentioned wiskostatin concentration. Conversely, in the presence of 10 μM wiskostatin or 10 μM ML141, the actin patterns within the cells appeared similar to the ones produced under control conditions.

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