Ferru-Clément Romain, Fresquet Fleur, Norez Caroline, Métayé Thierry, Becq Frédéric, Kitzis Alain, Thoreau Vincent
Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France.
Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France; Service de Génétique, Centre Hospitalier Universitaire de Poitiers, Poitiers, France.
PLoS One. 2015 Mar 13;10(3):e0118943. doi: 10.1371/journal.pone.0118943. eCollection 2015.
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.
囊性纤维化跨膜传导调节因子（CFTR）是一种氯离子通道，在上皮细胞的顶端质膜（PM）上表达。最常见的有害等位基因编码一种存在转运缺陷的突变蛋白，该蛋白会经历内质网相关降解（ERAD），并且在质膜上的稳定性较低。在本研究中，我们调查了Cdc42通路在表达野生型CFTR的人细支气管上皮细胞系（CFBE41o-）中CFTR周转和转运过程中的作用。Cdc42是Rho家族的一种小GTP酶，具有多种细胞功能，其中之一是通过肌动蛋白细胞骨架重塑参与内吞作用和再循环过程。当我们用针对Cdc42的化学抑制剂如ML141和针对下游效应器N-WASP的威斯科他汀处理细胞时，我们观察到CFTR通道活性受到抑制，这与细胞表面CFTR数量的减少以及依赖发动蛋白的CFTR内吞作用的增加相关。CFTR与皮质细胞骨架的锚定可能随后因肌动蛋白紊乱而受损。当我们通过小干扰RNA介导耗尽Cdc42时，肌动蛋白聚合未受影响，但我们观察到CFTR出现了不依赖肌动蛋白的结果。CFTR的总量和质膜CFTR量增加，导致CFTR的激活增强。脉冲追踪实验表明，虽然CFTR降解减缓，但CFTR通过高尔基体的成熟过程不受影响。此外，我们观察到CFTR在质膜中的稳定性增加，其胞吞作用减少。本研究强调了Cdc42通路在CFTR生物合成和转运的多个层面的作用：（i）Cdc42参与CFTR生物合成和加工的第一步；（ii）它通过锚定到皮质肌动蛋白上有助于CFTR在质膜中的稳定性；（iii）它促进CFTR内吞作用，并可能促进其向溶酶体降解的分选。
