Holst Mikkel R, Nejsum Lene N
Department of Clinical Medicine, Aarhus University , Aarhus , Denmark.
Am J Physiol Renal Physiol. 2019 Jul 1;317(1):F124-F132. doi: 10.1152/ajprenal.00150.2019. Epub 2019 May 15.
Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.
水通道蛋白2（AQP2）可根据抗利尿激素血管加压素精确调节尿液浓缩。此外，有研究表明AQP2可促进细胞迁移和上皮形态发生。此前一直缺乏一种能够对AQP2及其磷酸化模拟突变体的表达水平进行时间和定量控制的细胞系统，也没有一种能够表达荧光标记的AQP2用于延时成像的系统。在本研究中，我们构建并验证了一种Flp-In T-REx 麦迪逊-达比犬肾细胞系统，用于对AQP2及其磷酸化模拟突变体进行时间和定量控制。我们证实该系统的表达水平可在时间和数量上受到控制，并且AQP2会响应cAMP升高而转位至质膜，同时这也会诱导S256位点的磷酸化。磷酸化模拟突变体AQP2-S256A和AQP2-S256D的定位与之前描述的一致，分别主要定位于细胞内和质膜。诱导AQP2表达并结合瞬时低表达增强型绿色荧光蛋白标记的AQP2，能够实现其在不聚集的情况下表达，并在cAMP升高时正确转位。有趣的是，延时成像显示含有AQP2的形成微管的内体，并且在cAMP升高30分钟后微管形成显著减少。磷酸化模拟突变体AQP2-S256A和AQP2-S256D也呈现出类似情况，含有AQP2-S256A的内体形成微管，而含有AQP2-S256D的内体则不形成微管。因此，该细胞系统能够进行多种基于细胞的分析，有助于更深入地了解AQP2的调控机制及其对细胞迁移和上皮形态发生的影响。
