Abstract：
The shuttling of renal collecting duct aquaporin-2 (AQP2) between intracellular vesicles and the apical plasma membrane is paramount for regulation of renal water reabsorption. The binding of the circulating antidiuretic hormone arginine vasopressin (AVP) to the basolateral AVP receptor increases intracellular cAMP, which ultimately leads to AQP2 plasma membrane accumulation via a dual effect on AQP2 vesicle fusion with the apical plasma membrane and reduced AQP2 endocytosis. This AQP2 plasma membrane accumulation increases water reabsorption and consequently urine concentration. Conventional fluorescent microscopy provides a lateral resolution of ∼250 nm, which is insufficient to resolve the AQP2-containing endosomes/vesicles. Therefore, detailed information regarding the AQP2 vesicular population is still lacking. Newly established 4.5x Expansion Microscopy (ExM) can increase resolution to 60-70 nm. Using 4.5x ExM, we detected AQP2 vesicles/endosomes as small as 79 nm considering an average expansion factor of 4.3 for endosomes. Using different markers of the endosomal system provided detailed information of the cellular AQP2 itinerary upon changes in endogenous cAMP levels. Before cAMP elevation, AQP2 colocalized with early and recycling, but not late endosomes. Forskolin-induced cAMP increase was characterized by AQP2 insertion into the plasma membrane and AQP2 withdrawal from large perinuclear endosomes as well as some localization to lysosomal compartments. Forskolin washout promoted AQP2 endocytosis where AQP2 localized to not only early and recycling endosomes but also late endosomes and lysosomes indicating increased AQP2 degradation. Thus, our results show that 4.5 ExM is an attractive approach to obtain detailed information regarding AQP2 shuttling.NEW & NOTEWORTHY Renal aquaporin-2 (AQP2) imaged by expansion microscopy provides unprecedented 3-D information regarding the AQP2 itinerary in response to changes in cellular cAMP.
摘要：
肾集合管水通道蛋白2（AQP2）在细胞内囊泡和顶端质膜之间的穿梭对于调节肾水的重吸收至关重要。循环抗利尿激素精氨酸加压素（AVP）的结合，基底外侧AVP受体增加细胞内cAMP，最终通过对AQP2囊泡与顶端质膜融合和减少AQP2内吞作用的双重作用导致AQP2质膜积累。这种AQP2质膜的积累增加了水的重吸收，从而增加了尿液浓度。传统的荧光显微镜提供〜250nm的横向分辨率，这不足以解析含有AQP2的内体/囊泡。因此,关于AQP2囊泡群体的详细信息仍然缺乏.新建立的4.5x扩展显微镜（ExM）可以将分辨率提高到60-70nm。使用4.5xExM，我们检测到AQP2囊泡/内体小至79nm,考虑到内体的平均扩张因子为4.3.使用内体系统的不同标记物，可根据内源性cAMP水平的变化提供细胞AQP2路线的详细信息。在cAMP升高之前,AQP2与早期和回收共同本地化，但不是晚期内体。Forskolin诱导的cAMP增加的特征是AQP2插入质膜，AQP2从大的核周内体中退出，以及一些定位到溶酶体区室。Forskolin洗脱促进了AQP2的内吞作用，其中AQP2定位到早期和再循环内体，也定位到晚期内体和溶酶体，表明AQP2降解增加。因此，我们的结果表明，4.5ExM是获得有关AQP2穿梭的详细信息的有吸引力的方法。
