Key points Aquaporin‐2 (AQP2) is crucial for water homeostasis, and vasopressin (VP) induces AQP2 membrane trafficking by increasing intracellular cAMP, activating PKA and causing phosphorylation of AQP2 at serine 256, 264 and 269 residues and dephosphorylation of serine 261 residue on the AQP2 C‐terminus. It is thought that serine 256 is the master regulator of AQP2 trafficking, and its phosphorylation has to precede the change of phosphorylation state of other serine residues. We found that Src inhibition causes serine 256‐independent AQP2 membrane trafficking and induces phosphorylation of serine 269 independently of serine 256. This targeted phosphorylation of serine 269 is important for Src inhibition‐induced AQP2 membrane accumulation; without serine 269, Src inhibition exerts no effect on AQP2 trafficking. This result helps us better understand the independent pathways that can target different AQP2 residues, and design new strategies to induce or sustain AQP2 membrane expression when VP signalling is defective. Abstract Aquaporin‐2 (AQP2) is essential for water homeostasis. Upon stimulation by vasopressin, AQP2 is phosphorylated at serine 256 (S256), S264 and S269, and dephosphorylated at S261. It is thought that S256 is the master regulator of AQP2 trafficking and membrane accumulation, and that its phosphorylation has to precede phosphorylation of other serine residues. In this study, we found that VP reduces Src kinase phosphorylation: by suppressing Src using the inhibitor dasatinib and siRNA, we could increase AQP2 membrane accumulation in cultured AQP2‐expressing cells and in kidney collecting duct principal cells. Src inhibition increased exocytosis and inhibited clathrin‐mediated endocytosis of AQP2, but exerted its effect in a cAMP, PKA and S256 phosphorylation (pS256)‐independent manner. Despite the lack of S256 phosphorylation, dasatinib increased phosphorylation of S269, even in S256A mutant cells in which S256 phosphorylation cannot occur. To confirm the importance of pS269 in AQP2 re‐distribution, we expressed an AQP2 S269A mutant in LLC‐PK1 cells, and found that dasatinib no longer induced AQP2 membrane accumulation. In conclusion, Src inhibition causes phosphorylation of S269 independently of pS256, and induces AQP2 membrane accumulation by inhibiting clathrin‐mediated endocytosis and increasing exocytosis. We conclude that S269 can be phosphorylated without pS256, and pS269 alone is important for AQP2 apical membrane accumulation under some conditions. These data increase our understanding of the independent pathways that can phosphorylate different residues in the AQP2 C‐terminus, and suggest new strategies to target distinct AQP2 serine residues to induce membrane expression of this water channel when VP signalling is defective.

The use of decellularized skeletal muscle (DSM) as a cell substrate and scaffold for the repair of volumetric muscle loss injuries has shown therapeutic promise. The performance of DSM materials motivated our interest in exploring the chemical and physical properties of this promising material. We suggest that these properties could serve as a blueprint for the development of next generation engineered materials with DSM mimetic properties. In this study, whole human lower limb rectus femoris (n=10) and upper limb supraspinatus muscle samples (n=10) were collected from both male and female tissue donors. Skeletal muscle samples were decellularized and nine property values, capturing key compositional, architectural, and mechanical properties, were measured and statistically analyzed. Mean values for each property were determined across muscle types and genders. Additionally, the influence of muscle type (upper versus lower limb) and donor gender (male versus female) on each of the DSM material properties was examined. The data suggests that DSM materials prepared from lower limb rectus femoris samples have an increased modulus and contain a higher collagen content then upper limb supraspinatus muscles. Specifically, lower limb rectus femoris DSM material modulus and collagen content was approximately twice that of lower limb supraspinatus DSM samples. While muscle type did show some influence on material properties, we did not find significant trends related to gender. The material properties reported herein may be used as a blueprint for the data-driven design of next generation engineered scaffolds with muscle mimetic properties, as well as inputs for computational and physical models of skeletal muscle.

We previously showed that in polarized Madin-Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.Cells 2020, 9, 1057 2 of 18 mechanisms of water channel accumulation in the apical plasma membrane of collecting duct cells have been of particular interest, the regulation of basolateral AQP2 has also been investigated in several studies [22][23][24][25][26]. In the cortical connecting segment, basolateral AQP2 is usually readily detectable [23], and AQP2 has varying levels of basolateral expression in collecting duct principal cells from kidney cortex to medulla, with the most obvious basolateral expression in the inner medulla. In MDCK cells, derived from canine kidney, transfected AQP2 usually accumulates in the apical membrane upon intracellular cAMP elevation. However, AQP2 instead concentrates on the basolateral membrane after forskolin (FK) treatment under hypertonic conditions [26]. Importantly, Sec6, Sec8, and RalA, all components of the exocyst, which targets proteins to the basolateral plasma membrane [27], were associated with AQP2-containing vesicles upon mass spectrometry analysis [28]. Furthermore, we showed that AQP2 is necessary for cell migration and tubule morphogenesis through its RGD-regulated interaction with basolateral integrin-β1 [29]. These previous findings all pointed to the existence of an AQP2 basolateral targeting pathway, and indeed we demonstrated the existence of a transcytotic pathway for AQP2 in MDCK cells by blocking basolateral AQP2 during its transit through this membrane domain using low temperature [30]. After initial basolateral targeting, AQP2 is retrieved into Rab5-positive vesicles, then undertakes microtubule-dependent transcytosis to apical recycling vesicles [30]. This novel trafficking concept is supported by an apical plasma membrane proteomics study using mpkCCD cel...