Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel

Abstract

targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel. Am J Physiol Cell Physiol 304: C38-C48, 2013. First published September 26, 2012 doi:10.1152/ajpcell.00109.2012.-The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

“…Mal2 is a protein with four membrane-spanning helices that is involved in trafficking of apical membrane proteins via transcytosis (57), in which the apical membrane proteins are first delivered to the basolateral membrane before they are endocytosed and targeted to the apical membrane. AQP2 has been reported in the basolateral membrane in native collecting duct cells and cultured epithelial cells (58,59). It is possible that Mal2 may be involved in vasopressin-induced apical AQP2 trafficking via the transcytosis mechanism (59).…”

“…AQP2 has been reported in the basolateral membrane in native collecting duct cells and cultured epithelial cells (58,59). It is possible that Mal2 may be involved in vasopressin-induced apical AQP2 trafficking via the transcytosis mechanism (59).…”

Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin

“…Culturing of mpkCCD-C14 collecting duct-derived cells was done as described previously (21,22). AQP2-MDCK cells were cultured in DMEM supplemented with 10% fetal bovine serum and G418 (500 g/ml) (23). For transient expression, cells were transfected with the appropriate cDNAs using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions unless otherwise mentioned.…”

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

“…Second is that following the cells in real time allows researchers to observe multiple aspects of the phagocytic process simultaneous and so effects on migration, sensing and binding particles might be easily discerned here, whereas it might be overlooked with population based methods. Additionally, synchronization of the initiation of phagocytosis, often performed by placing cells at 4 °C, which might perturb other trafficking events due to cold shock 40,41 , is not necessary as time zero can be discerned directly. Indeed, when performing live microscopy, this 4 °C synchronization technique is not recommended if capturing periods of time close to the initiation of phagocytosis as the condensation that forms on the bottom of the dish during the temperature shift can mix with the objective immersion oil and temperature differences between the warming dish and microscope components can cause focus shifts.…”