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Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.

Rice WL, Li W, Mamuya F, McKee M, Păunescu TG, Lu HA - PLoS ONE (2015)

Bottom Line: Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking.Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2.The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Program in Membrane Biology, Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, United States of America.

ABSTRACT
In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

No MeSH data available.


Related in: MedlinePlus

Constitutive recycling of AQP2 is detected in the basolateral domain of the MDCK-AQP2 cyst.In the MDCK-AQP2 cyst, blockade of endocytosis by addition of mβCD for 20 minutes led to predominant enrichment of AQP2 in the basolateral domain. (A) Immunofluorescence staining reveals basolateral accumulation of total AQP2 with mβCD treatment. (B) In transmission electron micrographs, some 15nm gold AQP2-labeled particles are found in the apical/subapical domain (left panel) but they are more predominantly presented on the basolateral membranes of two neighboring cells. Bar = 500 nm
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pone.0131719.g004: Constitutive recycling of AQP2 is detected in the basolateral domain of the MDCK-AQP2 cyst.In the MDCK-AQP2 cyst, blockade of endocytosis by addition of mβCD for 20 minutes led to predominant enrichment of AQP2 in the basolateral domain. (A) Immunofluorescence staining reveals basolateral accumulation of total AQP2 with mβCD treatment. (B) In transmission electron micrographs, some 15nm gold AQP2-labeled particles are found in the apical/subapical domain (left panel) but they are more predominantly presented on the basolateral membranes of two neighboring cells. Bar = 500 nm

Mentions: Previously, using a less well-polarized cell culture model [41], we have shown that, in addition to regulated trafficking, AQP2 is also constitutively recycling inside cells. Acute or chronic blockade of endocytosis by mβCD in these cells causes membrane accumulation of AQP2 by inhibiting endocytosis. In the 3D epithelial model, treatment with mβCD for 20 minutes results in accumulation of AQP2 in the basolateral membrane, without detectable accumulation of total AQP2 signal in the apical membrane by immunofluorescence staining (Fig 4A). Increased accumulation of AQP2 in the basolateral membrane after treatment with mβ-CD is further revealed by immunogold electron microscopy (Fig 4B). This data suggests that active insertion and endocytotic removal of AQP2 occurs in the basolateral domain of polarized MDCK cells grown in the 3D culture which is consistent with our observation of active clathrin coated vesicles in the basolateral domain of the cyst and with our recent observations in MDCK cells grown on filters [12]. The lack of observed apical membrane accumulation of AQP2 in mβCD treated cysts may be due to the fact that mβCD first encounters the basolateral membranes of the cysts followed later by the organelle and apical membrane dmains. Thus AQP2 is probably sequestered in the basolateral domain (and is unavailable for further trafficking) while apical endocytosis is still ongoing to deplete the apical domain of the remaining AQP2.


Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.

Rice WL, Li W, Mamuya F, McKee M, Păunescu TG, Lu HA - PLoS ONE (2015)

Constitutive recycling of AQP2 is detected in the basolateral domain of the MDCK-AQP2 cyst.In the MDCK-AQP2 cyst, blockade of endocytosis by addition of mβCD for 20 minutes led to predominant enrichment of AQP2 in the basolateral domain. (A) Immunofluorescence staining reveals basolateral accumulation of total AQP2 with mβCD treatment. (B) In transmission electron micrographs, some 15nm gold AQP2-labeled particles are found in the apical/subapical domain (left panel) but they are more predominantly presented on the basolateral membranes of two neighboring cells. Bar = 500 nm
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493001&req=5

pone.0131719.g004: Constitutive recycling of AQP2 is detected in the basolateral domain of the MDCK-AQP2 cyst.In the MDCK-AQP2 cyst, blockade of endocytosis by addition of mβCD for 20 minutes led to predominant enrichment of AQP2 in the basolateral domain. (A) Immunofluorescence staining reveals basolateral accumulation of total AQP2 with mβCD treatment. (B) In transmission electron micrographs, some 15nm gold AQP2-labeled particles are found in the apical/subapical domain (left panel) but they are more predominantly presented on the basolateral membranes of two neighboring cells. Bar = 500 nm
Mentions: Previously, using a less well-polarized cell culture model [41], we have shown that, in addition to regulated trafficking, AQP2 is also constitutively recycling inside cells. Acute or chronic blockade of endocytosis by mβCD in these cells causes membrane accumulation of AQP2 by inhibiting endocytosis. In the 3D epithelial model, treatment with mβCD for 20 minutes results in accumulation of AQP2 in the basolateral membrane, without detectable accumulation of total AQP2 signal in the apical membrane by immunofluorescence staining (Fig 4A). Increased accumulation of AQP2 in the basolateral membrane after treatment with mβ-CD is further revealed by immunogold electron microscopy (Fig 4B). This data suggests that active insertion and endocytotic removal of AQP2 occurs in the basolateral domain of polarized MDCK cells grown in the 3D culture which is consistent with our observation of active clathrin coated vesicles in the basolateral domain of the cyst and with our recent observations in MDCK cells grown on filters [12]. The lack of observed apical membrane accumulation of AQP2 in mβCD treated cysts may be due to the fact that mβCD first encounters the basolateral membranes of the cysts followed later by the organelle and apical membrane dmains. Thus AQP2 is probably sequestered in the basolateral domain (and is unavailable for further trafficking) while apical endocytosis is still ongoing to deplete the apical domain of the remaining AQP2.

Bottom Line: Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking.Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2.The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Program in Membrane Biology, Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, United States of America.

ABSTRACT
In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

No MeSH data available.


Related in: MedlinePlus