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First published online 27 May 2008
doi: 10.1242/jcs.022210

Journal of Cell Science 121, 2097-2106 (2008)
Published by The Company of Biologists 2008
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AQP2 exocytosis in the renal collecting duct – involvement of SNARE isoforms and the regulatory role of Munc18b

Giuseppe Procino1, Claudia Barbieri1, Grazia Tamma1, Leonarda De Benedictis2, Jeffrey E. Pessin3, Maria Svelto1 and Giovanna Valenti1,*

1 Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, Bari 70126, Italy
2 Department of Pharmacology and Human Physiology, University of Bari Medical School, Bari 70124, Italy
3 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA


Figure 1
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  		Fig. 1. AQP2 redistribution in response to FK in MCD4 cells. AQP2 trafficking was analyzed in polarized MCD4 cells grown on permeable support to full confluence. Cells were stained with antibodies against AQP2 and ZO-1 (both in green) and counterstained with WGA-TRITC (red) to visualize the plasma membrane. Confocal scans were taken in both the xy and yz plane. (A) In resting cells, AQP2 was mainly detectable in sub-apical vesicles. (B) After FK treatment, AQP2 localized to the apical membrane with a substantial co-localization with the plasma membrane marker. (C) MCD4 cells cultured on permeable support to full confluence display a positive staining for the tight junction marker ZO-1, indicating a full polarization.

 

Figure 2
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  		Fig. 2. Subcellular distribution of SNARE and SNARE-regulator Munc18 proteins in MCD4 cells. Subcellular localization of SNARE and Munc18 isoforms was analyzed in MCD4 cells by CLSM and western blotting. (A-G) Polarized MCD4 cells were fixed and stained with antibodies against VAMP2, VAMP3, Stx3, Stx4, SNAP23, Munc18b and Munc18c (all in green) and counterstained with WGA-TRITC (red). Confocal scans were taken in the yz plane to analyze the apical vs basolateral distribution of each protein. (A'-G') MCD4 total homogenates, plasma membrane (P.M.), intracellular vesicles (I.V.) and mouse kidney homogenates were separated by NuPAGE electrophoresis and analyzed by western blotting using the same antibodies as above. VAMP2 and VAMP3 were found almost equally distributed between plasma membranes and intracellular vesicles, whereas Stx3, Stx4 localized at the apical and basolateral membrane respectively. SNAP23 was abundantly expressed at both the plasma membrane and intracellularly. Munc18b and Munc18c were both associated with the apical plasma membrane. Same results were obtained in at least three independent experiments.

 

Figure 3
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  		Fig. 3. Immunoisolated AQP2-expressing vesicles contain VAMP2 and VAMP3. Immunoblotting of MCD4 lysate, total intracellular vesicles (200k vesicles), immunoisolated AQP2-bearing vesicles (I° and II° Ab) and negative control (II° Ab alone) probed with antibodies against AQP2, VAMP2 and VAMP3. AQP2, VAMP2 and VAMP3 were expressed and enriched in the immunoisolated vesicles. Results are representative of three independent experiments.

 

Figure 4
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  		Fig. 4. Apical SNARE complex in MCD4 cells. Anti-Stx3 antibodies or normal rabbit IgG were used for co-immunoprecipitation studies from MCD4 cell lysates. (A) Immunocomplexes were resolved by NuPAGE and immunoblotted with antibodies against Stx3, VAMP2, VAMP3, SNAP23, Munc18b and Munc18c. Anti-Stx3 antibody was very efficient in immunoprecipitating Stx3 protein from MCD4 lysates and no bands were seen in samples incubated with normal rabbit serum (Rb IgG). Immunoblotting performed on the same samples revealed that equal amounts of VAMP2 and VAMP3 co-precipitated with Stx3 in both resting or FK-stimulated MCD4 cells, whereas the association of SNAP 23 or Munc18b with Stx3 was FK-dependent. No band for Munc18c was detectable in Stx3 immunoprecipitates, even after long exposure. (B) Densitometric analysis of the amount of each protein precipitated from contol- (ctr; set as 100%) or FK-stimulated cells. Experiments were performed three times with comparable results.

 

Figure 5
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  		Fig. 5. Selective SNARE and Munc18 isoforms knockdown (K.D.). (A) Endogenous VAMP2, VAMP3, Stx3, Stx4, SNAP23, Munc18b and Munc18c were selectively knocked down using specific siRNAs. Cells were transfected with siRNA specifically targeting each protein (indicated on top of each lane) or with control siRNA (Mock), lysed and analyzed at 48 hours after transfection. 15 µg of each cell lysate were probed with all antibodies against SNARE and Munc18 isoforms (indicated on the left) to confirm the specificity of the knockdown. Protein expression levels were decreased by at least 75%. Actin was used as a loading control.

 

Figure 6
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  		Fig. 6. Effect of SNARE and Munc18 knockdown on AQP2 translocation to the apical surface. Cells silenced for SNARE (left panel) or Munc18 (right panel) isoforms, were subjected to apical surface biotinylation in resting condition (ctr) of after FK stimulation (FK). The amount of biotinylated AQP2 was estimated by western blotting (biot. AQP2). siRNA transfection did not affect the total amount of AQP2 detectable in each sample (tot. AQP2). (C) Densitometric analysis of the 29 kDa biotinylated AQP2 band. Results are expressed as mean values ± s.e. of the values obtained in three independent experiments. The amount of AQP2 detected in mock-transfected cells in resting condition was set as 100%.

 

Figure 7
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  		Fig. 7. CLSM analysis of AQP2 translocation in MCD4 cells silenced for Munc18b and Munc18c. AQP2 trafficking was analyzed in polarized MCD4 mock-transfected or after transfection with siRNA targeting Munc18b ad Munc18c. Cells were stained with antibodies against AQP2 and counterstained with WGA-TRITC (red). Confocal scans were taken in the yz plane. In mock transfected and in Munc18c-silenced cells AQP2 was found mainly associated with subapical vesicles and translocated towards the apical membrane after FK treatment. Munc18b knockdown produced a clear accumulation of AQP2 at the apical plasma membrane, even without FK stimulation.

 

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