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Characterization of Aquaporin‐2 Containing Vesicle Trafficking in mpkCCD Cells
Author(s) -
Yip KayPong,
Amin Jahanshah,
Cha Byeong
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.809.9
Subject(s) - vesicle , forskolin , aquaporin 2 , microbiology and biotechnology , chemistry , organelle , biophysics , biology , biochemistry , water channel , membrane , in vitro , mechanical engineering , engineering , inlet
Vasopressin triggers the trafficking of aquaporin‐2 (AQP2) from cytoplasm to apical membrane in the principal cells of collecting duct. To gain insight into the trafficking process of AQP2, the properties of AQP2‐containing vesicles were characterized in mpkCCD cells. Cells were transfected with mouse AQP2 tagged with photoconvertible green fluorescent protein (mEos‐AQP2). The mean diameter of AQP2 vesicles is 0.84 ± 0.029 μm (n=27). These vesicles displayed trafficking at 0.17 ± 0.015 μm/s (n=25), regardless of forskolin stimulation. Their movement seemed random, but the net direction of trafficking was biased toward the minus end of microtubule in the presence of forskolin. Partial photoconversion of mEos‐AQP2 in a single vesicle resulted in both green and red fluorescent AQP2 in the same vesicle. The intensity of both AQP2 fluorescence signals remained relatively constant, suggesting that the AQP2‐containing vesicle does not exchange the composition during trafficking. In baseline condition, most of the mEos‐AQP2 fluorescence was localized in acidic organelles, which was evidenced by the Forster Resonance Energy Transfer (FRET) between mEos‐AQP2 and Lysotracker ( fluorescent marker of acidic organelles). Blocking of vacuolar H + ‐ATPase with Bafilomycin A abolished FRET between mEos‐AQP2 and Lysotracker, decreased the trafficking speed of AQP2 vesicles to 0.07 ± 0.009 μm/s (n=25), and inhibited forskolin stimulated apical accumulation of mEos‐AQP2. These observations suggest that acidification of AQP2‐containing vesicle is necessary for cAMP‐dependent AQP2 trafficking.