Cytoskeletal and motor proteins facilitate trafficking of AQP1-containing vesicles in cholangiocytes.
BACKGROUND
We have previously showed that: (i) cholangiocytes contain AQP1 (aquaporin 1) water channels sequestered in intracellular vesicles; and (ii) upon stimulation with choleretic agonists such as secretin or dibutyryl-cAMP (dbcAMP), the AQP1 vesicles move via microtubules to the apical cholangiocyte membrane to facilitate osmotically driven, passive water movement (i.e. ductal bile secretion). The aim of the present study was to determine which proteins and mechanisms regulate AQP1 trafficking in cholangiocytes.


RESULTS
Using polarized cultured NMCs (normal mouse cholangiocytes) or NRCs (normal rat cholangiocytes) and affinity-purified antibodies, we performed immunofluorescent confocal microscopy on fixed cells or immunoblotting on cell lysates for actin, tubulin, kinesin and dynein, proteins known to regulate intracellular vesicle trafficking. By immunostaining, the appropriate orientation of the actin (i.e. sub-apical) and tubulin (i.e. generalized) cytoskeleton was apparent; kinesin and dynein displayed a homogeneous punctate distribution. Immunoblotting showed kinesin and dynein to be present in both cholangiocyte lysates and in isolated AQP1-containing vesicles. We utilized real-time fluorescence confocal microscopy of NMCs transfected with a GFP (green fluorescent protein)-AQP1 fusion construct in the presence and absence of dbcAMP.


CONCLUSIONS
Our results provide additional insights into the potential molecular mechanisms of ductal bile secretion.
Tietz
Pamela S
PS
Center for Basic Research in Digestive Diseases, Department of Internal Medicine, Mayo Medical School, Clinic and Foundation, Rochester, MN 55905, USA.
McNiven
Mark A
MA
Splinter
Patrick L
PL
Huang
Bing Q
BQ
Larusso
Nicholas F
NF
D000818:Animals; D051398:Aquaporin 1 / Q000378:metabolism*; D001652:Bile Ducts / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D004398:Dyneins / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008870:Microtubules / Q000502:physiology; D051381:Rats; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism
C496022:Aqp1 protein, rat; D003598:Cytoskeletal Proteins; D051398:Aquaporin 1; D004398:Dyneins; D016547:Kinesins