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Journal of cell science

Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells.


PMID 10574703

Abstract

The bile canalicular membrane contains several ATP-dependent transporters that are involved in biliary secretion. Canalicular transporters are synthesized in ER, modified in Golgi and transported to the apical plasma membrane. However, the route and regulation of intracellular trafficking of ATP-dependent transporters have not been elucidated. In the present study, we generated a translational fusion of mdr1 and green fluorescent protein and investigated bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells, a polarized liver derived cell line. Similar to hepatocytes, WIF-B cells secrete bile acids and organic cations (i.e. rhodamine-123) into the bile canaliculi. Canalicular secretion of fluorescein isothiocyanate-glycocholate was stimulated by taurocholate and a decapeptide activator of phosphoinositide 3-kinase and was decreased by wortmannin. WIF-B9 cells were transiently and stably transfected with a mdr1-GFP construct. Fluorescence was observed in the canalicular membrane, pericanalicular punctate structures and Golgi region. Time lapse microscopy revealed that mdr1-GFP is transferred from Golgi as tubular vesicular structures the majority of which traveled directly to the canalicular membrane. Recycling between the canalicular membrane and subapical region was also observed. At no time was mdr1-GFP detected in the basolateral plasma membrane. At 15 degrees C, mdr1-GFP accumulated in Golgi; after a shift to 37 degrees C, fluorescence moved directly to the canalicular membrane. This process was enhanced by taurocholate and blocked by wortmannin. In these studies as well, no mdr1-GFP fluorescence was observed at any time in basolateral membranes or other intracellular organelles. In conclusion, in WIF-B cells, there is a direct route from Golgi to the canalicular membrane for trafficking of mdr1, a bile canalicular ATP-dependent transporter of organic cations. As in normal hepatocytes, phosphoinositide 3-kinase regulates bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells. WIF-B cells stably transfected with mdr1-GFP provide an important model in which to study trafficking and regulation of canalicular transporters. Movies available on-line: http://www.healthsci.tufts.edu/LABS/IMArias++ + /Sai_F9.html

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