Sai Y, Nies A T, Arias I M
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Cell Sci. 1999 Dec;112 ( Pt 24):4535-45. doi: 10.1242/jcs.112.24.4535.
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
胆小管膜含有几种参与胆汁分泌的ATP依赖性转运蛋白。胆小管转运蛋白在粗面内质网中合成,在高尔基体中修饰,然后转运至顶端质膜。然而,ATP依赖性转运蛋白的细胞内运输途径和调控机制尚未阐明。在本研究中,我们构建了mdr1与绿色荧光蛋白的翻译融合体,并在WIF-B细胞(一种极化的肝源性细胞系)中研究了mdr1的胆汁酸分泌及细胞内运输。与肝细胞相似,WIF-B细胞将胆汁酸和有机阳离子(如罗丹明123)分泌到胆小管中。牛磺胆酸盐和磷酸肌醇3激酶的十肽激活剂可刺激异硫氰酸荧光素-甘氨胆酸盐的胆小管分泌,而渥曼青霉素可使其分泌减少。用mdr1-GFP构建体对WIF-B9细胞进行瞬时和稳定转染。在胆小管膜、胆小管周围的点状结构和高尔基体区域观察到荧光。延时显微镜显示,mdr1-GFP以管状囊泡结构从高尔基体转运,其中大部分直接转运至胆小管膜。还观察到在胆小管膜和顶端下区域之间的循环。在基底外侧质膜中未检测到mdr1-GFP。在15℃时,mdr1-GFP在高尔基体中积累;转移至37℃后,荧光直接转移至胆小管膜。牛磺胆酸盐可增强这一过程,而渥曼青霉素可阻断这一过程。在这些研究中,在基底外侧膜或其他细胞内细胞器中也未在任何时间观察到mdr1-GFP荧光。总之,在WIF-B细胞中,存在一条从高尔基体到胆小管膜的直接途径用于mdr1(一种胆小管有机阳离子ATP依赖性转运蛋白)的运输。与正常肝细胞一样,磷酸肌醇3激酶调节WIF-B细胞中胆汁酸的分泌及mdr1的细胞内运输。稳定转染mdr1-GFP的WIF-B细胞提供了一个研究胆小管转运蛋白运输和调控的重要模型。在线视频:http://www.healthsci.tufts.edu/LABS/IMArias++ + /Sai_F9.html