Zaal K J, Kok J W, Sormunen R, Eskelinen S, Hoekstra D
Department of Physiological Chemistry, University of Groningen, The Netherlands.
Eur J Cell Biol. 1994 Feb;63(1):10-9.
Studies in hepatoma cells and hepatocytes have revealed that the biogenesis of bile canalicular membrane involves microvilli-lined vesicles (MLV), which are formed in well differentiated cells. The vesicles grow as a function of time and are presumably vectorially transported to cell surface contact sites of attached cells. We demonstrate that a fluorescent head group-labeled lipid analog, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), after its exogenous insertion into the plasma membrane of HepG2 cells at 4 degrees C, accumulates in these microvilli-lined vesicles at 37 degrees C. This shows that the MLV are a target for plasma membrane-derived lipids. Furthermore, also the Golgi apparatus is involved in the formation of the vesicles. After initial accumulation of the fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (C6-NBD)-ceramide in the Golgi apparatus at 37 degrees C, prolonged incubation at 37 degrees C results in the appearance of NBD fluorescence in the microvilli-lined vesicles. The transport route for the Golgi-derived material to the developing bile canalicular vesicle is not an indirect pathway, i.e. involving transcytosis via the basolateral plasma membrane. This could be demonstrated by including bovine serum albumin (BSA) in the incubation media, a lipid scavenger that will remove any C6-NBD-lipids exposed at the basolateral membrane. At these conditions, lipid trafficking between the Golgi complex and MLV still occurred. We further demonstrate that the targeting from the Golgi apparatus to the bile canaliculus is also operational in isolated human hepatocytes. The latter results suggests that the Golgi complex is involved in both the formation of bile canaliculi and in bile secretion in fully differentiated cells.
对肝癌细胞和肝细胞的研究表明，胆小管膜的生物发生涉及微绒毛内衬囊泡（MLV），其在分化良好的细胞中形成。这些囊泡随时间增长，并可能通过向量运输至附着细胞的细胞表面接触位点。我们证明，一种荧光头部基团标记的脂质类似物，N-（丽丝胺罗丹明B磺酰基）磷脂酰乙醇胺（N-Rh-PE），在4℃下外源插入HepG2细胞的质膜后，在37℃下积聚在这些微绒毛内衬囊泡中。这表明MLV是质膜衍生脂质的靶标。此外，高尔基体也参与了囊泡的形成。在37℃下荧光鞘脂前体6-[N-（7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基）氨基]己酸（C6-NBD）-神经酰胺在高尔基体中最初积聚后，在37℃下长时间孵育导致微绒毛内衬囊泡中出现NBD荧光。高尔基体衍生物质向正在发育的胆小管囊泡的运输途径不是间接途径，即不涉及通过基底外侧质膜的转胞吞作用。这可以通过在孵育培养基中加入牛血清白蛋白（BSA）来证明，BSA是一种脂质清除剂，可去除基底外侧膜上暴露的任何C6-NBD-脂质。在这些条件下，高尔基体复合体和MLV之间的脂质运输仍然发生。我们进一步证明，从高尔基体到胆小管的靶向作用在分离的人肝细胞中也起作用。后一结果表明，高尔基体复合体参与了完全分化细胞中胆小管的形成和胆汁分泌。
