WIF-B cells: an in vitro model for studies of hepatocyte polarity.
WIF-B cells: an in vitro model for studies of hepatocyte polarity.
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WIF-B细胞：用于研究肝细胞极性的体外模型。
DOI：
10.1083/jcb.123.6.1761
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发表时间：
1993-12
影响因子：
7.8
通讯作者：
Hubbard, A L
中科院分区：
文献类型：
--
作者：
Ihrke, G;Neufeld, E B;Meads, T;Shanks, M R;Cassio, D;Laurent, M;Schroer, T A;Pagano, R E;Hubbard, A L
我们已经评估了肝癌衍生的杂交细胞系，WIF-B，极化肝细胞功能的体外研究的效用。汇合培养物中的大多数（> 70%）细胞与相邻细胞形成封闭空间。这些胆小管样空间（BC）积累荧光素，这是体内胆小管的特性。通过间接免疫荧光，6质膜（PM）蛋白显示类似于大鼠肝细胞原位极化分布。四种顶端PM蛋白集中在WIF-B细胞的BC膜上。微管辐射从BC（顶端）膜，肌动蛋白和焦点的γ-微管蛋白集中在这个区域。紧密连接相关蛋白ZO-1呈带状存在，标记顶侧和基底侧PM域之间的边界。我们探讨了在活细胞中使用荧光膜脂质类似物和可溶性示踪剂，这一边界的功能特性。当细胞与鞘磷脂的荧光类似物在4 ℃下孵育时，仅基底外侧PM被标记。相反，当两个PM域标记从头合成的荧光鞘磷脂从神经酰胺，荧光脂质只能从基底外侧域。这些数据表明，PM域之间的脂质的横向扩散的障碍的存在。然而，小的可溶性FITC-葡聚糖（4，400摩尔/重量）能够扩散到BC中，而较大的FITC-葡聚糖取决于其大小和孵育温度而受到不同程度的限制。在4 ℃时，表面标记试剂sNHS-LC-生物素（557 mol wt）可接近整个PM，但与生物素化分子结合的链霉亲和素（60，000 mol wt）仅限于基底外侧结构域。良好表征的探针的这种差异可及性可用于单独标记每个膜结构域。这些结果表明，WIF-B细胞是一个合适的模型，研究膜运输和靶向肝细胞在体外。
We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction- associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC- dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC- dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.