THE FUNCTION OF TIGHT JUNCTIONS IN MAINTAINING DIFFERENCES IN LIPID-COMPOSITION BETWEEN THE APICAL AND THE BASOLATERAL CELL-SURFACE DOMAINS OF MDCK CELLS
THE FUNCTION OF TIGHT JUNCTIONS IN MAINTAINING DIFFERENCES IN LIPID-COMPOSITION BETWEEN THE APICAL AND THE BASOLATERAL CELL-SURFACE DOMAINS OF MDCK CELLS
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DOI：
10.1002/j.1460-2075.1986.tb04382.x
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发表时间：
1986-07-01
期刊：
影响因子：
11.4
通讯作者：
SIMONS, K
中科院分区：
文献类型：
--
作者：
VAN MEER, G;SIMONS, K
上皮细胞的紧密连接被认为是抑制脂质和蛋白质在顶端和基底侧质膜结构域之间横向扩散的屏障。为了更详细地研究紧密连接的栅栏功能，我们将含有荧光磷脂N‐Rh‐PE的脂质体融合到MDCK细胞的顶质膜中。低pH诱导脂质体融合，由流感病毒血凝素介导，病毒感染后在根尖细胞表面表达。通过荧光显微镜在0℃下监测到的N - Rh - PE在基底侧表面的重新分布，似乎依赖于质膜中荧光脂质的跨双层取向。磷脂酶A2测定显示，外双分子层小叶中含有85%以上N - Rh - PE的不对称脂质体是通过辛基β - D -糖苷透析产生的。当这些不对称脂质体与根尖质膜融合时，荧光脂质不会向基底外侧移动。用冻融法或反相蒸发法获得了含有标记物的对称脂质体。当这些膜与根尖膜融合后，立即重新分布到基底外膜。只有在无Ca2+培养基中孵育打开紧密连接时，才能观察到不对称脂质体的重新分布。在正常的实验操作中，紧密连接保持完整，因为在细胞单层上保持了高的跨上皮电阻。我们得出结论，紧密连接在质膜的外质小叶中充当荧光磷脂N - Rh - PE的扩散屏障，而在细胞质小叶中不起作用。
Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N‐Rh‐PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N‐Rh‐PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N‐Rh‐PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta‐D‐glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze‐thawing asymmetric liposomes or by reverse‐phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+‐free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans‐epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N‐Rh‐PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.