Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells
Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells
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DOI：
10.1038/ncb917
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发表时间：
2003-02-01
影响因子：
21.3
通讯作者：
Rodriguez-Boulan, E
中科院分区：
文献类型：
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作者：
Kreitzer, G;Schmoranzer, J;Rodriguez-Boulan, E
靶向递送蛋白质至不同的质膜结构域对于上皮细胞中极性的发展和维持至关重要。我们使用共聚焦和时间推移全内反射荧光显微镜（TIR-FM），研究在定位和胞吐部位的后高尔基体转运中间体（PGTI）携带GFP标记的顶端或基底外侧膜蛋白在上皮极化的变化。在非极化Madin达比犬肾（MDCK）细胞中，顶端和基底外侧PGTI存在于整个细胞质中，并观察到与质膜的基底域融合。在极化过程中，顶端和基底侧PGTI被限制在细胞质的不同区域，它们与基底膜的融合被完全废除。定量分析表明，基底外侧，但不是顶端，PGTI融合与侧膜极化细胞，相关的限制性本地化的突触融合蛋白4和3侧和顶端膜域，分别。微管破坏诱导Syntaxin 3去极化和融合的顶端PGTI与基底膜，但既不影响侧定位的Syntaxin 4或Sec 6，也不促进融合的基底外侧PGTI与基底膜。
Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.