Caveolae and endoplasmic reticulum: Immunofluorescence microscopy and time-lapse analysis
Caveolae and endoplasmic reticulum: Immunofluorescence microscopy and time-lapse analysis
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DOI：
10.1267/ahc.30.593
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发表时间：
1997-01-01
影响因子：
2.4
通讯作者：
Fujimoto, T
中科院分区：
文献类型：
--
作者：
Kogo, H;Shioya, M;Fujimoto, T
Caveolae已被假设参与Ca 2+信号传导。电镜下可见胞膜小窝与内质网并列，内质网是细胞内主要的钙池。在本研究中，我们研究了当微管解聚改变ER的分布时，小窝和ER之间的关系。采用免疫荧光双镜检测小窝蛋白和ER抗原，并延时观察绿色荧光蛋白（GFP）标记的小窝蛋白。在正常的人成纤维细胞和PtK 2细胞中，ER被视为一个遍布细胞质的网络，大多数小窝蛋白出现在沿着细胞边缘的斑块中。当微管被秋水仙胺或诺考达唑解聚时，ER从细胞外周收缩并聚集在细胞核周围;在相同的细胞中，小窝蛋白沿着细胞边缘没有看到，但沿着沿着收缩的ER的边缘排列。通过延时分析，观察到在PtK 2细胞中表达的GFP-小窝蛋白在秋水仙胺处理的细胞中从细胞周边向细胞中心移动。结果表明，小窝和内质网的并列即使在总体分布的变化，并建议之间的机械联系的两个细胞器。
Caveolae have been hypothesized to be involved in Ca2+ signaling. By electron microscopy, caveolae were observed to be apposed to the endoplasmic reticulum (ER), which is a major intracellular Ca2+ pool. In the present study, we examined the relationship between caveolae and the ER when the distribution of the latter was changed by depolymerization of microtubules. Double immunofluorescence microscopy for detection of caveolin and the ER antigens, and time-lapse observation of green fluorescent protein (GFP)-tagged caveolin were employed. in normal human fibroblasts and PtK2 cells, the ER was seen as a network extending throughout the cytoplasm, and most caveolin occurred in patches along the cell edge. When microtubules were depolymerized by Colcemid or nocodazole, the ER became retracted from the cell periphery and aggregated around the nucleus; in the same cells, caveolin was not seen along the cell edge, but was aligned along the edge of the retracted ER. By time-lapse analysis, GFP-caveolin expressed in PtK2 cells was observed to move from the cell periphery toward the cell center in Colcemid-treated cells. The result shows that the apposition of caveolae and the ER is maintained even after the gross distributional change, and suggests a mechanical linkage between the two organelles.