A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique
A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique
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DOI：
10.1073/pnas.0900216106
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发表时间：
2009-06-09
影响因子：
11.1
通讯作者：
Fujimoto, Toyoshi
中科院分区：
文献类型：
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作者：
Fujita, Akikazu;Cheng, Jinglei;Fujimoto, Toyoshi
已经假定在细胞膜中存在多个功能独立的磷脂酰肌醇4，5-二磷酸[PI（4，5）P-2]池，但现有技术缺乏足够的分辨率来明确证实其存在。为了分析PI（4，5）P-2在纳米尺度上的分布，我们开发了一种电子显微镜技术，通过磷脂酶C-δ 1的普列克斯特林同源结构域来探测冷冻断裂膜的制备。该方法不需要人工探针的化学固定或表达，适用于体内、外任何细胞，并能定量测定PI（4，5）P-2的分布。用这种方法，我们发现PI（4，5）P-2在体内培养的成纤维细胞和小鼠平滑肌细胞中高度集中在小窝的边缘。PI（4，5）P-2也在被膜纹孔中富集，但在平坦的未分化膜中仅观察到低水平的聚集。当细胞用血管紧张素II处理时，未分化膜中的PI（4，5）P-2水平在10秒内下降到37.9%，然后恢复到初始水平。值得注意的是，小窝中的PI（4，5）P-2水平显示出较慢但更剧烈的变化，并在40秒时下降至20.6%，而包被窝中的PI（4，5）P-2水平相对恒定，仅在10秒时下降至70.2%。这些结果表明，不同的PI（4，5）P-2池的存在下，在细胞膜上，并建议一个独特的作用，在磷酸肌醇信号小窝。
Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P-2] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of PI(4,5)P-2 at the nanoscale, we developed an electron microscopic technique that probes the freeze-fractured membrane preparation by the pleckstrin homology domain of phospholipase C-delta 1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the PI(4,5)P-2 distribution quantitatively. By using this method, we found that PI(4,5)P-2 is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. PI(4,5)P-2 was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the PI(4,5)P-2 level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the PI(4,5)P-2 level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the PI(4,5)P-2 level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct PI(4,5)P-2 pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling.