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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique
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A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique

机译：纳米级标记技术揭示了海绵体中磷脂酰肌醇4,5-二磷酸的独特集合

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摘要

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 f reeze-f ractured membrane preparation by the pleckstrin homology domain of phospholipase C-δ1. 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 undif-ferentiated 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.
机译：假定磷脂酰肌醇4，5-双磷酸[PI（4,5）P_2]的多个功能独立的池发生在细胞膜中，但是现有技术缺乏足够的分辨率来明确确认其存在。为了分析PI（4,5）P_2在纳米级的分布，我们开发了一种电子显微镜技术，该技术通过磷脂酶C-δ1的pleckstrin同源结构域探测微带断裂膜的制备。该方法不需要化学固定或人工探针的表达，它适用于体内和体外的任何细胞，并且可以定量定义PI（4,5）P_2的分布。通过使用此方法，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都高度集中在小窝的边缘。 PI（4,5）P_2也富集在包被的凹坑中，但是在平坦的未扩散膜中仅观察到低水平的聚集。当用血管紧张素II处理细胞时，未分化膜中的PI（4,5）P_2水平在10秒内降至37.9％，然后恢复到初始水平。值得注意的是，小凹洞中的PI（4,5）P_2水平显示出较慢但剧烈的变化，并在40秒时下降至20.6％，而包膜凹坑中的PI（4,5）P_2水平相对恒定，仅下降至10秒时为70.2％。这些结果表明细胞膜中存在独特的PI（4,5）P_2池，并提示小窝在磷酸肌醇信号传导中的独特作用。

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