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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Metabolic labeling and direct imaging of choline phospholipids in vivo
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Metabolic labeling and direct imaging of choline phospholipids in vivo

机译：体内胆碱磷脂的代谢标记和直接成像

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摘要

Choline (Cho)-containing phospholipids are the most abundant phospholipids in cellular membranes and play fundamental structural as well as regulatory roles in cell metabolism and signaling. Although much is known about the biochemistry and metabolism of Cho phospholipids, their cell biology has remained obscure, due to the lack of methods for their direct microscopic visualization in cells. We developed a simple and robust method to label Cho phospholipids in vivo, based on the metabolic incorporation of the Cho analog propargylcholine (propargyl-Cho) into phospholipids. The resulting propargyl-labeled phospholipid molecules can be visualized with high sensitivity and spatial resolution in cells via a Cu(I)-catalyzed cycloaddition reaction between the terminal alkyne group of propargyl-Cho and a labeled azide. Total lipid analysis of labeled cells shows strong incorporation of propargyl-Cho into all classes of Cho phospholipids; furthermore, the fatty acid composition of propargyl-Cho-labeled phospholipids is very similar to that of normal Cho phospholipids. We demonstrate the use of propargyl-Cho in cultured cells, by imaging phospholipid synthesis, turnover, and subcellular localization by both fluorescence and electron microscopy. Finally, we use propargyl-Cho to assay microscopically phospholipid synthesis in vivo in mouse tissues.
机译：含胆碱（Cho）的磷脂是细胞膜中最丰富的磷脂，在细胞代谢和信号传导中起着基本的结构以及调节作用。尽管人们对Cho磷脂的生物化学和代谢知之甚少，但由于缺乏在细胞中直接进行显微镜观察的方法，其细胞生物学仍不清楚。我们基于将Cho类似物炔丙基胆碱（propargyl-Cho）代谢整合到磷脂中的方法，开发了一种简单而强大的方法来在体内标记Cho磷脂。经由炔丙基-Cho的末端炔基与标记的叠氮化物之间的Cu（I）催化的环加成反应，可以在细胞中以高灵敏度和空间分辨率可视化所得的炔丙基标记的磷脂分子。标记细胞的总脂质分析显示，炔丙基-Cho强烈掺入所有种类的Cho磷脂中；此外，炔丙基-Cho标记的磷脂的脂肪酸组成与正常Cho磷脂的非常相似。我们通过成像磷脂合成，营业额和通过荧光和电子显微镜的亚细胞定位证明了炔丙基-Cho在培养细胞中的使用。最后，我们使用炔丙基-Cho显微镜观察小鼠体内体内磷脂的合成。

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