Metabolic labeling and direct imaging of choline phospholipids in vivo
Metabolic labeling and direct imaging of choline phospholipids in vivo
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DOI：
10.1073/pnas.0907864106
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发表时间：
2009-09-08
影响因子：
11.1
通讯作者：
Salic, Adrian
中科院分区：
文献类型：
--
作者：
Jao, Cindy Y.;Roth, Mary;Salic, Adrian
胆碱磷脂是细胞膜中含量最丰富的磷脂，在细胞代谢和信号转导中起着重要的结构和调节作用。尽管对Cho磷脂的生物化学和代谢有很多了解，但由于缺乏在细胞中直接显微观察的方法，它们的细胞生物学仍然模糊不清。我们开发了一种简单而强大的方法来标记Cho磷脂在体内，基于代谢纳入的Cho类似物炔丙基胆碱（炔丙基-Cho）的磷脂。通过炔丙基-Cho的末端炔基和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到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强烈纳入所有类别的Cho磷脂;此外，炔丙基-Cho标记的磷脂的脂肪酸组成与正常Cho磷脂非常相似。我们证明了使用炔丙基-胆碱在培养的细胞中，通过成像磷脂合成，营业额，和亚细胞定位的荧光和电子显微镜。最后，我们使用炔丙基-Cho在显微镜下测定小鼠组织中体内磷脂的合成。
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.