Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation
Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation
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DOI：
10.1039/c005362g
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发表时间：
2011-01-01
影响因子：
3.4
通讯作者：
Allbritton, Nancy Lynn
中科院分区：
文献类型：
--
作者：
Jiang, Dechen;Sims, Christopher Eldridge;Allbritton, Nancy Lynn
开发了一种单细胞测定法，使用微量分析化学分离和荧光标记的脂质底物来测量磷酸肌醇 3-激酶 (PI3K) 的激活。使用Bodipy荧光素(Bodipy F1 PIP2)在其酰基链上标记的磷脂酰肌醇4,5二磷酸用作体外和基于细胞的测定的底物。 PI3K 反应的底物和产物的检测限为 10 至 20 zeptomol。使用或不使用药物抑制剂的 PI3K 体外测定表明，Bodipy F1 PIP2 转化为磷脂酰肌醇 3,4,5 三磷酸（Bodipy F1 PIP3）。 Bodipy F1 PIP3 可以通过磷酸酶 PTEN 返回转化为 Bodipy F1 PIP2。当将 Bodipy F1 PIP2 添加到细胞裂解物中时，细胞质提取物中每 ng 蛋白质在 10 分钟内产生 1.4 fmol Bodipy F1 PIP3。将Bodipy F1 PIP3添加至细胞裂解物中，在8分钟内产生每ng蛋白质3fmol的Bodipy F1 PIP2。 Bodipy F1 PIP2 和 Bodipy F1 PIP3 均可在单细胞中测量，并且两个物种可以相互转化。在适当的条件下，还在单细胞中检测到荧光二酰基甘油。当负载有荧光脂质的细胞上的Fc epsilon R1受体交联时，每个细胞产生的Bodipy F1 PIP3的量比未刺激的细胞增加4倍。 Bodipy F1 PIP3 的这种产生被渥曼青霉素阻断。利用荧光脂质的化学细胞术对于了解单细胞水平的脂质代谢具有重要意义。
A single-cell assay was developed to measure the activation of phosphoinositide 3-kinase (PI3K) using microanalytical chemical separations and a fluorescently labeled lipid substrate. Phosphatidyl-inositol 4,5 bisphosphate labeled on its acyl chain with Bodipy fluorescein (Bodipy Fl PIP2) was utilized as a substrate for both in vitro and cell-based assays. Detection limits for the substrate and product of the PI3K reaction were 10 to 20 zeptomol. In vitro assays with PI3K with and without pharmacologic inhibitors demonstrated that Bodipy Fl PIP2 was converted to phosphatidyl-inositol 3,4,5 trisphosphate (Bodipy Fl PIP3). Bodipy Fl PIP3 could be back converted to Bodipy Fl PIP2 by the phosphatase PTEN. When Bodipy Fl PIP2 was added to a cell lysate, 1.4 fmol of the Bodipy Fl PIP3 were produced per ng of protein in the cytoplasmic extract in 10 min. Addition of Bodipy Fl PIP3 to a cell lysate yielded 3 fmol of Bodipy Fl PIP2 per ng of protein in 8 min. Both Bodipy Fl PIP2 and Bodipy Fl PIP3 were measureable in single cells and the two species could be inter-converted. Under the appropriate conditions, a fluorescent diacylglycerol was also detected in single cells. When the Fc epsilon R1 receptor on the cells loaded with the fluorescent lipid was cross-linked, the amount of Bodipy Fl PIP3 generated per cell increased 4-fold over that of unstimulated cells. This production of Bodipy Fl PIP3 was blocked by wortmannin. Chemical cytometry utilizing the fluorescent lipids will be of value in understanding lipid metabolism at the single-cell level.