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首页> 外文期刊>Faraday discussions >Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation
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Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation

机译：磷酸肌醇3-激酶和磷酸酶与张力蛋白同源物激活的单细胞分析

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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 Fc3R1 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.
机译：开发了一种单细胞测定法，以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）的活化。在其酰基链上用Bodipy荧光素（Bodipy F1 PIP2）标记的磷脂酰肌醇4,5二磷酸酯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出限为10至20个zeptomol。在有和没有药理抑制剂的情况下，用PI3K进行的体外测定表明，Bodipy F1 PIP2被转化为磷脂酰肌醇3,4,5三磷酸酯（Bodipy F1 PIP3）。 Bodipy F1 PIP3可以通过磷酸酶PTEN逆转为Bodipy F1 PIP2。当将Bodipy F1 PIP2添加到细胞裂解物中时，在10分钟内每ng蛋白质在细胞质提取物中产生1.4fmol Bodipy F1 PIP3。在细胞裂解物中添加Bodipy F1 PIP3在8分钟内每ng蛋白质产生3 fmol Bodipy F1 PIP2。 Bodipy F1 PIP2和Bodipy F1 PIP3均可在单个细胞中测量，并且两个物种可以相互转换。在适当的条件下，还可以在单​​个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Fc3R1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将有助于理解单细胞水平的脂质代谢。

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