Heterogeneity of Phosphatidic Acid Levels and Distribution at the Plasma Membrane in Living Cells as Visualized by a Forster Resonance Energy Transfer (FRET) Biosensor
Heterogeneity of Phosphatidic Acid Levels and Distribution at the Plasma Membrane in Living Cells as Visualized by a Forster Resonance Energy Transfer (FRET) Biosensor
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DOI：
10.1074/jbc.m110.153007
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发表时间：
2010-11-12
影响因子：
4.8
通讯作者：
Kiyokawa, Etsuko
中科院分区：
文献类型：
--
作者：
Nishioka, Teruko;Frohman, Michael A.;Kiyokawa, Etsuko
磷脂酸（Phosphatidic acid，PA）是细胞膜中的主要磷脂之一。虽然已经报道了PA在细胞存活和形态中起关键作用，但仍然不知道PA在活细胞中何时何地产生。基于Forster共振能量转移（FRET）原理，我们制备了PA生物传感器，并命名为Pii（phosphatidic acid indicator）。在这些生物传感器中，DOCK 2的脂质结合结构域与青色荧光蛋白和黄色荧光蛋白夹在一起，并标记有K-Ras的质膜靶向序列。合成PA的加入，或磷脂酶D或二酰基甘油激酶在质膜的激活，改变了表达Pii的细胞中FRET的水平，证明了Pii对PA的响应。该生物传感器还检测到不同的PA含量之间的各种细胞系，以及在一个细胞系。有趣的是，生长因子诱导的PA含量的增加与刺激前的基础PA含量呈负相关，这表明在质膜的PA浓度存在一个上限阈值。该生物传感器还揭示了不均匀的PA分布在细胞内，即基础水平和生长因子诱导的PA积累是在无细胞的边缘高于在细胞-细胞接触区域。PA增加不足可能是细胞-细胞接触区域Ras活化无效的原因。总之，PA生物传感器Pii是一种多功能的工具，用于检查PA在单细胞以及不同细胞中的含量和分布的异质性。
Phosphatidic acid (PA) is one of the major phospholipids in the plasma membrane. Although it has been reported that PA plays key roles in cell survival and morphology, it remains unknown when and where PA is produced in the living cell. Based on the principle of Forster resonance energy transfer (FRET), we generated PA biosensor, and named Pii (phosphatidic acid indicator). In these biosensors, the lipid-binding domain of DOCK2 is sandwiched with the cyan fluorescent protein and yellow fluorescent protein and is tagged with the plasma membrane-targeting sequence of K-Ras. The addition of synthetic PA, or the activation of phospholipase D or diacylglycerol kinase at the plasma membrane, changed the level of FRET in Pii-expressing cells, demonstrating the response of Pii to PA. The biosensor also detected divergent PA content among various cell lines as well as within one cell line. Interestingly, the growth factor-induced increment in PA content correlated negatively with the basal PA content before stimulation, suggesting the presence of an upper threshold in the PA concentration at the plasma membrane. The biosensor also revealed uneven PA distribution within the cell, i.e. the basal level and growth factor-induced accumulation of PA was higher at the cell-free edges than at the cell-cell contact region. An insufficient increase in PA may account for ineffective Ras activation at areas of cell-cell contact. In conclusion, the PA biosensor Pii is a versatile tool for examining heterogeneity in the content and distribution of PA in single cells as well as among different cells.