Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Anal Biochem. 2011 Jun 15;413(2):192-9. doi: 10.1016/j.ab.2011.02.021. Epub 2011 Feb 23.
Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (Förster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P(2) was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P(2) accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP(3) distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.
Pippi（磷酸肌醇磷酸盐指示剂）是一种基于 FRET（荧光共振能量转移）原理的生物传感器，由一对荧光蛋白，CFP（青色荧光蛋白）和 YFP（黄色荧光蛋白），夹在它们之间的 PH 结构域以及用于质膜定位的 K-Ras C 末端序列组成。由于 YFP 在用于激发 CFP 的条件下的显著交叉激发，通过 TPE（双光子激发）显微镜获得的初始 FRET 图像的信噪比低，阻碍了对三维结构中脂质的观察。为了解决这个问题，原始 Pippi-PI(3,4)P(2)中的 YFP 和 CFP 分别被 sREACh（超共振能量接受色蛋白）和 mTFP1（单体青色荧光蛋白）取代。该生物传感器还与内部对照蛋白 mKeima 融合，其中 Keima/mTFP1 表示 FRET 效率，并且表皮生长因子刺激确实增加了 HeLa 细胞中的 Keima/mTFP1。该生物传感器成功地在三维环境中培养的 MDCK 类器官中显示了侧向膜上的 PI(3,4)P(2)积累。此外，还基于该方法开发了用于 PIP(3)分布和用于酪氨酸激酶活性的其他基于 FRET 的生物传感器，表明其广泛应用于通过 TPE 显微镜可视化信号转导事件。
