論文 - 詳細
| RRC ID | 10960 |
|---|---|
| 著者 | Kumagai Y, Kamioka Y, Yagi S, Matsuda M, Kiyokawa E. |
| タイトル | A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy. |
| ジャーナル | Anal Biochem |
| Abstract |
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. |
| 巻・号 | 413(2) |
| ページ | 192-9 |
| 公開日 | 2011-6-15 |
| DOI | 10.1016/j.ab.2011.02.021 |
| PII | S0003-2697(11)00108-4 |
| PMID | 21352796 |
| MeSH | Animals Biosensing Techniques / methods* Cell Line Dogs Fluorescence Fluorescence Resonance Energy Transfer* Humans Luminescent Proteins / chemistry* Luminescent Proteins / metabolism Microscopy, Fluorescence, Multiphoton / methods Phosphatidylinositol Phosphates / chemistry* Phosphatidylinositol Phosphates / metabolism Protein-Tyrosine Kinases / chemistry Protein-Tyrosine Kinases / metabolism* Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / metabolism |
| IF | 2.877 |
| 引用数 | 4 |
| WOS 分野 | CHEMISTRY, ANALYTICAL BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | |
| 各媒体での言及数の合計 | 1 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| ヒト・動物細胞 | MDCK(RCB0995) |