title: A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy.
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.
authors.affiliation: Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Kumagai
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamioka
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Yagi
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Matsuda
authors.forename: Etsuko
authors.initials: E
authors.lastname: Kiyokawa
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D004285:Dogs; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases