A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy

Yuka Kumagai, Yuji Kamioka, Shunsuke Yagi, Michiyuki Matsuda, Etsuko Kiyokawa

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    4 Scopus citations

    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)P2 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)P2 accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP3 distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.

    Original languageEnglish (US)
    Pages (from-to)192-199
    Number of pages8
    JournalAnalytical Biochemistry
    Volume413
    Issue number2
    DOIs
    StatePublished - Jun 15 2011

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    Keywords

    • Cyst
    • Förster resonance energy transfer
    • Phosphoinositides
    • TFP
    • sREACh

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology
    • Cell Biology

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