| 研究課題/領域番号 |
16F16712
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
宮脇 敦史 国立研究開発法人理化学研究所, 脳科学総合研究センター, チームリーダー (80251445)
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| 研究分担者 |
ROSENDALE MORGANE 国立研究開発法人理化学研究所, 脳科学総合研究センター, 外国人特別研究員
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| 研究期間 (年度) |
2016-07-27 – 2019-03-31
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| キーワード | Calcium signalling / Long term potentiation / FRET / Biosensors / Protein engineering |
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| 研究実績の概要 |
We aim to develop FRET biosensors to study the effect of dendritic spine calcium transients on the molecular events underlying synaptic long term potentiation.
Following the in vitro screen for N-WASP biosensors (see Progress Report for FY2017), we have performed ratiometric live-cell imaging to validate the best candidate of the library in vivo. The aim was to observe a dynamic change in FRET signal upon stimulation of the N-WASP pathway.
The best Cyan/Yellow FRET pair obtained in Cos7 cells stimulated with EGF was mTurquoise2/cp173V. High FRET signals could clearly be observed in growing lamellipodia where N-WASP is expected to drive cytoskeletal remodelling. This is consistent with the in vitro results. Indeed, this pair was the second best candidate from the in vitro screen but the best in vitro candidate itself (mTurquoise2/mNeonGreen) could not be tested in a live ratiometric assay because of the close emission wavelengths of the donor and acceptor. To circumvent this technical issue, we have tried spectral scanning imaging but the frame rate obtainable with this method was too slow to hope to monitor the expected activation pattern.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Unfortunately, promising the Cyan/Yellow sensor was, results were difficult to reproduce and overall response had a low dynamic range. To further validate it, we set up a semi-automated assay capable of screening up to 12 cells at a time within an hour, greatly increasing the number of data points and reliability of the results. To cope with the increased amount of data resulting from this assay,we wrote a Matlab script to automatize the analysis process, reducing the analysis time from several days to an afternoon and abolishing the risk of user bias. With these optimised conditions, we screened the Cyan/Yellow library in Bradykinin stimulated NIH3T3 fibroblasts and EGF stimulated RPE-1 cells but with no greater success.
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| 今後の研究の推進方策 |
We moved on to diversify the library to Green/Red FRET pairs. This diversification step had always been part of the initial strategy because longer wavelengths are more suitable for deep tissue imaging, a property that will be required for the project goal: imaging the sensors in brain slices. we screened this new library in EGF stimulated Cos7 or RPE-1 cells and obtained the highest dynamic change and best reproducibility using the mClover3/mKate2 fluorescent protein pair.
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