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 |
研究代表者 |
林 康紀 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 (90466037)
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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 |
研究実績の概要 |
I aim to develop FRET biosensors to study the effect of dendritic spine calcium transients on the molecular events underlying synaptic long term potentiation. To date, I created a library of candidate N-WASP activity sensors optimising 3 axes: i) diversify fluorophore pairs to improve the dynamic range of the probe ii) vary the position for fusing the acceptor to improve sensitivity as the protein changes conformation and iii) introduce a floppy linker to modulate the flexibility of the probe. In vitro screening shows that the FRET pair mNeonGreen/mTurquoise2 has the best dynamic range, that fusing mNeonGreen at position 469 confers high sensitivity to the presence of an upstream activator and that presence of a linker doesn’t further improve it. In parallel to the library, I set up an in vivo assay in which N-WASP activity could be detected at steady state in cells lines. However, I was so far unable to observe dynamic activation of N-WASP.
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現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The experiments are going as planned.
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今後の研究の推進方策 |
The future plan consists of 4 subtasks: -Diversify the library even more extensively by investigating on i) non-fluorescent acceptors for fluorescence lifetime imaging ii) red-shifted or iii) single fluorophore based probes for dual FRET imaging and iv) domain based probes (as opposed to full length) to avoid overexpression issues. -Based on knowledge acquired for N-WASP engineering possibilities, develop sensors for other proteins of the WASP/WAVE family such as WASP and WAVE. -Set up an in vivo screening assay based on physiological stimulation in 96-well plates rather than 1-by-1 in vitro spectrometer measurements. -Monitor the spatiotemporal activity patterns of the probes in neurons and their interplay with upstream regulators. The requirements of WAVE and N-WASP for early and late LTP will be investigated as their respective activating GTPases Rac1 and Cdc42 have been implicated in early and late long term potentiation.
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