シナプス可塑性の基盤となるカルシウムシグナルの可視化

2016 Fiscal Year Annual Research Report

• Project/Area Number: 16F16712
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: 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)
• Co-Investigator(Kenkyū-buntansha): 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
• Project Period (FY): 2016-07-27 – 2019-03-31
• Keywords: Calcium signalling / Long term potentiation / FRET / Biosensors / Protein engineering

Outline of Annual Research Achievements

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.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The experiments are going as planned.

Strategy for Future Research Activity

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.