The Yasuda lab specializes in making and refining fluorescent sensors for second messenger activity, most famous among them Ras, CaMKII, and Rho-GTPases (I remember one grant proposal where Ryohei proposed making sensors for all GTPases). My corner of the kingdom was to develop a FRET/FLIM sensor for PI3K. PI3K is a second messenger involved in LTP, and works by phosphorylating the third carbon of phosphoinositol, turing PIP2 into PIP3.
FRET nor FLIM here, but FRET is used to measure the proximity between two fluorophores. When the two fluorophores are distant, FRET is low, and when they are close, FRET is high. In this case, the donor fluorophore was mEGFP tagged to the plasma membrane via a CAAX box. The acceptor fluorophore was mCherry tagged to the PH domain of Btk, which selectively binds to PIP3. Under normal conditions, there is a lot of PIP2 at the plasma membrane, but little PIP3, which means the Btk-PH-GFP floats about in the cytosol, and FRET is low. When PI3K is active, however, it recruits Btk-PH-GFP to the plasma membrane, bringing the mCh and GFP into proximity, and causing FRET.
Most of the initial sensor development was done by an undergrad, Wei Leong Chew, under my supervision. We tried a variety of different donor-acceptor combinations, including using Akt-PH instead of Btk-PH; trying different acceptors like dimYFP or tandem-mCh. It's important to have the donor at the plasma membrane instead of the acceptor (to be honest, one year later, I can't remember the specific, technical reason why; certainly it's helpful to have a consistent donor fluorescence).
Testing in HEK cells
In any case, we got the sensor to work. To test the sensor in HEK cells, we applied EGF, which activates a wide-ranging signaling cascade, including PI3K (see below). When we did so, the cells underwent significant morphological changes, and the FRET fraction increased. Then, to reverse the process, we applied the PI3K antagonist LY294002, which caused the FRET fraction to decrease.
Testing in neurons
Having tested the sensor in HEK cells, we next moved to neurons. In addition to developing FRET sensors, the Yasuda lab specializes in two-photon glutamate uncaging, so we uncaged on spines to see how the sensor would react. During uncaging, we measured spine size as a proxy for synaptic strength. It was especially tricky here to get Btk expression high enough to make the sensor work. The downside of high Btk expression, however, was that it inhibited long-term structurally plasticity (below, left). (We also did uncaging experiments in the presence of LY, and found that LY partially blocked late phase structural plasticity.)
That's the last of my unpublished data from the Yasuda lab. In retrospect, the story doesn't seem far from some version of complete. The neuronal data just needed some refinement to get more consistent results (the n above is quite low). At the time, though, after seven years in Durham, I was impatient start the next phase in my career. If I was more strategic, I probably would have finished it. I know a couple people are still working on the sensor, trying to get it to work without interfering with structural plasticity. You can probably look forward to a more expanded result in the near future (1-2 years).