Time-lapse FRET imaging as demonstrated by the visualization of Akt activation
|author||Haruko Miura, Matsuda Michiyuki, Kazuhiro Aoki|
|所属||Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Japan; Imaging Platform for Spatio-Temporal Information, Graduate School of Medicine, Kyoto University, Japan|
|Keyword||FRET, imaging, Akt|
This protocol provides a step-by-step description of time-lapse Förster resonance energy transfer (FRET) imaging by using the specific example of the FRET biosensor Eevee-iAkt (Miura et al., 2014). Eevee-iAkt is a genetically encoded FRET biosensor for the visualization of the spatiotemporal dynamics of Akt activity in living cells. As an intramolecular FRET biosensor, Eevee-iAkt comprises, starting from the N-Terminus, YPet as FRET donor, the phosphopeptide-binding domain FHA1, a substrate sequence derived from the Akt substrate GSK3β, ECFP as FRET acceptor, and a nuclear export sequence. Phosphorylation of the substrate sequence by Akt leads to a conformational change due to binding of the FHA1 domain to the phosphorylated substrate sequence, leading to an increase in the FRET efficiency (Figure 1).
- Plate HeLa cells on a 35 mm glass base dish and let cells grow for one day at 37°C, 5% CO2.
- Transfect cells with 1 μg plasmid DNA encoding Eevee-iAkt using 293fectin according to manufacturer’s instructions.
- Culture transfected cells for 1 to 2 days at 37 °C, 5% CO2.
FRET time-lapse imaging
- Starve cells in 2 ml M199 medium 20 mM HEPES 0.1% BSA for 3 hours.
- Transfer 500 μl of the conditioned medium to a microtube and add EGF (final concentration 10 ng/ml).
- Set up the microscope and pre-warm the imaging chamber to 37 °C, and place the glass bottom dish onto the stage.
- Focus cells, set the autofocus, and select around 5 positions with cells expressing the FRET biosensor.
- Wait for 1 to 2 minutes to allow the YFP to recover from reversible photo-bleaching and then start image acquisition.
- After 10 minutes pause image acquisition, add the EGF containing conditioned medium to the dish, and resume image acquisition for further 30 minutes. Note: Do not touch the dish!
- Subtract background from each plane of the image stack file.
- Create FRET/CFP ratio images in the intensity modulated display (IMD) mode.
- Export background subtracted FRET and CFP intensities of single cells to Excel software.
- Average the intensities over the cell area, calculate the FRET/CFP ratio, and normalize to the average FRET/CFP ratio before stimulation.
- Plot the FRET/CFP ratio versus elapsed time. Typical results are shown in Figure 2.