Discussion
Protein-protein interactions play a key role in almost all biological functions. Therefore much research is spend in this field. Conventional investigation methods, like the yeast two-hybrid system or Co-IP, are highly time consuming. Foerster resonance energy transfer (FRET) is often used to identify protein-protein interactions by confocal microscopy. However, this requires expert knowledge and produces vast amounts of data.
In order to overcome those limitations, we developed a program, which automatically measures and determines the FRET efficiency on cell by cell basis on the MACSQuant flow cytometer. The amount of transferred energy is calculated very accurately according to Nagy et al. (1998) by registering relative changes of donor and acceptor fluorescence intensities.
We could validate the sensitivity and accuracy of the FRET program by measuring FRET standard beads with defined linker length between the fluorochromes. FRET efficiency outputs of the program were compared to established manual FRET calculations. Due to the high correlation between those two methods we could prove the functionality of this program.
The FRET program was applied to measure successfully the dynamic clustering of the CD3 and CD4 receptors on the cell surface after T cell activation with staphylococcal enterotoxin B (SEB). The clustering could be validated by confocal colocalization analysis. Furthermore the homoclustering of the CD3 receptor after T cell activation could be shown by the use of the FRET program. The T cell activation which led to a strong increase in FRET efficiency could be validated by staining dephosphorylated Fyn as an activation marker. T cell activation and the consequential increase in FRET could also be inhibited by applying a blocking antibody to SEB.
As a conclusion, we developed a program which automatically measures FRET in living cells on the flow cytometer. This allows the identification of protein-protein interactions on large cell numbers in a minimum of time, in high throughput screenings and is easy to use.
