Background and Objective: These studies have solved how to simply calculate FRET efficiency when FRET data are collected using flow cytometry and then demonstrate FRET's unique applications in determining protein-protein interaction and caspase activity in living cells. We used CFP->YFP FRET technique that has been established using flow cytometry and confocal microscopy over last a few years in our Section. For a protein-protein interaction, two independent proteins were tagged either CFP or YFP while for caspase monitoring, caspase target sequence LEVD was inserted between CFP and YFP fusion. When two independent proteins are in close proximity (10 nm), YFP will emits itself due to gain energy from excited CFP. Therefore, protein-protein interaction in living cells can be monitored by FRET technique. However, there is NO method available of how to calculate CFP->YFP FRET efficiency on flow cytometry. We developed a simplest method in which donor fluorochrome (CFP) quenches during FRET process. When FRET is used to monitor caspase activity in living cells, we generated a caspase-sensitive FRET probe CFP-LEVD-YFP that will be cleaved when caspases are activated. Thus, caspase activation triggers FRET loss. Results: 1. Protein-protein interaction: FRET was rapidly and easily visualized by observing donor CFP quench during FRET process and thus the quench was used to calculate FRET efficiency of CFP->YFP FRET. In order to apply it to protein-protein interactions, tumor necrosis factor receptor-associated factors, such as TRAF3 and TRAF5, were chosen to examine the system. We did calculate molecular distance based on FRET efficiency between two molecules of TRAF3 and TRAF3 homodimers or of TRAF3-TRAF5 heterodimers. 2. Caspase monitoring in living cells: When the caspase-sensitive FRET probe was transfected into Hela cells, two FRET populations of FRET-positive and FRET-diminished cells were seen. The first one indicated minimal caspase activity whereas the latter indicated caspase activation. We also validated that this method has the exact same property as other methods, such as fluorigenric substrates and FLICAs to monitor caspase activity. Based on these data, two publications have been published: 1. Cytometry Part A, 55A: 71-85, 2003. 2. American Journal of Pathology, 164: 1901-1913, 2004. Conclusions: we have achieved methods to monitor caspase activity and determine protein-protein interactions in living cells using flow cytometry and CFP-YFP FRET system. And also we apply this technique to explore signal transduction complexes during CD40 receptor signaling.
