FRET technique development in flow cytometry facility

Arthritis, Musculoskeletal, Skin Dis

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Abstract

Summary: Background: A molecular interaction between two proteins has been successfully determined in living cells on flow cytometry in term of FRET (fluorescence resonance energy transfer), using cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). However, it requires very expensive Krypton-UV laser that is not easily accessible to some of flow cytometry facilities. Moreover, there is no simple way to calculate FRET efficiency on flow cytometric data. Objective of present studies: To overcome that an expensive Krypton-UV laser has been used to measure FRET signals, we have developed a method in which a single argon-ion laser at 458 nm excitation can be used to measure FRET signals. And furthermore, we have developed another method in which donor CFP quenching is directly visualized and then further is used to calculate FRET efficiency on flow cytometric data. Results during the past year: A 458 nm lines emitted from an argon-ion laser was used to successfully detect FRET signals by showing the enhancement of the acceptor YFP signal and the decrease of the donor CFP signal as well as FRET signal detected at 564-606 nm in the cells transfected with a CFP-YFP fusion plasmid. Confocal microscopy and spectrofluorometry were also used to confirm our flow cytometric data. In order to calculate FRET efficiency, we firstly constructed a caspase-sensitive CFP-LEVD-YFP probe that will be cleaved upon caspase activation and the cleavage in turn results in loss of FRET signal in cells. Comparison of the CFP intensity in the FRET-positive and FRET-negative populations was used to document the relationship of FRET to donor quenching and permit the calculation of FRET efficiency. Similar results were obtained when cells transfected with this caspase-sensitive probe (in the presence of z-VAV) were mixed with those expressing the CFP-YFP construct with the 232-aa linker (FRET-negative control). Moreover, using the same approach, we confirmed that members of TNF receptor-associated factor (TRAF) family engaged in both homotypic and heterotypic interactions. Conclusion and significance: 1. We have developed a novel method using 458 nm excitation to detect FRET signals. This successful measurement of FRET by a single laser excitation at 458 nm should make FRET a more generally available flow cytometric technique. 2. We have developed a novel flow cytometric method to assess donor CFP quenching during CFP->YFP FRET, which can be sued for the calculation of FRET efficiency and relative biological molecular distance between CFP and YFP moieties.