Figure 5

FRET/FLIM-based reporters of synaptic signaling. (A) FRET/FLIM-based systems use a donor and acceptor fluorophore to monitor conformational changes within a protein. One such system, which reports CAMKII activity, uses monomeric enhanced GFP (mEGFP) for the donor fluorophore and a variant of YFP for the acceptor fluorophore; the fluorophores are fused, respectively, to the N- and C-terminal tail of the protein. The conformational change that accompanies CAMKII activation increases the distance between the closely apposed N- and C-terminal tails, resulting in a reduction of FRET (YFP/mEGFP fluorescence) and an increase in the fluorescence lifetime of mEGFP; either measure can be used to monitor CAMKII activity. (B) FRET/FLIM based systems can also be designed to report protein-protein interactions. One such system, which reports Ras activation, uses a donor fluorophore (mEGFP) fused to the Ras protein and an acceptor fluorophore (mRFP) fused to a Ras-binding domain; when active, Ras binds to the Ras-binding domain, and the co-localization of the mRFP and mGFP fluorophores results in an increase of FRET (mRFP/mGFPfluorescene), and a decrease in the fluorescence lifetime of mGFP. (C) LTP induction at single spines (arrow head) by repetitive glutamate uncaging (t = 0) is associated with a sustained activation of Cdc42, which remains restricted to the spine and (D) RhoA, which diffuses away from the spine (<10 μm). Figure 5C,D: Reprinted by permission from Macmillan Publishers Ltd: [NATURE] (Murakoshi et al., 472(7341):100-4) copyright (2011).