Microscopy, FLIM. First, the baseline lifetime of the GFP donor was measured in GFP-PS1 transfected cells in the absence of RFP acceptor (Table 1). As expected, GFP lifetime significantly shortens in the presence of the RFP acceptor in G-PS1-R transfected cells, compared to that in the G-PS1 expressing cells. This indicates FRET, i.e. close proximity between GFP-PS1 NT and RFP-PS1 loop. Since PS1 dimers have been previously reported, [22-24] we tested how much of the FRET signal is contributed by inter-molecular (PS1 dimers) versus intra-molecu- lar (single PS1 molecule) interaction. For this, dilution of the G- PS1-R probe with untagged PS1 DNA was prepared for the transfection. We reasoned that dilution of the G-PS1-R probe by the wt PS1 would reduce inter-molecular FRET via competitive inhibition of the G-PS1-R/G-PS1-R dimer formation, whereas G- PS1-R intra-molecular FRET would not be affected. GFP-PS1 NT to RFP-PS1 loop FRET efficiency (GFP lifetime shortening) was monitored by FLIM in cells transfected with G-PS1-R only (presumed mixture of intra- and inter-molecular FRET), co- transfected with G-PS1-R and untagged wild type PS1 in 1:3 ratio (mostly intra-molecular FRET), and in cells co-transfected with GFP-PS1 and PS1-RFP constructs (inter-molecular FRET only). We found that the GFP lifetime did not significantly change when G-PS1-R was diluted by unlabeled PS1, indicating that the vast majority of the detected FRET signal was due to intra-molecular FRET (Table 1). In addition, GFP-PS1 lifetime in GFP-PS1 and PS1-RFP co-transfected cells (inter-molecular FRET) was signif- icantly longer, comparing to that in the G-PS1-R transfected cells. This further supports the notion that the FRET signal detected with the G-PS1-R probe originates mostly from intra-molecular FRET. Manipulation of the c-Secretase Components Leads to Altered Ab42/40 Ratio and Correlates with PS1 Conformational Change Based on our previous findings in fixed cells, we have hypothesized that mature PS1 molecules may exist in at least two conformational states: ‘‘open’’ and ‘‘closed’’ [20,21]. Accord- ing to this model, familial Alzheimer’s disease (FAD) PS1 mutations shift the equilibrium to favor a ‘‘close’’ NT-CT conformation (with NT-CT closer together) and stabilize an alignment of the PS1/c-secretase active site with the APP substrate to favor predominant cleavage of APP at the Ab42 position. Now we test whether the G-PS1-R probe can monitor changes in PS1 conformation in live cells. First, to determine if the G-PS1-R probe adopts a different conformation in the presence of FAD-linked mutation, either wild- type G-PS1-R probe or G-PS1-R probe incorporating the L166P Table 1. Detection of inter- vs. intra-molecular FRET using G-PS1-R probe. Condition G-PS1 G-PS1-R G-PS1-R:wtPS1 GFP-NT (1:3) G-PS1+PS1-R (1:1) Donor GFP-NT GFP-NT GFP-NT *: p,0.001 vs G-PS1, #: p,0.05 vs G-PS1. doi:10.1371/journal.pone.0007893.t001 Acceptor Donor lifetime (psec) none RFP-loop RFP-loop RFP-loop 2097678 (n = 11) 17836197 (n = 14)* 17526117 (n = 14)* 1983662 (n = 13)# PS1 Conformation and Ab PS1 mutation (L166P G-PS1-R) was transfected into CHO cells and the NT-loop proximity was analyzed by FLIM. We found that the GFP lifetime in the L166P G-PS1-R expressing cells was noticeably shorter (Table 2), compared to that in the wt G-PS1-R transfected cells, indicating that L166P G-PS1-R adopts a ‘‘close’’ NT-loop conformation. Isoo et al. [13] reported recently that introducing a structural change to the N-terminus of one of the c-secretase complex components, Pen2, leads to an increase in the Ab42 production. Since we have previously observed that a change in the Ab42/40 ratio due to familial AD mutations correlates with a change in the PS1 NT-CT proximity we hypothesized that an elongation of the Pen2 NT may have an effect on PS1 conformation similar to that of FAD PS1 mutations. To test this, CHO cells were co-transfected with G-PS1-R and either wt Pen2 or Pen2 with elongated NT due to a flag tag (flag- Pen2). We found that in the conditioned media of the flag-Pen2 transfected cells the Ab42/40 ratio was increased by ,1.6 fold, compared to that in the Pen2 transfected cells, confirming the data by Isoo et al. [13]. Correspondingly, when we monitored G-PS1-R conformation in living cells by the FLIM assay we found that cells co-transfected with flag-Pen2 displayed closer PS1 NT-loop proximity compared to that in the wt Pen2 transfected cells, as reflected by the shortening of the average donor fluorophore lifetime in flag-Pen2 expressing cells (Table 3). We used an alternative approach to confirm these findings; our previously validated PS1 NT-CT conformation FLIM assay in fixed and immunostained cells. The cells were double-immunostained with the previously reported PS1 NT-CT pair of antibodies [21] as well as with the PS1 NT-loop pair of antibodies, a new assay that matches the G-PS1-R NT-loop conformation assay. The primary antibodies were fluorescently labeled with Alexa488 (A488) as a donor and Cy3 as an acceptor fluorophores. We found a statistically significant shortening of the donor lifetime per cell in the flag-Pen2 transfected cells in both PS1 NT-CT and PS1 NT- loop assays (Table 3). This indicates that elongation of the Pen-2 N-terminus correlates with the change in PS1 conformation, which in turn leads to an increase in Ab42 generation. We have recently reported that different isoforms of another c- secretase component, Aph1 (Aph1A vs. Aph1B), support differen- tial c-secretase activity, with Aph1B containing c-secretase complexes yielding a higher Ab42/40 ratio [14]. Using the antibody-based FLIM assay we showed that PS1 has shorter distance between its NT and CT in cells containing Aph1B alone, compared to that in Aph1A expressing cells [14]. Thus, we asked whether conformational changes in the PS1 molecule induced by different Aph1 isoforms could be detected in live cells by the G- PS1-R probe. MEF cells expressing either all Aph1 isoforms (wt Aph1), or exclusively Aph1A or Aph1B containing c-secretase complexes were transfected with G-PS1-R, and NT-loop distance was evaluated by the FLIM analysis. We found that the lifetime of Table 2. The effect of L166P mutant on G-PS1-R conformation. Condition G-PS1 G-PS1-R L166P G-PS1-R Donor GFP-NT GFP-NT GFP-NT *: p,0.001 vs G-PS1-R. doi:10.1371/journal.pone.0007893.t002 Acceptor Donor lifetime (psec) none RFP-loop RFP-loop 2074627 (n = 7) 17426244 (n = 15) 14886145 (n = 15)* PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7893