is that it allows monitoring PS1 conformational changes in living cells while monitoring cell toxicity of the candidate compounds at the same time. Finally, the relatively high temporal resolution of the spectral FRET assay (2–3 seconds per image, compared to 3–4 min per image in the FLIM assay) enables near ‘‘real time’’ monitoring of changes in the PS1/c-secretase conformation during experimental manipulations. These data show directly that changes in PS1 conformation can be detected following various manipulations of PS1 itself, Pen2, Aph1, APP, and pharmacological agents known as c-secretase modulators. Together these results strongly support the hypothesis that c-secretase catalytic specificity can be allosterically modified via conformational changes in the catalytic PS1 moiety. Therefore, monitoring PS1 conformation using the G-PS1-R probe could be a powerful approach to uncover pathological mechanisms leading to increased Ab42/40 ratio in vitro and in mouse brain in vivo, as well as a promising tool for therapeutic drug screening of c-secretase modulating agents. Materials and Methods Cell Lines and Pharmacological Treatments Chinese hamster ovary (CHO) cells and Human embryonic kidney (HEK) 293 cells were obtained from ATCC (Manassas, VA). PS1/PS2 double knockout mouse embryonic fibroblasts (PS1/2 dKO MEF) as well as Aph1ABC2/2 cells, reconstituted with either Aph1AL or Aph1B component were previously described [14,34]. CHO, HEK293 and MEF cells were maintained in Opti-MEM (Invitrogen) supplemented with 5% fetal bovine serum. The cells were plated into four-chamber slides or 35 mm glass-bottom dishes, transfected or infected with various constructs, and were used for biochemical (Western blot and ELISA) or microscopy (FLIM, spectral FRET, TIRFM) analyses. The cells were treated for 24 hours with 100 mM fenofibrate or 400 mM ibuprofen to evaluate the effect of Ab42 –modulating compounds on PS1 conformation. Dimethylsulfoxide (DMSO) or ethanol were used as respective vehicle controls. Constructs and Lenti-Viral Infections The GFP-PS1-RFP (G-PS1-R) construct was generated as follows: Human PS1 (NM_000021) was cloned into EGFP-C3 vector using XhoI and HindIII restriction sites to generate N- terminally (NT) labeled GFP-PS1 construct (G-PS1). A NotI restriction site was introduced after the G-PS1 codon 351 within the cytoplasmic loop between transmembrane domains 6 and 7 (TM6-7), resulting in G-PS1NotI construct (based on the cloning strategy by Drs. Haass and Kaether, Munich, Germany). NotI- RFP-NotI tag was produced by polymerase chain reaction and sub-cloned into G-PS1NotI to generate the GFP-PS1-RFP (G-PS1- R) construct. The familial Alzheimer’s disease L166P PS1 mutation was introduced into the G-PS1-R construct using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. RFP was also inserted into the loop domain of wtPS1 construct to generate PS1- R plasmid. The integrity of constructs was confirmed by sequencing. To increase the efficiency of G-PS1-R construct delivery to cells, it was packaged into lentivirus (MGH Viral Core Facility, Director Dr. Miguel Sena-Esteves). For the fluorescent lifetime imaging microscopy (FLIM) analysis, G-PS1-R lenti-virus multiplicity of infection, (MOI), was 0.2-1 for CHO, HEK293 and MEF cells. For the Western blotting of G-PS1-R infected CHO or MEF cells we used G-PS1-R lentis at MOI 10. Alternatively, a G- PS1-R encoding plasmid was transfected or co-transfected with other plasmid constructs as indicated in the manuscript using PS1 Conformation and Ab Lipofectamine 2000 (Invitrogen). N-terminally truncated Notch 1 receptor construct (NEC), which does not require ligand binding for its activation and represents an immediate substrate of c- secretase was a gift from Dr. R. Kopan [35]. Pen2 construct tagged with Flag on its N-terminus was a gift from Dr.Selkoe (BWH, Boston, MA). G-PS1 construct as well as EGFP-C3 empty vector (Clontech, Madison, WI) was used as negative controls for the Forster resonance energy transfer (FRET) based experiments. An RFP-GFP fusion (R-G fusion) plasmid in which RFP is fused to the N-terminus of GFP with a short linker was used as a positive control in the FRET-based assays. Western Blot Analysis For the Western blot analysis CHO or MEF cells plated on 100 mm cell culture dishes were infected with G-PS1-R for 48– 72 hrs. Where NEC needed to be co-expressed, the MEF cells were co-transfected with NEC 24 hrs prior to the cell harvesting. The cells were lysed in 1% CHAPSO, and the lysates were resolved on a 4–20% Tris-Glycine gel. The following antibodies were used for detection: rabbit anti-APP C-terminus [36](C8, Selkoe et al., 1988), rabbit anti-GFP (Clontech); mouse anti-PS1 loop (Chemicon), rabbit anti-NICD (Val1744, Cell Signaling). Enzyme-Linked ImmunoSorbent Assay (ELISA) For the detection of Ab production, we used PS70 cells, CHO cells stably transfected with wild type human APP and PS1 [37]. PS70 cells growing in 35 mm cell culture dishes were treated with drugs or transiently transfected with plasmids encoding either wt Pen-2 or Flag-Pen-2 using Lipofectamine 2000 reagent (Invitrogen). Sixteen hours after the transfection cells culture media was exchanged to 1 ml of Opti-MEM (Invitrogen) supplemented with 2% fetal bovine serum, and the cells were incubated for additional 24 hours. The cell culture media for each transfection condition was subjected to the analysis of human Amyloid b (40) and (42) using ELISA assay kit (WAKO), according to the manufacturer’s instructions. Fluorescence Lifetime Imaging Microscopy (FLIM) A FLIM assay was employed to monitor proximity between the PS1 NT and TM6-7 loop as an indicator of PS1 conformation in live cells expressing the G-PS1-R construct. A donor fluorophore lifetime is an indicator of the proximity between the donor and an acceptor fluorophore. If the fluorophores are in close proximity to each other (,10 nm), the GFP donor lifetime decreases due to a non-radiative transfer of part of its emission energy to the RFP acceptor (FRET present). GFP fused to the PS1 NT and RFP located in the PS1 TM6-7 loop serve as FRET donor and acceptor fluorophores, respectively. The GFP-PS1 construct, in which the donor lifetime is measured in the absence of an acceptor (FRET absent), was used as a negative control. GFP-RFP fusion construct was used as FRET positive control. Live cells were imaged 2–4 days post-transfection/infection on a Zeiss LSM510 microscope equipped with 37uC heating chamber containing 5% CO2. A Chameleon Ti:Sapphire laser was used to excite the donor fluorophore (two-photon excitation wave length: 840 nm for GFP donor and 800 nm for Alexa488 donor), a high- speed Hamamatsu (MCP R3809; Hamamatsu, Hamamatsu City, Japan) detector with very high temporal resolution optimized for fluorescence lifetime imaging, and Becker&Hickl TCSPC hardware/ software (Becker&Hickl, Berlin, Germany) were used to acquire donor lifetime information on a pixel-by-pixel basis. Data analysis was performed using SPC Image (Becker&Hickl, Berlin, Germany), in which donor fluorophore lifetimes are determined by fitting the data to one (negative control) or two (experimental conditions) exponential decay curves. A 1286128 pixel matrix was created to PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7893