display donor lifetimes on a pixel-by-pixel basis in pseudocolored images, in which red pixels represent molecules in close proximity (FRET present), whereas blue pixels represent molecules that are greater than 10 nm apart from each other (FRET absent). Alternatively, PS70 cells or CHO cells transfected with untagged wild type PS1 were fixed in 4% paraformaldehyde and incubated with antibodies against PS1 NT and C-terminus (CT) or TM 6–7 loop domain as previously described [21]. PS1 NT was labeled with Alexa488 (donor) and PS1 CT/PS1 loop with Cy3 (acceptor) to be subjected to the FLIM analysis as described above. The following antibodies were used for immunostaining; goat anti- PS1 NT (Sigma), rabbit anti-PS1 CT (Sigma), mouse anti-PS1 loop (Chemicon), donkey anti-goat Alexa 488 (Molecular probe), donkey anti-rabbit Cy3 (Molecular probe), and donkey anti-mouse Cy3 (Molecular probe). Total Internal Reflection Fluorescence Microscopy (TIRFM) To test whether G-PS1-R protein traffics through the secretory pathway and reaches the plasma membrane, we performed a TIRFM assay. TIRFM detects fluorescent proteins within or in close proximity (#100 nm) to the plasma membrane of living cells. The high resolution is achieved by laser beam being totally reflected on a cell-substrate interface. Thus, only a very thin layer of the cell immediately adjacent to the interface between two media is illuminated by the evanescent electromagnetic field. Cells were transfected with the G-PS1-R constructs and then re-plated a day before the imaging on a glass cover slip at 30% confluency using media that did not contain phenol red. The cells were placed in a CO2 and temperature controlled chamber of Carl Zeiss system for TIRF imaging. The excitation of EGFP was used to study presence of the GFP-PS1-RFP expression product within the proximity of the plasma membrane. Modified Carl Zeiss system was used to record the images as previously described [38]. Spectral FRET HEK293 cells in 35 mm glass-bottom dishes were transfected with PS1 constructs fused with living color tags (G-PS1, G-PS1-R or L166P G-PS1-R). RFP-GFP fusion construct was used as a FRET positive control. Cells were imaged 24–48 hours after the transfection. Spectral imaging was performed on the Zeiss LSM510 microscope equipped with 37uC heating chamber containing 5% CO2. The Argon laser at 488 nm was used to excite GFP, and emitted fluorescence was detected by 7 channels of the Metadetector with 21.4 nm spectral bandwidth for each channel that was set to detect fluorescence in the 502–651 nm wavelength range. The images were processed and quantified using LSM image browser as follows: average pixel intensity of the fluorescence in the entire cell was determined for each spectral window. G-PS1 transfected cells (negative control) showed maximal fluorescence emission at 513 nm. When FRET occurs, the energy is transferred from GFP (donor) to RFP (acceptor), thus the GFP emission peak weakens, and the fluorescence intensity of RFP increases. The FRET positive control probe (R-G fusion) showed robust increase in the fluorescence intensity at 598 nm, compared to that of the FRET negative control (G-PS1). Thus, we used the ratio of 598 nm (RFP) to 513 nm (GFP) as a readout of the FRET efficiency, which reflects relative proximity between the GFP and RFP fluorophores. The ratio value for the whole cell was References PS1 Conformation and Ab calculated as a ratio of average pixel fluorescence intensity at the 598 nm spectral window to that at the 513 nm, after subtraction of the background fluorescence. We used 10x and 25x objectives for the average-per-cell analysis, and 63X objective for studying sub- cellular distribution of PS1. The detector gain and an amplifier offset were adjusted to make sure that the intensities of cells in the image were not saturated and the setting maintained the same for all experimental manipulations. Ratiometric image was produced by dividing the intensity of the image from the 598 nm spectral window by that from the 513 nm window after background subtraction, using Image J. After adjustment of brightness/ contrast, a Look-Up Table was applied by mapping image values to color-scale (16 colors) resulting in pseudo-color image. Statistical Analysis StatView for Windows, Version 5.0.1 (SAS Institute, Inc) was employed to perform statistical analysis using Fisher’s PSLD analysis of variance (ANOVA). Samples were considered signif- icantly different at p,0.05. For the comparison of 598/513 ratio in spectral FRET assay, a t-test was used for the analysis. Supporting Information Figure S1 Expression of G-PS1-R probe in HEK 293 cells. (A– C) G-PS1-R probe was transfected in HEK293 cells and observed by conforcal microscopy. GFP (A) as well as RFP signal (B) was observed mainly in perinuclear area in reticular pattern, reminiscent of ER distribution. (C) Merged image. Found at: doi:10.1371/journal.pone.0007893.s001 (1.56 MB TIF) Figure S2 Conformational diversity of the G-PS1-R molecules in different subcellular compartments. (A) - Confocal image of the GFP fluorescence in HEK293 cell infected with G-PS1-R-lenti viral construct. Several ROIs (circles) were randomly chosen and 598/ 513 ratio was calculated for each ROI. The 598/513 ratio in the each ROI was shown next to the circle. Higher ratio represents closer GFP-PS1 NT to RFP-PS1-loop proximity. (B)- Spectral FRET pseudo-colored image is produced by dividing fluorescence intensity of the image in the 598 spectral window by that in the 513 window on a pixel-by-pixel basis after background subtraction. The colorimetric scale bar shows 598/513 ratios from 0 (black, GFP and RFP far apart) to 0.5 (white, GFP-RFP close together). (C) - FLIM pseudo-color image of the GFP lifetime distribution in HEK293 cells transfected with G-PS1-R. The colorimetric scale bar shows lifetime in picoseconds. Note red pixels (shorter lifetime) are predominantly located at/near the cell surface. Found at: doi:10.1371/journal.pone.0007893.s002 (1.56 MB TIF) Acknowledgments We are very grateful to Drs. Tomas Kirchhausen and Dr. Saveez Saffarian (Harvard Medical School) for assistance and advice with the TIRF experiments, Dr. Rebecca Betensky for helpful discussions of the statistical analysis, and Mary Banks and Lauren Herl for technical assistance. Author Contributions Conceived and designed the experiments: BTH OB. Performed the experiments: KU CML XL JAP AI ZF. Analyzed the data: KU. Contributed reagents/materials/analysis tools: BD BJB. Wrote the paper: KU. 1. Yu G, Nishimura M, Arawaka S, Levitan D, Zhang L, et al. (2000) Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature 407: 48–54. 2. Francis R, McGrath G, Zhang J, Ruddy DA, Sym M, et al. (2002) aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev Cell 3: 85–97. PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7893