FRET–FLIM analysis of ERTGoCS. (A) Schematic representation of the TGN (TGN46-GFP; top construct) and ER (C tail of CytB5, Cb5; bottom construct) membrane reporter proteins used in this study (see Materials and methods for a detailed description of individual domains). (B) Graphic representation of the topology of the donor (TGN46-GFP) and the acceptor (mCherry-Cb5) fluorophores at the ERTGoCS under different conditions: left, normal-length linkers (16 amino acids used in the majority of the experiments; Várnai et al., 2007); middle, longer helical linkers (61 amino acids; Várnai et al., 2007); and right, 2 min treatment with 200 nM rapamycin. The mean FRET efficiency measured in the ROI is indicated for each condition. (C) The FLIM showing donor (TGN46-GFP) mean lifetime in the presence of the acceptor (mCherry-Cb5) in a defined ROI (top) compared with the whole TGN area (bottom) in cells expressing reporter proteins with normal-length linkers. The τ values are represented by a pseudocolor scale ranging from 1.8 to 2.5 ns. (D) FLIM (in color) and immunofluorescence (IF; gray) images of HeLa cells expressing the TGN46-GFP (donor) alone (top), TGN46-GFP and mCherry-Cb5 (donor+acceptor; middle), or TGN46-GFP with free cytosolic mCherry (bottom). The FLIM images show the spatial variation of the mean fluorescence lifetime (τ) of TGN46-GFP: τ values are represented by a pseudocolor scale ranging from 1.8 to 2.5 ns. (E) Mean τ value distribution curves of TGN46-GFP (donor) in cells expressing TGN46-GFP alone (top; red line), TGN46-GFP and mCherry-Cb5 (middle; green line), or TGN46-GFP with free mCherry (bottom; orange line). (F) The spatial distribution of the FRET efficiency (FRET [%] map) is depicted (color scale ranges from 0 to 30%).