FIG 5.
FRET between CLAG3 and RhopH2 at distinct sites within infected cells. (A) Live-cell fluorescence microscopy of a C3R2-DKI schizont-infected cell before and after acceptor photobleaching (PB). Right panels show RGB color scaling of mCerulean fluorescence intensity; increased red labeling of individual rhoptries in the bottom panels indicates increased mCerulean intensity after mVenus photobleaching. Color scale bar at right, 4 to 103 AU linear gradient. (B and C) Identical photobleaching experiments with LmC-R2mV negative-control parasite (RGB scale bar, 6 to 163 AU) and R3-C5V positive-control parasites (RGB scale bar, 0 to 298 AU). Images representative of 31 to 151 cells for each line. (D) Mean FRET efficiencies ± the SEM for indicated parasites using photobleaching FRET (PB-FRET; calculated from the donor mean fluorescence intensity [MFI] pre- and post-acceptor photobleaching) or PB-FLIM-FRET efficiencies (calculated from donor lifetimes pre- and post-acceptor photobleaching). *, P < 10−4. (E) Host membrane colocalization of CLAG3-mCerulean and RhopH2-mVenus in a trophozoite-stage C3R2-DKI-infected cell. Colocalization at the host membrane is highlighted (boxed region and dashed line). (F) Corresponding line scan with yellow and cyan lines representing mVenus and mCerulean intensities. (G) Three-dimensional surface plot from the along the dashed line in panel F. (H) Live-cell images of trophozoite-stage C3R2-DKI parasite before and after acceptor photobleaching to estimate FLIM-FRET. Right panels, RGB color scaling of mCerulean intensity (scale bar, 77 to 125 AU). (I) Mean FLIM-FRET efficiencies ± the SEM at the host membrane using C3R2-DKI parasites, calculated using both photobleaching and standard methods (PB and St, respectively; n = 2 cells each).