 | Figure 2
Nature Structural & Molecular Biology
11, 1114 - 1121 (2004)
Published online: 3 October 2004; | doi:10.1038/nsmb837
PML bodies control the nuclear dynamics and function of the CHFR mitotic checkpoint proteinMatthew J Daniels, Alexander Marson
& Ashok R Venkitaraman | | | | Figure 2. Intermolecular FRET between CFP-CHFR and PML-YFP.
(a) A schematic of intermolecular FRET. CFP excited with 405-nm light usually emits at 470 nm (no FRET, left). If correctly oriented CFP and YFP fluorophores are within nanometer proximity, FRET occurs, quenching the usual 470-nm emission from CFP, but exciting 535-nm emission from YFP (FRET, right). Note that 405-nm light cannot directly excite YFP (Supplementary Fig. 1 online). (b) Cells cotransfected with CFP-CHFR and PML-YFP were fixed and visualized using 405-nm laser light. Emission spectra for CFP (green) and YFP (red) are shown, as is the merged image. Scale bar, 5  m. (c) YFP was irreversibly photobleached with 514-nm light in the areas bound by the white line, before visualization of CFP and YFP emission excited by 405-nm light. Scale bar, 5 m. (d) A pseudo-colored FRET image (right) shows the ratio of donor (CFP) emissions before (left) and after (middle) YFP photobleaching, according to ref. 12. Scale bar, 5 m. The pseudo color scale marks the FRET ratio from 0 (blue) to 6.0 (red). The image depicts the magnitude of interaction between donor and acceptor fluorophores within PML bodies, and also reveals the geography of that interaction as heterogeneities in the pseudo-colored FRET ratios. Results are typical of at least three independent experiments.
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