FIG. 4.

Analysis of Gag-Gag interactions by FRET microscopy and spectral analysis. (A) Gag-CFP and Gag-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. The arrow indicates the selected plasma membrane region to be bleached. (B) The same cell as that shown in panel A is depicted following photobleaching at 514 nm. (C) Emission scans were obtained from the selected region of interest, with excitation at 458 nm (CFP excitation), before (solid line) and after (dashed line) photobleaching of YFP. Peak emission wavelengths for CFP (secondary peak) and YFP are indicated. (D) Gag-YFP distribution before bleaching. (E) Gag-YFP after photobleaching of indicated region. (F) Spectra obtained before (solid line) and after (dashed line) photobleaching of cells from panels D and E. (G) Cotransfection of Myr⁻ Gag-YFP and Myr⁻ Gag-CFP before photobleaching. (H) The same cell as that shown in panel G is depicted after photobleaching of indicated region of the cytoplasm. (I) Spectral analysis of the cell shown in panels G and H.