Figure 5.

FRET-FLIM analysis of the interaction between gRNA and Gag at the PM. (A) MS2-eGFP HeLa cells were transfected with a combination of plasmids, and FLIM analysis in the cytoplasm was carried out 24 h post-transfection. The fluorescence lifetime of MS2-eGFP-gRNA was determined by using a single exponential model and was color coded, ranging from red (2.0 ns) to blue (2.4 ns). Shown are FLIM images of gRNA in the presence of unlabeled Gag and free mCH (1), Gag-mCH (2), GagΔZF1-mCH (3), or GagΔZF2-mCH (4). (B) Shown are corresponding plots representing FRET efficiencies for Gag-mCH (circles), GagΔZF1-mCH (squares), and GagΔZF2-mCH (triangles). We performed three independent experiments on at least 30 cells. Above the threshold value (5%), FRET efficiencies can be considered as corresponding to a direct interaction between fluorescently labeled gRNA and Gag proteins. (44) FRET efficiency values were calculated as described in Materials and Methods (Eq. 1). Individual data points, corresponding mean values, and SEM are indicated. The statistical analysis was realized by a Student’s t-test with significant differences represented by ^∗p < 0.05. All images were acquired using a 50 × 50 μm scale and 128 pixels × 128 pixels. To see this figure in color, go online.