Figure 1

Dynamic of Tat and Cdk9 at HIV-1 transcription sites. A-Accumulation of Tat, but not the C22G mutant, at HIV-1 transcription sites. U2OS_HIV-1 cells were transfected with Tat-GFP or Tat(C22G)-GFP, and then induced 7h with PMA/ionomycin. Cells were then fixed and hybridized in situ with a Cy3-labelled oligo probe against the MS2 repeat. The HIV-1 transcription site corresponds to the focal accumulation labelled by the MS2 probe. Blue: dapi. Each field is 22 × 22 μm. B-Dynamic of Tat at HIV-1 transcription sites. U2OS_HIV-1 cells were transfected with vectors expressing Tat-CFP and MS2-YFP. Tat-CFP was then bleached, and recovery was analyzed by tracking transcription sites in 3D with a wide-field microscope. Upper panel: colocalization of Tat-CFP and MS2-YFP in living cells (30 × 25μm). Middle panels: image sequence from a FRAP experiment (time in second; each field is 30 × 25 μm). Graph: recovery curves in the nucleoplasm of transfected U2OS cells (pink), or at the HIV-1 transcription site (blue). The best fit is shown in green. C-Dynamics of Cdk9 at HIV-1 transcription sites. U2OS_HIV-1 cells were transfected with vectors expressing Cdk9-GFP and MS2-mCherry. Cdk9-GFP was then bleached, and recovery was analyzed by tracking transcription sites in 3D with a wide-field microscope. Upper panel: colocalization of Cdk9-GFP and MS2-mCherry in living cells (30 × 25 μm). Middle panels: image sequence from a FRAP experiment (time in second; each field is 30 × 25 μm). Graph: recovery curves in the nucleoplasm of transfected U2OS cells (pink), or at the HIV-1 transcription site. Blue: cells were transfected with Tat; Green: Tat was absent but cells were induced by PMA/ionomycin.