Fig. 3

The invariant R540 residue in PFV Gag regulates nucleolar localization and binding to mitotic chromosomes. Living HeLa cells expressing the indicated GFP-GRs (a) or GFP-Gag (c) constructs and stained with Hoechst 33342 were observed on a confocal microscope 24 h after transfection (left panels). Merged images correspond to GFP, nucleic acid staining and differential interference contrast to visualize the cell shape. The “% nucleolar” column indicates the percentage of transfected cells displaying GFP staining in the nucleolus (−, < 1%; +, 1–25%; ++, 26–50%; +++, 51–75%; ++++ , 76–100%). To study the interaction of GFP-fusion proteins with chromatin (right panels), cells ectopically expressing indicated proteins were arrested in metaphase by treatment with colcemid (0.1 µg/mL, 2 h) and chromosome spreads counterstained with DAPI. Images were acquired as described in Fig. 1b. The chromatin binding column indicates whether the GFP-fusion protein was exclusively localized onto chromosomes (++), both on chromosomes and throughout the cell (+), or was distributed throughout the cell and did not associate with chromosomes (−). Representative images from two independent experiments are shown. Between 100 and 120 cells were analyzed for each condition. Scale bars represent 10 µm. b HeLa cells expressing the indicated GFP-GRs were stained with DAPI. Images were acquired as described in Fig. 1b. Scale bars represent 10 µm