Figure 3.

FRAP analysis of GFP-HP1 shows that HP1 proteins are dynamic components of heterochromatin. (A) Nuclear localization of GFP-tagged proteins. GFP-HP1 expressing L929 cells were fixed with paraformaldehyde and localization of fusion protein was visualized with fluorescence microscopy. Dense foci seen by Hoechst staining represent pericentric heterochromatin regions. (B) Detection of GFP-HP1 and endogenous HP1 isoforms in stable cell lines and in L929 cells by Western blotting using specific antibodies for each isoform (α-HP1β, α-HP1γ and α-HP1α). (C) Time-lapse series of confocal images of living cells expressing GFP-HP1β, GFP-HP1γ or GFP-HP1α, as indicated. Heterochromatic foci were selected and photobleached. Images were recorded before (Prebleach) and at different time intervals after the bleach. Arrows indicate the photobleached areas. (B) Parameters of HP1 protein dynamics from FRAP curves. The half-time of fluorescence recovery (t1/2), the percentage of fluorescence recovery after 30 s, and the number of FRAP curves used are indicated. (E) Representation of the number of cells characterized by a given t1/2 value. A single area was photobleached per cell.