Figure 4
The mobilities of H2A.Z.1 and H2A.Z.2 are different in HeLa cells. (a) GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S and GFP-H2A were stably expressed in HeLa cells. Fluorescence images of HeLa cells stably expressing GFP-H2A (clone 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S are presented in the upper panels. The scale bar indicates 10 µm. The lower panel shows the distribution of the fluorescence intensities of GFP-H2A (clones 4 and 6), GFP-H2A.Z.1 (clones 2 and 5), GFP-H2A.Z.2 (clones 3 and 4), GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S represented in arbitrary units. (b) HeLa cells expressing GFP-H2A.Z.1, GFP-H2A.Z.2, GFP-H2A, GFP-H2A.Z.1 S38T and GFP-H2A.Z.2 T38S were subjected to FRAP analysis. The mobility of GFP-histones in living cells was analyzed by bleaching one-half of the nucleus in the presence of 100 µg ml-1 cycloheximide. Representative images before bleaching (left column), upon bleaching (0 min, centre column) and 180 min after bleaching (right column) are shown. The images for GFP-H2A, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in the top, middle and bottom rows, respectively. The scale bar indicates 10 µm. (c) The average relative fluorescence intensities of the bleached areas were plotted with their standard deviations (n = 11-36). The FRAP curves of GFP-H2A.Z.1, GFP-H2A.Z.2 and GFP-H2A are presented in blue, magenta and green, respectively. (d) Salt-resistance assay. The H2A nucleosomes (lanes 1-4), H2A.Z.1 nucleosomes (lanes 5-8) or H2A.Z.2 nucleosomes (lanes 9-12) were incubated in the presence of 0.4 M (lanes 1, 5 and 9), 0.6 M (lanes 2, 6 and 10), 0.7 M (lanes 3, 7 and 11) and 0.8 M NaCl (lanes 4, 8 and 12) at 328 K for 1 h. The samples were then analyzed by nondenaturing 6% PAGE with ethidium bromide staining. Bands corresponding to nucleosome monomers and nucleosome-nucleosome aggregates are indicated. Asterisks represent bands corresponding to non-nucleosomal DNA-histone complexes. (e) FRAP analysis of the H2A.Z.1 S38T and H2A.Z.2 T38S mutants. The average relative fluorescence intensities of the bleached areas were plotted with the standard deviations (n = 10-15). The FRAP curves of GFP-H2A.Z.1 S38T, GFP-H2A.Z.2 T38S, GFP-H2A.Z.1 and GFP-H2A.Z.2 are presented in dark blue, red, blue and magenta, respectively.  [article HTML]