Fig. 2.
MECP2e1 and MECP2e2 are mobile in vivo and demonstrate identical kinetics. (A) FRAP was performed on EGFP-tagged MECP2e1 and MECP2e2. Individual pericentromeric heterochromatin foci were photobleached and the recovery was monitored over time. The intensity in the photobleached region was normalized to total nuclear intensity and plotted as a function of time. The recovery curves indicate rapid binding kinetics of both protein isoforms and nearly complete recovery of fluorescence within the bleached area. (B) Early recovery kinetics for the two protein isoforms were examined by performing FRAP experiments at a high scan speed of 323 mseconds. The rapid recovery kinetics of MECP2e2 in euchromatin are also shown. HC, heterochromatin; EU, euchromatin. (C) The upper panels demonstrate a bleaching sequence of a heterochromatic focus in a MECP2e2-expressing cell. The lower panels show a similar sequence in a euchromatic area of the nucleus. Bar, 5 μm. (D) The t50 values were determined by fitting the recovery curves. The t50 values of the two isoforms in heterochromatin are not statistically significant (MECP2e1=20.8±5.6 and MECP2e2=21.3±5.4). By contrast, the mobility of MECP2e2 in euchromatin is extremely rapid (t50=0.125±0.003; *P<0.0001). The number of nuclei photobleached is shown within each bar.