Figure 1.

The distribution and mobility of DSBs in living WT and H2AX^−/− MEFs. (A) WT and H2AX^−/− MEFs expressing H2B-PAGFP were photoactivated with UV laser microirradiation in specific regions (circles and lines) within the nucleus (first row). DNA DSBs were introduced when cells were incubated with Hoechst 33342 DNA-binding dye (fourth row), as shown by γ-H2AX staining in WT cells (second row) and Nbs1 staining in WT and H2AX^−/− cells (third row). Bar, 30 μm. (B) WT MEFs expressing H2B-PAGFP were UV laser irradiated to photoactivate PAGFP and introduce DNA DSBs in subnuclear regions that were monitored over a 60-min time period. In the pre-UV panel, the green outline denotes the boundary of the nucleus, and the red circles denote UV laser–irradiated regions. Bar, 15 μm. (C) Mean squared displacement of the center of mass intensities of circular photoactivated and DSB-containing or solely photoactivated regions from their original position immediately after exposure to UV laser microirradiation until 10 min after irradiation. Image series were corrected for background and overall cellular migration by image registration before the calculation of center of mass intensities. Displacement values were calculated for WT with DSBs (triangles), WT without DSBs (circles), H2AX^−/− with DSBs (Xs), and H2AX^−/− without DSBs (squares). At least 40 cells were examined for each genotype and treatment. No significant difference was found between the mean squared displacement of regions containing or lacking DSBs (P > 0.25). Error bars represent SD. (D) WT MEFs expressing GFP-53BP1 were irradiated with 10 Gy γ irradiation and immediately placed on a heating stage of the LSM microscope. Foci, which appeared within 5 min, were tracked for 50 min. The yellow box denotes the region zoomed in the top right corner inset. The insets show multiple IRIF within close proximity to each other that frequently interact but then subsequently separate (yellow arrow). Bar, 5 μm.