Fig. 1. Mobility of MAPPs during mitosis. Live U-2 OS cells stably expressing GFP-PML IV were imaged for the movement of MAPPs by DIC and fluorescence microscopy. (A) A cell in late G2 was followed into mitosis and fluorescence and DIC images were taken every 4 minutes (see Movie 1 in supplementary material). (B) A cell in mitosis was imaged by fluorescence and DIC microscopy and images were collected every 20 seconds. An initial DIC image is shown for t=0. Mobile MAPPs are indicated with arrows and relatively immobile MAPPs are indicated by arrowheads. (C) Single particle tracks of MAPPs in mitosis. The left panel shows a single frame of the movie of a mitotic cell in which several MAPPs (numbered 1-4) were tracked over time at 20 second intervals for 3 minutes. The relative position of each MAPP, corrected for cellular movement, was noted in each frame and plotted in the right panel. (D) PML protein dynamics in PML nuclear bodies in interphase and in MAPPs during mitosis. A single GFP-PML containing body or MAPP, respectively, was selected for FRAP analysis. An initial image was collected before a PML nuclear body (interphase, open circle) or MAPP (mitosis, closed square) was selected for bleaching. The selected PML nuclear body or MAPPs was then bleached and images collected every minute for 9 minutes. The intensity of the region of interest was normalised to the change in fluorescence intensity of the control, unbleached PML nuclear body, and expressed as a percent of the initial fluorescence (not shown). Several experiments were averaged (interphase n=20; mitosis n=13) and the mean percentage of fluorescence recovery was plotted over time in minutes. Bars, 5 µm (A,B); 1 µm (C).