ATP levels reversibly alter the chromatin compaction balance. (A) Effects of ATP depletion on chromatin organization. HeLa^H2B-2FP cells were imaged, and the distribution of both H2B-EGFP (green)– and mCherry-H2B (red)–tagged histones before and after 30 min ATP depletion or after subsequent washing away of inhibitors after the 30-min depletion followed by a 30-min recovery was addressed. Arrows indicate dense chromatin regions after ATP depletion. The boxed areas delineate particular ROIs in the field of cells, which are magnified in the panels to the right. (B) Transcription assay using 5-FU incorporation. Images show micrographs of HeLa cells pulse labeled for 30 min with 5-FU and immunolabeled with a mouse anti-BrdU antibody to visualize nascent transcripts either before (top) or after 30 min of ATP depletion (middle) or after subsequent washing and recovery for 30 min (bottom). (C) TEM micrographs of HeLa cells before and after ATP depletion. Untreated control cells (left), HeLa cells ATP depleted for 30 min (middle), and HeLa cells after 30-min ATP depletion followed by washing and recovery for 30 min (right) are shown. Closed arrowheads indicate heterochromatin at the nuclear periphery, and open arrowheads indicate heterochromatin around the nucleolus (Nol). Insets show higher magnification of ROIs in the field of cells (boxed areas). Cy, cytoplasm; Nu, nucleus. (D) Time-lapse FLIM–FRET measurements on HeLa^H2B-2FP cells during ATP depletion and recovery. The red boundaries surround micrographs showing HeLa^H2B-2FP cells at 5–30 min after ATP depletion, and the yellow boundaries surround micrographs showing HeLa^H2B-2FP cells during 10–30 min of recovery. The top panels show MPLSM images of H2B-EGFP fluorescence, and the bottom panels show the calculated FRET efficiency (percentage) over time in the selected ROI. In this case, the ROI includes the two interphase nuclei. Arrows indicate high FRET levels. (E) High magnification views of micrographs showing the spatial distribution of FRET efficiency. (F) Graph showing the time course of enrichment in high FRET pixels (red pixels) of two individual cells during ATP depletion and recovery (see Materials and methods). Bars: (A, B, D, and E) 10 µm; (C) 1 µm; (C, insets) 0.1 µm.