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Fig. 5. Sucrose-induced chromatin compaction proceeds gradually with incubation time. Chromatin compaction during incubation with 320 mM sucrose medium was studied by live observation of GFP-tagged histone H2B in a HeLa cell nucleus. (A) Chromatin compaction became visible within seconds of hypertonic treatment, proceeded substantially over the first minute and slowly approached its steady state (pre, H2B-GFP signal of cell in DMEM before start of sucrose treatment; other labels show duration of sucrose incubation in seconds). (B) Upon re-incubation in normal growth medium, the chromatin distribution recovered to close to its initial state. In particular, the original dense domains appeared to be preserved (arrowheads). (C) Density analysis of the two image series in A and B (colors code for time points as indicated, images were corrected for gain and offset, and normalized to equal summed intensity to account for constant total amount of histones). Chromatin compaction in 320 mM sucrose is shown in the graph on the left and recovery in normal growth medium on the right. The density distributions are monophasic and no distinct intermediate or terminal states of chromatin compaction are revealed. Higher compaction states correlate with a larger width of distribution. Bar, 10 µm.
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