For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1–5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6–17], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [18–28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like “beads on a string” by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion . However, technical limitations to measure intracellular free divalent cations, but not total cations , especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+  and adenosine triphosphate (ATP)  indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell . ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance. How the negatively charged long genomic DNA is organized into mitotic chromosome remains unclear. Using a newly developed Mg2+ indicator, Maeshima et al. demonstrate a transient rise in free Mg2+ released from ATP-Mg during mitosis and suggest that the rise contributes to mitotic chromosome condensation by charge neutralization.
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