Orientation-Independent-DIC imaging reveals that a transient rise in depletion force contributes to mitotic chromosome condensation

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Abstract
Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion force/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the “chromosome milieu” during mitosis of living human cells using orientation-independent-differential interference contrast (OI-DIC) module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prometaphase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro , native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like, with further condensation. These results suggest that a transient rise in depletion force, likely triggered by the relocation of macromolecules (proteins, RNAs and others) via nuclear envelope breakdown and also by a subsequent decrease in cell-volumes, contributes to mitotic chromosome condensation, shedding light on a new aspect of the condensation mechanism in living human cells. Significance Statement: Mitotic chromosome condensation is an essential process to transmit replicated chromosomes into two daughter cells during cell division. To study the underlying physical principles of this process, we focused on depletion force/macromolecular crowding, which is a force that attracts large structures in crowded cell environments. Using newly developed special light microscopy, which can image the molecular density of cellular environments, we found that crowding around chromosomes increases during cell division. In vitro , higher concentrations of macromolecules condense chromatin and make it stiffer and more solid-like. Our results suggest that the rise in depletion force renders chromosomes more rigid, ensuring accurate chromosome transmission during cell division.
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