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

Abstract

Genomic information must be faithfully transmitted into two daughter cells during cell division. 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. Macromolecular crowding/depletion force, 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 macromolecular crowding (proteins and RNAs), likely triggered by the relocation of macromolecules 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 StatementMitotic 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 macromolecular crowding/depletion force, 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 macromolecular crowding renders chromosomes more rigid, ensuring accurate chromosome transmission during cell division.

Live-cell imaging under centrifugation characterized the cellular force for nuclear centration in theCaenorhabditis elegansembryo

“…From the OPD and the thickness of the object, the refractive index can be calculated. Because the refractive index is proportional to the dry mass and because the refractive increments of most substances in cells are approximately the same 26–28 , mass density in the living cell can be calculated from the refractive index 29 .…”

Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells

“…Chromatin puri cation was carried out as described by (Ura and Kaneda 2001;Iida et al 2023), with some modi cations. The nuclei (equivalent to ~ 2 mg of DNA) in nuclei isolation buffer (10 mM Tris-HCl, pH 7.5, 1.5 mM MgCl 2 , 1.0 mM CaCl 2 , 0.25 M sucrose, and 0.1 mM PMSF) were digested with 50 U of micrococcal nuclease (LS004797; Worthington) at 30°C for 1 h. In this digestion step, 1 µM HaloTag PEG-Biotin Ligand (G859A; Promega) or an equivalent amount of DMSO was added.…”

Estimation of the mass density of biological matter from refractive index measurements

“…The notion that biological function of cells and tissues is affected by their physical phenotype and vice versa has been validated in many experimental studies (see, e.g., ( 1 , 2 , 3 )). A fundamental property of matter, including living matter, is the mass density (MD) ( 4 ), which not only is associated with buoyancy, crowdedness ( 5 ), biomolecular condensation ( 6 ), and inherent dynamical processes of the sample of interest ( 7 , 8 ) but also plays a major role in elastography, particularly Brillouin microscopy ( 9 , 10 , 11 , 12 ) and transient optical coherence elastography (see, e.g., ( 13 , 14 )). However, measuring the in vivo MD distribution in a direct manner poses a challenge that has not been resolved so far.…”