Chromosomes undergo dramatic compaction during mitosis, but accurately measuring their volume has been challenging. Employing serial block face scanning electron microscopy, Cisneros-Soberanis et al. (https://doi.org/10.1083/jcb.202403165) report that mitotic chromosomes compact to a nucleosome concentration of ∼760 µM.

In the late 19th century, Walther Flemming reported a dramatic architectural conversion of chromatin at the transition from interphase nucleus to mitotic chromosomes, termed “Karyomitosis (meaning threadlike metamorphosis of the nucleus)” (1). This event now commonly referred to as mitotic “chromosome condensation” involves two distinct processes, chromatin condensation and chromosome individualization. Whereas chromatin condensation shortens the time to segregate separated sister chromatids into dividing cells, chromosome individualization ensures that the movement of each chromosome is not affected by the other so that they are distributed equally by attached microtubules. Emerging ideas indicate that chromatin condensation involves inter-nucleosome interaction via the positively charged histone tails, while condensin-mediated DNA loop formation and the topoisomerase II–mediated DNA strand passing drive chromosome individualization (2, 3). The two mechanisms must balance each other to form individualized chromosomes with proper size and width, as extensive chromatin condensation can limit the access of condensin and other factors important for mitotic processes. Then, how densely are nucleosomes packed in mitotic chromosomes? In this issue of JCB, Cisneros-Soberanis et al. use serial block face scanning electron microscopy (SBF-SEM) to determine the volume of mitotic chromosomes and report that nucleosome concentration reaches “near millimolar” levels from prometaphase to early anaphase, when chromosomes are most compacted and under the highest mechanical stress due to segregation forces (4).

In eukaryotes, chromosomal DNA is densely folded by the nucleosome, where ∼146 bp DNA wraps around the core histone octamers to form the “bead-like” 11 × 5.5-nm-size structure, and each nucleosome is separated by ∼50 bp linker DNA. This “beads on a string” configuration is further folded to form mitotic chromosomes, but the exact manner by which this folding occurs has been the subject of debate. Although it has been long depicted in textbooks that the nucleosome arrays organize into secondary 30-nm fibers, it is now accepted that nucleosomes are not arranged into such an ordered configuration in mitotic chromosomes (5). Instead, it has been suggested that nucleosomes exist in the polymer melt, the viscoelastic liquid-like state where a nucleosome does not necessarily interact with most adjacent nucleosomes within the same fiber. However, the liquid-like behavior of nucleosomes does not necessarily mean that liquid–liquid phase separation (LLPS) is the driving force of mitotic chromosome compaction, and mitotic chromosomes do not exist as spherical droplets that would form by LLPS (6). LLPS of chromatin would noncovalently fuse different chromosomes and interfere with chromosome individualization. Since LLPS depends on the concentration of the subject molecules (as well as many other factors, such as salt concentration and temperature), accurately determining the nucleosome concentration in mitotic chromosomes would be helpful in understanding the underlying mechanisms behind chromosome condensation.

Cisneros-Soberanis et al. addressed this question by measuring the volumes of mitotic human chromosomes in hTERT-immortalized retinal pigment epithelial (RPE1-hTERT) cells using SBF-SEM with the sampling resolution to 4 nm in the XY axis and 60 nm in the Z axis. As conducting EM analysis on multiple cells is technically challenging, they used a cell line expressing the “analog-sensitive” CDK1as, which allows for precise cell cycle synchronization during mitosis (4). They first reconstructed 3D structures of metaphase chromosomes and measured the physical parameters such as volume and length. By correlating these measurements with known DNA content, they calculated a DNA packing ratio of 66 ± 5.7 Mb/μm, corresponding to an overall DNA density of 84.3 ± 2.86 Mb/µm3 in metaphase chromosomes—equivalent to packing a 3-cm DNA fiber into a 1-µm3 box.

To calculate nucleosome packing in metaphase chromosomes, the authors estimated that there are 4.57 × 105 nucleosomes per µm3 in the RPE1 cells, assuming that a nucleosome is positioned every 195 bp (including linker DNA) and nucleosome-free regions represent 0.3% of the genome, based on the previously reported assay for transposase-accessible chromatin using sequencing analysis. This suggests that each nucleosome occupies 2,186 nm3 within metaphase chromosomes. Based on this value, they estimated the average nucleosome concentration in metaphase chromosomes to be ∼760 µM. The authors reported that the volume of the mitotic chromosomes decreased from ∼250 to 150 µm3 during prometaphase, achieving maximum compaction during metaphase to early anaphase (∼140 µm3), followed by gradual decompaction from mid-anaphase to telophase. From these observations, the authors estimated that the nucleosome concentration maintains >400 µM from prometaphase to early telophase, while it reaches a maximum concentration of ∼760 µM in metaphase and early anaphase. These numbers are in line with the nucleosome concentration of ∼500 µM (or 137 mg/ml) and protein/DNA density of 192 mg/ml that had been previously estimated in Indian Muntjac cells using laser scanning microscopy and orientation-independent differential interference contrast microscopy by the Maeshima group (7, 8).

According to in vitro biochemical studies, nucleosome concentration inside chromatin LLPS droplets is ∼340 µM (9), leading Cisneros-Soberanis et al. to consider the possibility that mitotic nucleosome concentration (>400 µM) is high enough to exhibit LLPS characteristics. However, the study by the Maeshima group also showed that nucleosome concentrations inside chromatin LLPS droplets can be increased to >1 mM by crowding agents such as polyethylene glycol (PEG) and bovine serum albumin (BSA), and those droplets became stiffer and resistant to fusing each other (8). Maeshima and colleagues proposed that increased density of mitotic cytoplasm (∼170 mg/ml) from interphase nucleoplasm (∼140 mg/ml) makes chromatin stiffer (i.e., solid-like) through enhancing the depletion attraction/macromolecular crowding effect. Notably, DNA digestion within mitotic chromosomes converts chromatin in spherical droplets, suggesting that the network of DNA acts as a physical constraint and helps maintain chromosome individualization together with condensin-mediated DNA looping (2, 10). Another study by the Maeshima group investigating the single-molecule dynamics of nucleosomes in mitotic chromosomes also suggested that nucleosome movement is more constrained in mitosis than in interphase, exhibiting gel-like behavior in a condensin-dependent manner (3). Such physical constraints are critical for resisting microtubule-mediated forces.

The study by Cisneros-Soberanis et al. also reports observations that are not consistent with published results on mitotic chromosomes. First, based on live microscopy measurement of mitotic chromosomes labeled with EGFP-H2B, the Ellenberg group has previously concluded that maximum chromosome compaction is achieved during anaphase (11), though Cisneros-Soberanis and colleagues did not observe this transient increase of chromosome compaction in anaphase. Second, Kakui and colleagues reported that the width of mitotic chromosomes correlates with chromosome arm length (12), whereas Cisneros-Soberanis et al. suggest that while such a correlation may exist, the difference is much more subtle. These discrepancies may be caused by differences in sample preparation methods (e.g., fixation), chromatin visualization methods, choice of cell lines, and low sample numbers.

During the short period of mitosis, numerous proteins must engage with compacted chromosomes to ensure faithful chromosome segregation in a crowded mitotic cellular milieu (∼170 mg/ml protein density) (8). The reported high nucleosome density within mitotic chromosomes may explain their stiffness, while it remains to be established how the LLPS-like behavior of nucleosomes is constrained to ensure mitotic chromosome individualization and accessibility.

H. Funabiki is supported by grants from the National Institutes of Health (R35GM132111), the Stavros Niarchos Foundation (SNF) as part of its grant to the SNF Institute for Global Infectious Disease Research at The Rockefeller University, and Anderson Center for Cancer Research at The Rockefeller University. H. Funabiki is affiliated with the Graduate School of Medical Sciences, Weill Cornell Medicine.

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