Near millimolar concentration of nucleosomes in mitotic chromosomes from late prometaphase into anaphase

Abstract

Chromosome compaction is a key feature of mitosis and critical for accurate chromosome segregation. However, a precise quantitative analysis of chromosome geometry during mitotic progression is lacking. Here, we use volume electron microscopy to map, with nanometer precision, chromosomes from prometaphase through telophase in human RPE1 cells. During prometaphase, chromosomes acquire a smoother surface, their arms shorten, and the primary centromeric constriction is formed. The chromatin is progressively compacted, ultimately reaching a remarkable nucleosome concentration of over 750 µM in late prometaphase that remains relatively constant during metaphase and early anaphase. Surprisingly, chromosomes then increase their volume in late anaphase prior to deposition of the nuclear envelope. The plateau of total chromosome volume from late prometaphase through early anaphase described here is consistent with proposals that the final stages of chromatin condensation in mitosis involve a limit density, such as might be expected for a process involving phase separation.

Figure S1.

Establishing the CDK1^as synchronization system in RPE1 hTERT cells and characterization of the clones. (A) Western blot to detect CDK1as protein in the RPE1 CDK1^as clones, parental RPE1 hTERT (left), and HeLa CDK1^as (right) are shown as control. (B) Palbociclib block and release treatment for 24 h showing cells in G0/G1 (blue), S phase (dark green), and G2/M (light green). (C) Synchronization protocol (top) with the percentage of cells in interphase (blue), mitosis (green), and apoptosis (red) at 0, 90, and 150 min after the 1NM-PP1 washout. (D) Mitotic index counting the relative number of various mitotic stages. (E) Counts of the number of chromosomes in the RPE1 hTERT (parental cell line) and the 2 RPE1 CDK1^as clones, n = 20–50. Scale bar = 10 µm. (F) Representative karyotype of RPE1 CDK1^as clone 2 showing an extra chromosome 12 and an insertion in chromosome X, n = 20. Source data are available for this figure: SourceData FS1.

Figure S2.

Excess blocking time in G₂ increases the number of multipolar cells. (A) Representative images of monopolar, bipolar, and multipolar RPE1 CDK1^as cells. Scale bar = 5 µm. (B) Frequency of number of spindle poles (mono/bipolar and multipolar) after 8, 10, 12, and 20 h incubation with 1NM-PP1. (C) Percentage of apoptotic cells (0, 1.5, 2, and 2.5 h) after release from a 20 h incubation with 1NM-PP1. Each represents the time after release.

Figure S3.

The resolution of the image has little effect on the volume but a substantial effect on the surface area of the chromosomes. High-resolution versus low-resolution models of the same cell are represented at the top. Scale bar = 2 µm. Voxel size, memory size, chromosome volume, and chromosome surface area information are represented below each image.

Figure 1.

Quantitative 3D electron microscopy shows that metaphase chromosomes have a reproducible volume. (A) Representative image of an electron microscopy section of a mitotic RPE1 CDK1^as (left) and the intensity grey-scale values between the cytoplasm and the chromosomes (right). The red line represents the analyzed region of the mitotic cell. Scale bar = 2 µm. (B) Volume quantification of four metaphases. The red line represents the average between the four cells analyzed. (C) Orthoslice and segmented model of chromosomes (left; scale bar = 5 µm), front view (middle), and side view (right) of a mitotic RPE1 CDK1^as cell. (D) 3D karyotype and identification of chromosomes 1–5 and chromosomes 19–22. (E) Plot of chromosome volume versus DNA content used to calculate the DNA density in Mb/µm³ (n = 3). (F) Plot of chromosome length versus DNA content used to calculate the amount of DNA per µm (n = 3). (G) Cohesed chromosome (top) and chromatid (bottom) width. Left: Representation of chromosome 1 and the measurement taken. Right: Quantifications of chromosome and chromatid width ordered by chromosome in order of decreasing size (Chr1–5 and Chr 19–22). An average of 10 measurements per chromosome was plotted. The average chromosome width and SD for all the chromosomes are summarized below the graph. The average width ± SD is represented above the graph for large and small chromosomes. Two-tailed Student’s t test, n = 3 cells, P = 0.01915 (chromosomes), P = 0.004483 (chromatids).

Figure S4.

Correlation between chromosome/chromatid width and chromosome/chromatid length in metaphase and anaphase cells. (A–C) Metaphase chromatid width, (B) Metaphase chromosome width, and (C) Anaphase chromatid width. Each point represents the average of 10 measurements per chromosome. The slope of the line (blue) and P values obtained from an ANOVA test are annotated in each graph.

Figure 2.

Chromosomes in early prometaphase are longer, have a more granular surface, and lack a primary constriction. (A) Representation of early (Prometaphase 1–2) and late (Prometaphase 3–4) prometaphase chromosomes and a metaphase chromosome complement for comparison. Total chromosome volume and pole-to-pole (P2P) distance (red line) are annotated underneath each image. Centrosomes are red. Scale bar = 2 µm. (B) Individual segmented chromosomes for the above models are identified by different colors. Scale bar = 2 µm. (C) Representation of the largest chromosome (Chromosome 1) for each of the above cells. Scale bar = 2 µm. (D) Correlation between chromosome volume and surface area of individual chromosomes. (E) Correlation between chromosome volume and length of individual chromosomes.

Figure 3.

Chromatid length and width vary as anaphase progresses. (A) Models of four anaphase and two telophase cells with the total chromosome volume of each model annotated underneath each image. Scale bar = 5 µm. (B) Chromosome segmentations and orthoslice (left; scale bar = 5 µm). Separated sister chromatids are represented in the same color. Identification of chromatids 1–5 and chromatids 19–22 (right). (C) Correlation between the chromatid volume and DNA content of the anaphase cells (n = 4). (D) Correlation between the chromatid length and DNA content of the anaphase cells (n = 4). (E) Correlation between the chromatid surface area and DNA content of the anaphase cells (n = 4). (F) Chromatid width of anaphase cells and telophase 1 cell. Each dot represents the average of 10 measurements per chromatid. (G) Chromatid width is ordered by identified chromosomes (Chr1–5 and Chr 19–22). The average of 10 measurements per chromatid is plotted at each point. The average chromatid width and SD for all the chromatids is represented below the graph. The average width ± SD of large and small chromosomes are represented above the graph. Two-tailed Student’s t test, n = 4 cells, P = 0.00496.

Figure 4.

Small chromatids usually occupy the spindle interior and approach the poles more closely than large chromatids. (A) Representative images of separated chromosomes in one metaphase and four anaphase cells. Pole to pole (P2P) distance is shown under each image. Centrosomes are colored in red (arrows). Scale bar = 2 µm. (B) Representation of chromosome 1 of each cell. Sister kinetochore to kinetochore distance (sK2K) and sister telomere to telomere distance (sT2T) are shown under each image. Scale bar = 2 µm. (C) Graphic representation of various distances measured: Kinetochore to kinetochore (K2K), telomere to telomere (T2T), kinetochore to pole (K2P), and pole to pole (P2P). (D) Correlation between K2K distance and T2T distance. (E) Correlation between K2K distance and K2P distance. Each individual cell is shown in a single color. Chromosomes 1–5 are represented by big dots while chromosomes 19–22 are represented by small dots. (F) Left: Graphic representation of the relative location of large and small chromosomes. Right: Anaphase 4 model showing large (blue) and small (yellow) chromosomes in side-view and end-on view. Centrosomes are represented in red, with arrows, in both panels.

Figure 5.

Nucleosome density in mitotic chromosomes reaches a plateau in the near-millimolar range that remains relatively constant from late prometaphase through early anaphase. This plateau density is well above that calculated for nucleosome droplets in an in vitro chromatin phase (340 µM, dotted line; Gibson et al., 2019). Each circle represents the nucleosome concentration in a cell in which the entire chromosome volume has been measured in a 3View tomogram. The temporal order of the cells in prometaphase and anaphase/telophase was established by measuring the centrosome separation. The temporal order of the four metaphase cells is not known. Y axis, nucleosome concentration (µM). X axis, ATU (arbitrary time units).