Figure 1. Chromosome scattering volume is reduced in early prometaphase, independently of spindle MTs.

(A) Diagrams show how the chromosome scattering volume (CSV) was defined. Left-hand diagram shows how the convex hull was generated in two dimensions (2D) to represent chromosome distribution. A ‘rubber band’ (green) shrinks around chromosomes (blue) in 2D to create a convex hull or a minimum polygon wrapping chromosomes. Right-hand diagram shows how the convex-hull was generated in three dimensions (3D) to represent chromosome distribution. A ‘balloon’ (green) shrinks around chromosomes (dark green) in 3D to create a convex hull or a minimal polyhedron wrapping chromosomes. The volume of the 3D convex hull (chromosome scattering volume; CSV) quantifies how widely chromosomes are scattered in space. (B) CSV decreases immediately after NEBD. Images (top) are z-projections (z-sections are projected to 2D images) of a representative cell stained with SiR-DNA to visualize chromosomes (white) alongside their perimeter contours at z-sections (green lines; see Materials and methods). Time is shown, relative to NEBD. Timing of NEBD was determined by observation that GFP-LacI-NLS spread out of the nucleus (Figure 1—figure supplement 1). Scale bars, 6 µm. Bottom shows corresponding CSV (green). The left-hand graph shows CSV measurements in individual cells aligned by the time relative to NEBD (n = 9). To make the right-hand graph, these CSV values were normalized to the CSV value at NEBD in each cell, and the mean of the normalized CSV values were plotted at each time point. Error bars, s.e.m. (C) CSV decreases after NEBD even in the absence of MTs. Images (z-projections) show representative cell with SiR-DNA-stained chromosomes, which entered mitosis in the presence of 3.3 µM nocodazole (Nocod., bottom) or DMSO (control, top). 0.5 µM MK-1775 Wee1 inhibitor was used in both conditions to allow cells to enter mitosis. Time is shown relative to NEBD. Timing of NEBD was determined by observation that GFP-LacI-NLS spread out of the nucleus (Figure 1—figure supplement 1). Scale bars, 10 µm. Left-hand and center graphs show CSV measurements in individual control and nocodazole-treated cells, respectively (n = 10 each). The right-hand graph compares the means of normalized CSV between the nocodazole-treated and control cells (error bars, s.e.m.), as in the right-hand graph in (B). Reduction of normalized CSV was not significantly different between control and nocodazole treatment, when all the time points were considered by two-way ANOVA (p=0.81). Nonetheless, normalized CSV was significantly different between the two groups at +4 min (t-test, p=0.0016), but not at other time points.

Figure 1—figure supplement 1. Method for estimation of the NEBD timing.

Images of the same cell shown in Figure 1B. Top-row images show SiR-DNA-stained chromosomes. Bottom-row images show GFP-LacI-NLS (nuclear localization signal). The timing of NEBD was determined through observation of GFP-LacI-NLS spreading out of nucleus. Scale bars, 10 µm.

Figure 1—figure supplement 2. CSV decreases and actin accumulates on the NE around NEBD in asynchronous wild-type U2OS cells (without cdk1-as).

Images show single z-sections of a U2OS cell with SiR-DNA-stained chromosomes (blue), expressing mCherry-Lifeact (red) and undergoing NEBD. Yellow arrowheads indicate location of the actin network. Time is shown, relative to NEBD. Timing of NEBD was determined by observation that GFP-LacI-NLS spread out of the nucleus (Figure 1—figure supplement 1). Scale bars, 10 µm.

Figure 1—figure supplement 3. Confirmation that MTs are absent after nocodazole treatment.

Immunostaining of fixed cells to test the presence or absence of microtubules (MTs) within cells. Cells were analyzed by live-cell imaging for Figure 1C, and then fixed and immunostained for MTs (and LaminB1 as a control) to confirm that MTs were depleted within cells, after nocodazole treatment (right) but not in control. Images are projections of 4 Z-sections at 1 µm interval. Scale bars, 10 µm.

Figure 1—figure supplement 4. The chromosome distribution in three-dimensional space with and without nocodazole treatment.

The 3D convex hull of the cell in Figure 1C (top) is shown over the time course. The 3D convex hull (green) is viewed on two planes, that is x-y and z-x/y planes. ‘a’ and ‘b’ show the corresponding orientation in images and the 3D convex hull. Scale bars in images, 10 µm. The result suggests that chromosomes were spread more widely along the z-axis during +12 min to +24 min in the control cell than in the nocodazole-treated cell. This was generally the case when control cells and nocodazole-treated cells were compared. It explains why CSV are similar on average between the two conditions even if chromosomes are spread more widely on x-y planes during this period in nocodazole-treated cells than in control cells (Figure 1C).

Figure 1—figure supplement 5. Chromosome mass volume did not considerably change after NEBD.

Diagram (left) shows how CSV (chromosome scattering volume) and CMV (chromosome mass volume) were determined. Graph (right) shows the mean of CMV (n = 3) and CSV (n = 8). CSV data are from Figure 3A. Error bars, s.e.m.