The outcome of selective and rapid suppression of perinuclear actomyosin network (PANEM) contraction.

(A) Time-lapse images show a representative cell passing through the early stages of mitosis (prophase and prometaphase). A stable cell line expressing mCherry-LifeAct and GFP-⍺Tubulin, with chromosomes visualized by SYTO deep-red was imaged every 1.5 minutes. The timing of nuclear envelope breakdown (NEBD) is indicated by the dotted line. The PANEM is indicated by yellow arrowheads. Scale bar is 5µm. (B) Diagram shows how the chromosome scattering volume (CSV) was defined. The convex-hull was fitted in three dimensions (3D) to represent chromosome distribution. A hypothetical ‘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 indicates how widely chromosomes are scattered in space. (C) Time-lapse images show representative cells passing through mitosis (prometaphase to metaphase). A stable cell line expressing GFP-⍺Tubulin, with chromosomes visualized by SiRDNA, was imaged every 4 minutes following treatment with or without azBB followed by nuclear irradiation using a 2-photon 860nm laser. NEBD occurred at 0 minute and was detected by diffusion of free GFP-⍺Tubulin into the nuclear space. Chromosomes that did not congress and lie behind the spindle poles are indicated by yellow arrowheads. Scale bar is 10µm. Time-lapse images, exemplified here, were used in the analyses in D–G. (D) Graphs in the upper panels show normalized chromosome scattering volume (CSV; see B) before and after NEBD (0 min) for cells treated with or without azBB. The data is normalized to the volume at 0 minutes (immediately after NEBD). Each red or blue line represents the measurements from an individual cell while the heavy black lines represent the mean measurement across the time points. In the lower left panel the mean of normalized values obtained above are presented here again with standard error of mean (SEM) shown for each time point for each condition. In the lower right panel, the graph plot the areas under the curves for normalized CSV, measured in individual cells. The bars represent the mean and SEM. The p value was obtained using t-test. The data without normalization is shown in Figure 1 – figure supplement 3. (E) Graph shows the percentage of cells, treated either with or without azBB, whose chromosomes were congressed before 30min (green) or not (orange). The number of cells for each group was 15. The p value was obtained using Fisher’s exact test. (F) Graph shows the time (from NEBD) taken for completion of congression in individual cells treated either with or without azBB. Open circle data points represent cells that had not completed congression at the end of the time lapse sequence. The bars represent the mean and SEM. The p value obtained using an unpaired Mann-Whitney test. (G) Graph shows the percentage of cells which exhibited chromosomes behind the spindle poles 20 minutes after NEBD. The cartoon shows microtubules (green), boundaries for polar regions (the black dotted lines), chromosomes (blue), and polar chromosomes (pink arrowheads). The number of cells analyzed for each group was 15. The p value was obtained using Fisher’s exact test.

Tracking kinetochores during early phases of mitosis reveals four major phases of motion leading to congression to the spindle mid-plane.

(A) To assess kinetochore motions during early mitosis (prometaphase), distances were calculated between the kinetochore and the nearest spindle pole, the spindle mid-point and the metaphase plate (mid-plane). To assess the location of the kinetochore relative to the spindle poles (polar or non-polar), the pivot angle was calculated. Designated color codes for each distance are indicated in the diagram. (B) Time-lapse images show a representative control cell passing through the early stages of mitosis (prometaphase). A stable cell line expressing CENPB-mCherry and GFP-⍺Tubulin was imaged every 30 seconds. The spindle poles are indicated by white arrowheads. The tracked kinetochore is indicated in each frame by a yellow arrowhead. The nearest spindle pole is indicated by a magenta star in the first image of the sequence. Scale bar is 10µm. The graph on the right-hand side indicates the distance of the indicated kinetochore to the nearest spindle pole (dark blue line), the spindle midpoint (orange line) and the spindle mid-plane (magenta line). The colored boxes on the graph, and above the images on the left-hand side, represent the different phases of motion (explained in C). (C) Kinetochore tracking revealed four distinct phases of motion as summarized in the cartoon on the left-hand side (described in detail in the main text). The bold lines in the theoretical graph on the right-hand side show the characteristic changes in distance to the different cellular locations (indicated in A) for the four phases. Phase 2 is characterized by rapid reduction in distance to the nearest spindle pole while Phase 4 by increase in distance to the spindle pole and reduction in distance to the midpoint and mid-plane. Color coding for each phase is indicated by the colored frames and boxes in the cartoon and the graph. (D) Time-lapse images show two representative cells passing through the early stages of mitosis (prometaphase). Cells were as described in B except they were treated with azBB. The features of the graphs on the right-hand side of the time-lapse sequences are as described in B.

Reduced PANEM contraction affects motions of peripheral (but not central) kinetochores during Phase 1.

(A) Image shows a cell in late prophase expressing CENPB-mCherry and GFP-⍺Tubulin. The white line indicates the position of the nuclear membrane. Kinetochores that fell within the yellow dotted line (positioned 2µm inside the nucleus) were considered central kinetochores, those between the two lines were considered peripheral kinetochores. There were a small number of non-kinetochore-derived mCherry signals, which localized outside the nucleus before NEBD and did not show any characteristic kinetochore motions, such as those towards a spindle pole and the spindle mid-plane, after NEBD. Scale bar is 10µm. (B) The motions of representative kinetochores are shown in 3D space over time in control cells (top) or azBB-treated cells (bottom). Kinetochores (colored dots) and spindle poles (turquoise circles) were tracked as in Figure 2 and positions were plotted relative to the nearest spindle pole (C2 in upper panel, C1 in lower panel) which was positioned at x=0, y=0, z=0. The position of the opposite spindle pole represents the average relative position of the pole during the time sequence. The colored lines of the kinetochore track represent Phase 1 (purple), Phase 2 (blue) as in Figure 2C. Phases 3 and 4 are shown in grey. The scales on all three axes are in µm. (C) Graph shows the duration of Phase 1 for individual kinetochores from control (red) or from azBB-treated (blue) cells. Kinetochores from the periphery are shown on the left-hand side and those from the centre on the right-hand side with a yellow-colored box. The p values were obtained by t-test. 26, 27, 20 and 14 kinetochores (from 9, 9, 6 and 7 cells) were analyzed from left to right. Note that in the peripheral azBB-treated column one kinetochore took 17.5 minutes before entering phase 2 and is excluded from this graph. (D) Graph shows the distance moved towards the spindle mid-point during Phase 1 for individual kinetochores from control (red) or from azBB-treated (blue) cells. In each panel, the graph on the left-hand side shows the net change in distance of individual kinetochores towards the spindle mid-point, while the graphs on the right-hand side show the net change in distance during the indicated time period (relative to NEBD) for the subset of kinetochores that had not interacted with MTs during that period. The p values were obtained by t-test. (E-G) Graphs show the duration of Phase 2 (E), movement towards the nearest pole during Phase 2 (F) and the average poleward speed during Phase 2 (G) for individual kinetochores from control (red) or from azBB-treated (blue) cells. The source data for these analyses (coordinates of kinetochores and spindle poles) can be found in Figure 3 – source data 1 - 4.

Reduced PANEM contraction leads to a delay in, or absence of, congressional movement of peripheral kinetochores.

(A) The motions of representative kinetochores are shown in 3D space over time in control cells (left) or azBB-treated cells (right), as in Figure 3B. The colors of the kinetochore track represent Phase 3 (green), Phase 4 (yellow) as described in Figure 2C. The scales on all three axes are in µm. The same kinetochores as in Figure 3B are shown here. (B–E) Graphs shows the start time of Phase 3 relative to NEBD (B), duration of Phase 3 (C), net change in distance relative to the nearest spindle pole (difference between the start and end of Phase 3 [or the end of observation if congression did not start during observation]; D), and the start of Phase 4 relative to NEBD (or the end of observation if congression did not start during observation; E) for individual kinetochores from control (red) or from azBB-treated (blue) cells. The p values were obtained by t-test. 26, 27, 20 and 14 kinetochores (from 9, 9, 6 and 7 cells) were analyzed from left to right. Open circles indicate kinetochores that did not start congression (the end of Phase 3 was not defined). (F–H) Graphs show the duration of Phase 4 (F), net change in distance relative to the spindle mid-plane during Phase 4 (G) and the average speed during Phase 4 (H) for individual kinetochores from control (red) or from azBB-treated (blue) cells. The p values were obtained by t-test. 23, 9, 19 and 14 kinetochores (from cells corresponding to B-E) were analyzed from left to right. (I) Summary of comparison between control and azBB-treated cells for peripheral kinetochore motions during early mitosis (prometaphase). Similarities (equal sign) or differences (greater than or less than sign) are shown between control and azBB-treated cells. When a phase starts later, it is indicated by a ‘greater’ sign. Red asterisks indicate presumed direct effects of reduced PANEM contraction.

PANEM contraction is important to reposition kinetochores in polar regions at NEBD for efficient congression.

(A) Image of a cell in late prophase expressing CENPB-mCherry and GFP-⍺Tubulin. The white line indicates the position of the nuclear membrane. The dotted green line is the line connecting the spindle poles. The magenta dotted lines are those on one spindle pole and perpendicular to the green dotted line. The polar regions are defined as the nuclear regions behind the pink dotted lines. Scale bar is 5µm. (B) The motions of representative kinetochores are shown in 3D space over time in control cells (top) or azBB-treated cells (bottom), as in Figure 3B. The colors of the kinetochore track represent Phase 1-3 (green), Phase 4 (yellow) as described in Figure 2C. The scales on all three axes are in µm. (C) Graph shows the percentage of polar kinetochores (at NEBD) that congressed in control (left) or azBB-treated (right) cells. The p values were obtained by Fisher’s exact test. Number of polar kinetochores was 23 and 22 from control and azBB-treated cells, respectively. (D) Plots show changes in pivot angles (defined as in Figure 2A) of polar kinetochores (at NEBD) over time after NEBD (time 0), from control cells (upper panel) and azBB-treated cells (lower panel). Individual colored lines indicate individual kinetochores. The dotted line indicates the angle at which a polar kinetochore (>90°) passed into the region between the poles (central region) (<90°). The number of polar kinetochores analyzed was 23 and 25 from 5 control and 2 azBB-treated cells, respectively. Figure 5 - figure supplement 2 shows the analyses of 35 polar kinetochores from 3 individual azBB-treated cells – the data only from the first two azBB-treated cells are shown in D to avoid overcrowding in the graph. (E) Plots show changes in pivot angles of polar kinetochores (at NEBD) over time, as in D but aligned according to start of congressional motion (time 0). Number of polar kinetochores analyzed was 23 and 10 from 5 control and 3 azBB-treated cells, respectively. For azBB-treated cells, plots show only the polar kinetochores that subsequently exhibited congression. (F) Graph shows the duration of Phase 4 for polar kinetochores (at NEBD) from control (red) or from azBB-treated (blue) cells. The p values were obtained by t-test. Number of polar kinetochores was 20 and 9 from 5 control and 3 azBB-treated cells, respectively. For azBB-treated cells, the graph includes only the polar kinetochores that subsequently showed congression. The source data for these analyses (coordinates of kinetochores and spindle poles) can be found in Figure 5 – source data 1 and 2.

PANEM contraction leads to a rapid reduction of the PANEM-inside volume and chromosome volume at polar regions in early mitosis.

(A) Time-lapse images show representative cells passing through the early stages of mitosis (prophase and prometaphase). A stable cell line expressing mCherry-LifeAct and GFP-⍺Tubulin, with chromosomes visualized by SiRDNA, were treated (or not) with azBB and were imaged every 2 minutes. Times shown were relative to NEBD. In the left-hand images the PANEM is indicated by yellow arrowheads. On the right-hand side, selected images have been reproduced to highlight segmentation. In the upper images, polar regions are designated by spindle poles (green dots) and their perpendicular planes (magenta dotted lines) (see Figure 5A). Chromosome (middle images) or PANEM (lower images) volumes behind or between the spindle poles are colored with magenta or green shading, respectively. In the middle images, solid white lines represent the cell nucleus (before NEBD) and white dotted lines represent the cell periphery. In the lower images, yellow lines represent the PANEM. Scale bars are 5µm. (B and C) Graphs show changes in PANEM-inside volume (B) and chromosome volume (C) behind the spindle poles as calculated for control (red lines) or azBB-treated (blue lines) cells. In the upper graph, the changes at individual polar regions are shown while in the lower graph, the change in mean is shown. The bars represent the SEM. The p value was obtained by t-test performed after regression analysis (see Figure 6 – figure supplement 1). In B and C, the same polar regions were analyzed.

Evidence that the contractile PANEM directly pushes both polar chromosomes and non-polar peripheral chromosomes inward during early stages of mitosis.

(A) Time-lapse images show a representative cell passing through the early stages of mitosis (prophase and prometaphase). A stable cell line expressing mCherry-LifeAct and GFP-⍺Tubulin, with chromosomes visualized by SYTO deep-red, were imaged every 30 seconds. NEBD is indicated at time 0. Scale bars 5µm. (B) Image 1.5min before NEBD from the time-lapse sequence in A to indicate the positions of line profiles. Dotted lines indicate the line profiles that pass the non-polar region (150°) and the polar region (270°) of the cell. (C) Graphs showing line profiles, offset in the y-axis according to time, for PANEM (upper panel; orange frame) or chromosomes (lower panel; blue frame) for the lines indicated in B. As time progresses peaks move to the left, which indicates movement closer towards the chromosome mass center. (D) Graphs show a time sequence of intensities calculated along the line profiles that pass the polar region of the cell shown in A. Time progresses upwards, and the colored lines indicate the intensities for Actin (PANEM; orange) or chromosomes (blue). (E) Graph shows the progression of the relationship between the PANEM peak and the chromosome front, over time, through the line profiles indicated in B.

PANEM formation and function in non-cancer and cancer cell lines with and without numerical chromosomal instability.

(A) Cell lines from this study (the U2OS cell line primarily used in this study and five cell lines in Figure 8 – figure supplement 1), from our previous study (HeLa and RPE1 20), and from a study conducted by another group (MCF10A 21) were classified according to N-CIN+ or N-CIN-, i.e. with and without numerical chromosomal instability, respectively 56. Note that the PANEM status reported here for HCT116, RPE1 and U2OS cell lines were also confirmed by studies carried out independently of those from our lab 21. (B) Time-lapse images show a representative RPE1 cell passing through the early stages of mitosis (prophase and prometaphase). A stable cell line expressing GFP-⍺Tubulin, with chromosomes visualized by SYTO deep-red was imaged every 2 minutes after release from G2/M boundary. The timing of NEBD is indicated by the dotted line. Scale bar is 5µm. (C) The graph on the left shows the average change in normalized CSV for RPE1 cells imaged in B before and after NEBD (0 min) for cells treated with or without pnBB. The data from each cell was normalized to the volume at 0 minutes (immediately after NEBD) with standard error of mean (SEM) shown for each time point for each condition. The graph on the right plots the areas under the curves, measured in individual cells (which are shown in Figure 8 – figure supplement 2). The bars represent the mean and SEM. The number of cells for each group was 10 and 8 for control and pnBB, respectively. The p value was obtained using t-test. (D) Immunofluorescence of ⍺Tubulin and chromosomes (DAPI) in mitotic cells fixed 50 minutes after release from G2/M boundary. Different cell lines are shown from top to bottom, and different outcomes for each were observed, shown with coloured frames, from left to right. Those in green frames represent cells with successful alignment of all chromosomes; in orange some chromosomes had not completely aligned but were between the spindle poles (yellow arrow heads); in red some chromosomes had not aligned and remained behind the spindle poles (red arrow heads). In the lower image panels, showing DNA, for each cell line, the white circles represent the position of the spindle poles. Scale bars 5µm. (E) Quantification of chromosome alignment outcomes in different cell lines following treatment with DMSO (control) or pnBB for cells exemplified in D. Outcomes are shown in green, orange and red bars, as defined and using the same colors as in D. The p values were obtained using chi-square test for trends. The numbers of analyzed cells were 50, 76, 52, 80, 41, 64, 60 and 79 (left to right).

Model showing the effect of PANEM on peripheral/polar chromosomes.

Left-hand side shows the model of how the PANEM contraction helps to prevent chromosome misalignment and subsequent missegregation (not shown) by pushing peripheral and polar chromosomes inward so that they can be efficiently captured on spindle MTs and subsequently transported to the spindle mid-plane where they become bioriented. With reduced PANEM contraction (yellow-coloured box), chromosomes often remain in polar regions. On the right-hand side, the cartoons framed in boxes show the three main effects of PANEM contraction: (1) initial capture of a kinetochore on a peripheral chromosome by a MT, emanating from one of the spindle poles (Phase 1), so that subsequent movement towards that pole starts efficiently; (2) second MT interaction of sister kinetochore on a peripheral chromosome (Phase 3) allowing the start of congression towards the spindle mid-plane; (3) relocation of a polar chromosome to the region between the spindle poles to facilitate productive MT interactions. Figure 9 – figure supplement 1 shows an alternative model of how PANEM contraction advances the onset of chromosome congression.