Contractile perinuclear actomyosin network promotes peripheral and polar chromosome interaction with the mitotic spindle
- Division of Molecular, Cell and Developmental Biology, School of Life Sciences, University of Dundee, United Kingdom
Figures
Figure 1 with 6 supplements
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 min. The timing of nuclear envelope breakdown (NEBD) is indicated by the dotted line. The PANEM is indicated by yellow arrowheads. Scale bar is 5 µm. (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 min following treatment with or without azBB followed by nuclear irradiation using a two-photon 860 nm laser. NEBD occurred at 0 min 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 10 µm. 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 are normalized to the volume at 0 min (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 is presented here again with standard error of mean (SEM) shown for each time point for each condition. In the lower right panel, the graph plots 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 a 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 30 min (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 was obtained using an unpaired Mann–Whitney test. (G) Graph shows the percentage of cells which exhibited chromosomes behind the spindle poles 20 min 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.
Figure 1—figure supplement 1
Chromosomes often show missegregation after PANEM contraction is inhibited.
(A) Time-lapse images show representative cells passing through the different stages of mitosis (late prophase to telophase). A stable cell line expressing H2B-mCherry and GFP-⍺Tubulin was treated (or not) with pnBB and imaged every 2 min. Times shown were relative to NEBD. In the lower panel of images, a yellow arrowhead indicates a chromosome that was not aligned at the metaphase plate and was subsequently missegregated. At nuclear envelope reformation, this chromosome formed a micronucleus (mn; pink arrowhead). Scale bars are 5 µm. (B) Graph shows the outcomes for mitotic cells as observed in time-lapse imaging exemplified in A. Cells that did not progress to anaphase after at least 2.5 hr after NEBD were considered arrested at metaphase (red). Many of these cells had apparent misaligned chromosomes locating behind spindle poles. An example of a cell that entered anaphase with chromosomes behind the spindle (orange) is shown in part A. Cells that progressed through anaphase normally (and did not show chromosome missegregation), exemplified in the upper panel of A, make up the remaining percentages of the graphs. Among the control cells, none of the cells became arrested at metaphase, and all progressed to anaphase after proper alignment of chromosomes at metaphase. The p values were obtained by Fisher’s exact test. The number of cells analyzed was 15 and 19 from control and pnBB-treated cells, respectively.
Figure 1—figure supplement 2
Inhibition of PANEM contraction does not lead to change in timing of mitotic cell rounding.
Graph showing the timing of the start of cell rounding after NEBD for control (red) or azBB-treated (blue) cells. Cell rounding was determined as the first moment that the cell morphology changed as the cell started to detach from the growing surface. The bars show mean and SEM. The p values were obtained by t-test. The number of cells analyzed was 14 for both control and azBB-treated cells.
Figure 1—figure supplement 3
Inhibition of PANEM contraction leads to delay in reduction of chromosome scattering volume (CSV) (data without normalization).
(A) Graphs show CSV before and after NEBD (0 min) for cells treated with or without azBB. This is from the same data shown in Figure 1D but before normalization. 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. (B) Graph plots the areas under the curves for CSV (in A), measured in individual cells. The bars represent the mean and SEM. The p value was obtained using t-test.
Figure 1—figure supplement 4
PANEM contraction and chromosome scattering volume (CSV) reduction occur in the absence of microtubules (MTs).
(A) Time-lapse images show representative cells passing through the early stages of mitosis (late prophase and prometaphase) following treatment with nocodazole and with or without azBB treatment. A stable cell line expressing mCherry-LifeAct and GFP-⍺Tubulin, with chromosomes visualized by SiRDNA, was imaged every 2 min. Times are relative to NEBD. The PANEM is indicated by yellow arrowheads. Scale bar is 10 µm. (B) Graphs show CSV after NEBD (0 min) for cells treated with nocodazole and with azBB (blue lines) or without azBB (red lines). Measurements were made from cells exemplified in A using methodology summarized in Figure 1B. The data from each cell was normalized to the volume at 0 min (immediately after NEBD). Here the mean, normalized values are presented with standard error of mean (SEM) shown for each time point for each condition. Normalized CSV mean, with nocodazole and without azBB (red line), slightly increased after 15 min, probably because chromosomes (not attached to spindle MTs) scattered again after the nuclear envelope remnants were lost. (C) The areas under the curves for CSV measurements in individual cells from data presented in B are shown here. The bars represent the mean and SEM. The p value obtained using t-test.
Figure 1—figure supplement 5
PANEM reduces its inside volume soon after NEBD.
(A) Time-lapse images show representative cells passing through the early stages of mitosis (late prophase to prometaphase). A stable cell line expressing mCherry-LifeAct and GFP-⍺Tubulin, with chromosomes visualized by SiRDNA, was treated (or not) with azBB and imaged every 2 min. Times are relative to NEBD. The PANEM is indicated by yellow arrowheads. Scale bars are 5 µm. (B) Graphs show PANEM-inside volume before and after NEBD (0 min) for cells treated with or without azBB (upper panels). Measurements were made from cells exemplified in A. In the lower panels the same data is normalized to the volume at 0 min (immediately after NEBD). Each red or blue line represents the measurements from an individual cell. (C) The mean of normalized values obtained in B for cells treated with or without azBB are presented here with SEM shown for each time point for each condition. (D) The areas under the curves for normalized PANEM-inside volume measurements in individual cells from data presented in B are shown here. The bars represent the mean and SEM. The p value obtained using t-test. Number of cells analyzed was 12 for both control and azBB-treated cells.
Figure 1—figure supplement 6
Distance between spindle poles is not affected by azBB treatment soon after NEBD.
On the left-hand side, a graph shows changes in distance between spindle poles of individual cells treated with (blue lines) or without azBB (red lines) during prometaphase. In the middle, the mean values are shown with bars representing the SEM. In these graphs, times are relative to NEBD. Measurements were made from cells imaged as in Figure 2B, D. We did not observe a significant difference in the spindle-pole distance between control and azBB-treated cells at any individual time points (the smallest p value was 0.094 at 6 min). On the right-hand side, the areas under the curves for individual cells (left-hand side graph) are shown. The bars represent the mean and SEM. The p value obtained using t-test. Number of cells analyzed was 8 and 11 for control and azBB-treated cells, respectively.
Figure 2 with 1 supplement
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 s. 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 white circle in the first image of the sequence. Scale bar is 10 µm. The graph on the right-hand side indicates the distance of the indicated kinetochore to the nearest spindle pole (dark blue line), the spindle mid-point (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 mid-point 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.
Figure 2—figure supplement 1
Examples of kinetochore tracking during the early stages of mitosis (prometaphase).
(A–C) Time-lapse images show representative cells passing through the early stages of mitosis (prometaphase). A stable cell line expressing CENPB-mCherry and GFP-⍺Tubulin was treated with or without azBB and imaged every 30 s. The merged images on the left-hand side show the cells of interest at NEBD (0 min). In each case, the central images show the changing position of the selected kinetochores, which were tracked through time. Here, the xy positions of the spindle poles are indicated by green circles while the tracked kinetochore is indicated in each frame by a yellow circle. For each time point, a single z-stack (or two z-stacks with maximum projection) containing the kinetochore of interest is shown. Scale bar is 5 µm. The graph on the right-hand side indicates the distance of the indicated kinetochore to the nearest spindle pole (dark blue line), the spindle mid-point (orange line), and the spindle mid-plane (magenta line). The colored boxes on the graph, and surrounding the central images, represent the different phases of motion (explained in Figure 2C). For part A, selected time points of this time-lapse sequence for this cell, with maximum projection of up to 10 z stacks for each time point, are shown in Figure 2B. In part A, the same graph shown in Figure 2B is reproduced here. In part C, in order to show the region of the cell containing both the kinetochore of interest and the spindle poles, the merged image on the left-hand side is a maximum projection of 6 z slices.
Figure 3
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 2 µm 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 toward a spindle pole and the spindle mid-plane, after NEBD. Scale bar is 10 µm. (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 gray. 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 center 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 min before entering Phase 2 and is excluded from this graph. (D) Graph shows the distance moved toward 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 toward 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 microtubules (MTs) during that period. The p values were obtained by t-test. (E–G) Graphs show the duration of Phase 2 (E), movement toward 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.
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Figure 3—source data 1
Raw coordinate data for peripheral kinetochores tracked in control cells.
Contains xyz coordinate data relative to time for spindle poles and peripheral kinetochores of control cells used for the analyses. The file is organized with the data from each cell grouped together. It also includes a single tab showing the timing of the different phases (described in Figure 2) for each kinetochore mentioned.
- https://cdn.elifesciences.org/articles/110952/elife-110952-fig3-data1-v1.xlsx
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Figure 3—source data 2
Raw coordinate data for peripheral kinetochores tracked in azBB-treated cells.
Contains xyz coordinate data relative to time for spindle poles and peripheral kinetochores of azBB-treated cells used for the analyses. The file is organized with the data from each cell grouped together. It also includes a single tab showing the timing of the different phases (described in Figure 2) for each kinetochore mentioned.
- https://cdn.elifesciences.org/articles/110952/elife-110952-fig3-data2-v1.xlsx
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Figure 3—source data 3
Raw coordinate data for central kinetochores tracked in control cells.
Contains xyz coordinate data relative to time for spindle poles and central kinetochores of control cells used for the analyses. The file is organized with the data from each cell grouped together. It also includes a single tab showing the timing of the different phases (described in Figure 2) for each kinetochore mentioned.
- https://cdn.elifesciences.org/articles/110952/elife-110952-fig3-data3-v1.xlsx
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Figure 3—source data 4
Raw coordinate data for central kinetochores tracked in azBB-treated cells.
Contains xyz coordinate data relative to time for spindle poles and central kinetochores of azBB-treated cells used for the analyses. The file is organized with the data from each cell grouped together. It also includes a single tab showing the timing of the different phases (described in Figure 2) for each kinetochore mentioned.
- https://cdn.elifesciences.org/articles/110952/elife-110952-fig3-data4-v1.xlsx
Figure 4 with 1 supplement
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. Graphs show 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]); and (D) 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). 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.
Figure 4—figure supplement 1
Comparison of non-polar peripheral kinetochore dynamics during each phase between control and azBB-treated cells.
(A–E) Graphs show the dynamics of non-polar peripheral kinetochores during Phases 1 and 2. The results were analyzed and presented as in Figure 3C–G, using the same data set, except that polar peripheral kinetochores were excluded. 22 and 19 kinetochores were analyzed from control and azBB-treated cells, respectively. The p values were obtained by t-test. A Mann–Whitney test was also performed on data for part A giving a p value of 0.044. (F–L) Graphs show the dynamics of non-polar peripheral kinetochores during Phases 3 and 4. The results were analyzed and presented as in Figure 4B–H, using the same data set, except that polar peripheral kinetochores were excluded. The p values were obtained by t-test. 22 and 19 kinetochores (from 9 and 9 cells) (F–I) and 19 and 6 kinetochores (from the corresponding cells) (J–L) were analyzed from control and azBB-treated cells, respectively.
Figure 5 with 3 supplements
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 5 µm. (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 Phases 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 before the end of the time-lapse sequence, or before tracking was no longer possible. 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 the start of congressional motion (time 0). The 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. The 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.
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Figure 5—source data 1
Raw coordinate data for polar kinetochores tracked in control cells.
Contains xyz coordinate data relative to time for spindle poles and polar kinetochores of control cells used for the analyses. The file is organized with the data from each cell grouped together. It also includes a single tab showing the timing of the start and end of congression for each kinetochore mentioned.
- https://cdn.elifesciences.org/articles/110952/elife-110952-fig5-data1-v1.xlsx
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Figure 5—source data 2
Raw coordinate data for polar kinetochores tracked in azBB-treated cells.
Contains xyz coordinate data relative to time for spindle poles and polar kinetochores of azBB-treated cells used for the analyses. The file is organized with the data from each cell grouped together. It also includes a single tab showing the timing of the start and end of congression for each kinetochore mentioned.
- https://cdn.elifesciences.org/articles/110952/elife-110952-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Further examples of kinetochore-spindle pole pivoting angles in azBB-treated cells.
Plot shows changes in pivot angles (defined as in Figure 2A) of polar kinetochores (at NEBD) over time after NEBD (time 0), from three different cells treated with azBB. Individual colored lines indicate individual kinetochores. The dotted line indicates the angle at which a polar kinetochore (>90°) passed into the non-polar (central) region (<90°). The number of polar kinetochores analyzed was 36. This graph contains the data from Figure 5D.
Figure 5—figure supplement 2
Dynamics of congression itself are not significantly affected by inhibition of PANEM contraction.
Graphs show the movement of polar kinetochores toward the spindle mid-plane during congression (left-hand side) or the average speed of motion toward the spindle mid-plane during congression (right-hand side) for individual polar kinetochores from control (red) or from azBB-treated (blue) cells. The p values were obtained by t-test. The 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 analyzed kinetochores are the same as those in Figure 5F.
Figure 5—figure supplement 3
The distance between the spindle poles of asynchronous U2OS cells rarely grows following NEBD.
(A) Time-lapse images show three representative cells passing through the early stages of mitosis (prophase and prometaphase), each one framed in a different color. A stable cell line expressing GFP-⍺Tubulin was imaged every 5 min. The timing of nuclear envelope breakdown (NEBD) is indicated by the dotted line. The cartoons below the images show the determined positions of the spindle poles (white circles) and the outline of the cells (white dashed lines). Scale bar is 5 µm. (B) Graph on the left-hand side shows the distances between spindle poles over time, with each line representing an individual cell. The distances of the spindle poles of the cells shown in A are the same color as the frames in A and represented with heavy lines and closed circles. On the right-hand side, the mean distance for all the data is shown with bars representing the SD. The number of cells measured is 26 cells.
Figure 6 with 1 supplement
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, was treated (or not) with azBB and were imaged every 2 min. 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 5 µm. 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.
Figure 6—figure supplement 1
PANEM contraction is important to reduce PANEM or chromosome volume behind the spindle poles following NEBD.
The left-hand side graphs show linear regression analysis of changes in PANEM-inside volumes (upper panel) or chromosome volumes (lower panel) over time, behind the spindle poles, for individual control (red lines and shapes) or azBB-treated (blue lines and shapes) cells. The same data as in Figure 6B, C were analyzed. The right-hand side shows the slopes of the lines plotted for control (red) or azBB-treated (blue) cells. The bars represent the mean and SEM. The p value was obtained by t-test.
Figure 7 with 1 supplement
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, was imaged every 30 s. NEBD is indicated at time 0. Scale bars: 5 µm. (B) Image 1.5 min before NEBD from the time-lapse sequence in A to indicate the positions of line profiles. Dotted lines, plotted from the cell center (for determination see Materials and methods), 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 toward 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.
Figure 7—figure supplement 1
More examples showing that the contractile PANEM directly pushes both polar and non-polar peripheral chromosomes inward during early stages of mitosis.
(A) Time-lapse images show another cell passing through the early stages of mitosis. Images were acquired and presented as in Figure 7A. NEBD is indicated at time 0. Scale bars: 5 µm. (B) Images 1 min before NEBD from the time-lapse sequence in A to indicate the positions of line profiles. Dotted lines, plotted from the cell center (for determination see Materials and methods), indicate the line profiles that pass the non-polar region of the cell (50° and 220°) and the polar region (100°) of the cell. In this cell, both spindle poles were close to the PANEM, so there is no polar volume. (C) Graphs show 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 to the chromosome mass center. (D) Graphs show a time sequence of intensities calculated along the line profiles that pass a non-polar region of the cell shown in A. Time progresses upwards and the color of the lines indicates the intensities seen 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. (F) Time-lapse images show a representative cell passing through the early stages of mitosis. Images were acquired and presented as in Figure 7A. In this cell, one of the poles is close to the PANEM, so there is only one pole displaying polar volume. NEBD is indicated at time 0. Scale bars: 5 µm. (G) Graphs show 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 F. As time progresses, peaks move to the left, which indicates movement closer toward the chromosome mass center.
Figure 8 with 2 supplements
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; Booth et al., 2019), and from a study conducted by another group (MCF10A; Stiff et al., 2020) were classified according to N-CIN+ or N-CIN−, that is with and without numerical chromosomal instability, respectively (Cohen-Sharir et al., 2021). Note that the PANEM status reported here for HCT116, RPE1, and U2OS cell lines was also confirmed by studies carried out independently of those from our lab (Stiff et al., 2020). (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 min after release from G2/M boundary. The timing of NEBD is indicated by the dotted line. Scale bar is 5 µm. (C) The graph on the left shows the average change in normalized chromosome scattering volume (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 min (immediately after NEBD) with standard error of the 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 a t-test. (D) Immunofluorescence of ⍺Tubulin and chromosomes (DAPI) in mitotic cells fixed 50 min after release from the G2/M boundary. Different cell lines are shown from top to bottom, and different outcomes for each were observed, shown with colored 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 arrowheads); in red, some chromosomes had not aligned and remained behind the spindle poles (red arrowheads). In the lower image panels, showing DNA, for each cell line, the white circles represent the position of the spindle poles. Scale bars: 5 µm. (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 a chi-square test for trends. The numbers of analyzed cells were 50, 76, 52, 80, 41, 64, 60, and 79 (left to right).
Figure 8—figure supplement 1
Examination of several human cancer cell lines for the presence or absence of PANEM during prophase.
Images show representative prophase cells of five cancer cell lines. The cancer cell lines were fixed, and Actin (left) and DNA (middle) were visualized using phalloidin and Hoechst, respectively. Actin/DNA merged images are also shown (right). Prophase cells were identified by visualizing early stages of chromosome condensation. PANEM was identified in the left-hand side panel of cells (indicated by yellow arrowheads), but not identified in the right-hand panel of cells.
Figure 8—figure supplement 2
PANEM contraction is required for reducing chromosome scattering volume (CSV) in the non-cancerous RPE1 cell line.
Graph shows CSV before and after NEBD (0 min) for RPE1 cells treated with (blue) or without (red) pnBB. Here the data from each cell is normalized to the volume at 0 min (immediately after NEBD). Each red or blue line represents the measurements from an individual cell. The mean for each condition is shown in Figure 8C (left-hand side). The number of cells for each group was 10 and 8 for control and pnBB, respectively.
Figure 9 with 1 supplement
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-colored 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 an MT, emanating from one of the spindle poles (Phase 1), so that subsequent movement toward that pole starts efficiently; (2) second MT interaction of sister kinetochore on a peripheral chromosome (Phase 3) allowing the start of congression toward the spindle mid-plane; and (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.
Figure 9—figure supplement 1
Model showing the effect of PANEM on peripheral/polar chromosomes in the situation where biorientation precedes congression.
Left-hand side shows a model of how the PANEM contraction helps to prevent chromosome misalignment by pushing a peripheral chromosome inward to a region of the cell with increased density of astral microtubules emanating from the opposite cell pole. The box below indicates how directed growth of astral microtubules promotes efficient capture and biorientation before congression to the cell mid-plane. With defective PANEM contraction (right-hand side), peripheral chromosomes may remain in regions with lower density of astral microtubules from the opposite cell pole. The box below indicates how less focused growth leads to less efficient capture and biorientation.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Human) | U2OS cdk1-as; male | Rata et al., 2018 | ||
| Cell line (Human) | U2OS (wild type); male | ATCC; HTB-96 | RRID:CVCL_0042 | |
| Cell line (Human) | HeLa; female | ATCC; CCL-2 | RRID:CVCL_0030 | |
| Cell line (Human) | hTERT RPE1; female | ATCC; CRL-4000 | RRID:CVCL_4388 | |
| Cell line (Human) | MCF7; female | ATCC; HTB-22 | RRID:CVCL_0031 | |
| Cell line (Human) | WI38; female | ATCC; CCL-75 | RRID:CVCL_0579 | |
| Cell line (Human) | HT29; female | ATCC; HTB-38 | RRID:CVCL_0320 | |
| Cell line (Human) | HCT116; male | ATCC; CCL-247 | RRID:CVCL_0291 | |
| Cell line (Human) | Caco2; male | ATCC; HTB-37 | RRID:CVCL_0025 | |
| Cell line (Human) | TT215; male | This paper | U2OS cdk1-as cells stably transfected with pGFP-⍺Tubulin; see Materials and methods section | |
| Cell line (Human) | TT113; male | This paper | U2OS cdk1-as cells stably transfected with pGFP-⍺Tubulin and pH2B-mCherry; see Materials and methods section | |
| Cell line (Human) | TT124; male | This paper | U2OS cdk1-as cells stably transfected with pGFP-⍺Tubulin and pmCherry-LifeAct; see Materials and methods section | |
| Cell line (Human) | TT230; male | This paper | TT215 cells stably transfected with pT3570; see Materials and methods section | |
| Cell line (Human) | TT352; female | This paper | hTERT RPE1 cells stably transfected with pGFP-⍺Tubulin; see Materials and methods section | |
| Recombinant DNA reagent | pCENPB-mCherry | Addgene; 45219 | RRID:Addgene_45219 | A gift from the laboratory of Michael Lampson |
| Recombinant DNA reagent | pGFP-⍺Tubulin | A gift from the laboratory of Jason Swedlow | ||
| Recombinant DNA reagent | pH2B-mCherry | A gift from the laboratory of Jason Swedlow | ||
| Recombinant DNA reagent | ploxBlastR | Arakawa et al., 2001 | A gift from the laboratory of Jean-Marie Buerstedde | |
| Recombinant DNA reagent | pmCherry-Lifeact-7 | Addgene; 54491 | RRID:Addgene_54491 | A gift from the laboratory of Michael Davidson |
| Recombinant DNA reagent | pT3570 | This paper | pCENPB-mCherry with the G418 resistance marker replaced with a blasticidin resistance marker from ploxBlastR | |
| Antibody | anti-Tubulin (Rat monoclonal) | Merck; mab1864 | RRID:AB_2210391 | Used at 1:1000 dilution |
| Antibody | A488 donkey anti-Rat | Invitrogen; A21208 | RRID:AB_2535794 | Used at 1:1000 dilution |
| Chemical compound, drug | 1NMPP1 | Sigma; 529581-1MG | Used at 1 µM | |
| Chemical compound, drug | RO-3306 | Selleck Chemicals; S7747 | Used at 8 µM | |
| Chemical compound, drug | Nocodazole | Sigma; M-1404 | Used at 3.3 µM | |
| Chemical compound, drug | MK-1775 | Selleck Chemicals; S1525 | Used at 0.5 µM | |
| Chemical compound, drug | para-nitroBlebbistatin (pnBB) | Motorpharmacology | Used at 10–50 µM | |
| Chemical compound, drug | para-nitroBlebbistatin (pnBB) | Tocris; 8804 | Used at 10–50 µM | |
| Chemical compound, drug | azido-Blebbistatin (azBB) | Motorpharmacology | Used at 5 µM | |
| Other | SiR-DNA | Spirochromoe; SC007 | Used at 100 nM | |
| Other | SYTO-deep red | Thermo Fisher; S34900 | Used at 1 µM | |
| Other | Hoechst 33342 | Sigma; 14533 | Used at 0.5 µg/ml | |
| Other | Prolong Gold Antifade containing DAPI | Thermo Fisher; P36935 | ||
| Other | phalloidin DyLight 650 | Cell Signalling Technology; 12956 | Used at 0.002 Units/µl | |
| Software, algorithm | Imaris | Bitplane | RRID:SCR_007370 | |
| Software, algorithm | Imaris, spot function | Bitplane | ||
| Software, algorithm | Imaris, 3D surface tool | Bitplane | ||
| Software, algorithm | Imaris, Convex Hull generation tool | Bitplane | Custom Convex Hull tool from filament tool adapted for 3D surface tool; https://doi.org/10.5281/zenodo.19822203 | |
| Software, algorithm | Imaris, Surface cut at defined spots tool | Bitplane | Custom surface cut tool adopted to cut at a perpendicular at user defined spots; https://doi.org/10.5281/zenodo.19822203 | |
| Software, algorithm | Fiji | https://www.fiji.sc | RRID:SCR_002285 | |
| Software, algorithm | Fiji, macro for tracking PANEM progress with respect to the cell center | Custom made; https://doi.org/10.5281/zenodo.19822203 | ||
| Software, algorithm | GraphPad | https://www.graphpad.com | RRID:SCR_000306 | |
| Software, algorithm | RStudio | https://posit.co/ | RRID:SCR_000432 | |
| Software, algorithm | Python script, fixes spindle pole position and recalculates raw kinetochore coordinates around this | Custom made; https://doi.org/10.5281/zenodo.19822203 |
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Contractile perinuclear actomyosin network promotes peripheral and polar chromosome interaction with the mitotic spindle
eLife 15:RP110952.
https://doi.org/10.7554/eLife.110952.3
