Fig. 2.

The spindle is more mobile in cells at the periphery of ESC colonies than in cells dividing inside the colonies. (A) Representative time-lapse spinning-disk confocal microscopy images of H2B–RFP (red)-expressing naïve ESCs labeled with CellMask™ Deep Red (cyan), with a cell dividing inside the colony (top panels) and a cell dividing at the periphery of the colony (bottom panels). One picture is shown every 2 min. 0 min corresponds to metaphase. One Z-plane is shown. Scale bars: 10 µm. (B) Schematic showing a 2D projection of the angle describing the orientation of the metaphase plate (see also panel D; the angle is measured in 3D). (C) Graph showing the 3D mean square displacement of the center of the metaphase plate (see Materials and Methods section) for cells dividing inside (gray) or at the periphery (red) of the colony, as a function of time interval. 0 min represents anaphase. The mean±s.e.m. are shown (N=4, n=11 inside, n=19 at the periphery). (D) Representative example of the dynamics of the angle between the metaphase plate and line connecting the center of the metaphase plate to the center of the colony (see panel B) for a cell dividing inside a colony (black) and a cell dividing at the periphery of the colony (red) as a function of time. 0 min represents anaphase. (E) Dot plot showing the angular motion (the difference in angle between consecutive time points) of the metaphase plate averaged over the last 12 min before anaphase for cells dividing inside the colony (gray) or at the periphery with radial (red) or orthoradial (orange) orientation. The mean±s.e.m. are shown (N=3). (F) Dot plot showing the volumes of the cell and of the spindle for cells dividing inside the colony (gray) or at the periphery (red) in H2B–RFP ESCs. The mean±s.d. are shown (N=3). (G) Dot plot showing the size asymmetry ratio between daughter cells for ESCs dividing isolated or in 3D colonies (inside or at periphery, as highlighted in legend) as a function of their volume in metaphase. N=3. (H) Representative images of H2B–RFP (red) expressing naïve ESCs labelled with CellMask™ Deep Red (cyan) for a cell dividing at the periphery of the colony (left) and a cell dividing inside the colony (right) 10 min before entry into mitosis (defined by nuclear envelope breakdown) (top) and in metaphase (bottom). One Z plane is shown. Scale bars: 10 µm. (I) Dot plot showing the relative volume change before and during mitosis in ESCs dividing inside (left) and at the periphery (right) of the colony. The mean±s.d. are shown. (J) Representative images of H2B–RFP (red)-expressing naïve ESCs labelled with CellMask™ Deep Red (cyan) for a cell treated with DMSO (top) and a cell treated with 100 nM Reversine (bottom). One Z-plane is shown. Scale bars: 10 µm. (K) Dot plot showing cell division duration (from nuclear envelope breakdown until anaphase onset) for naïve H2B–RFP ESCs treated with DMSO (left) or 100 nM Reversine (right). Cells dividing inside the colony are plotted in gray, cells dividing at the periphery of the colony are plotted in red. The mean±s.d. are shown N=3. (L) Contingency plot showing the percentages of cells dividing with (black) or without (gray) lagging chromosomes after treatment with DMSO (left) or 100 nM Reversine (right). N=3. P-values were calculated using a Welch's t-test (E), Student's t-test (F, cell; K, control inside versus Reversine outside), Mann–Whitney test (F, spindle, I,K) and Fisher's test (L).