Fig. 4: Chromosomes separate microtubule bundles and increase spindle width.

a Left, Vertical prometaphase spindle midplane in a HeLa-Kyoto cell expressing PRC1-GFP (white), before (t = 0) and after antiparallel bundle formation (t = 4.5 min). Right, trajectories of all bundles from time 0 (free end of each curve) until 4.5 min (arrowheads), where the points between the end points were smoothed for better visibility and raw data are shown in Supplementary Movie 5. Two large bundles visible at the top at time 0 are fixed in space (central points). Time interval between images is 2.7 s. Similar results were obtained in 8 independent experiments. b Midplane of a vertical prometaphase spindle, showing DNA stained with SiR-DNA (left block) or PRC1-GFP (right block), as indicated, in a HeLa-Kyoto cell expressing PRC1-GFP, at t = 2 min (magenta) and t = 4.5 min (green). Individual images are shown in white at the right of the merged images. Encircled areas marked “new” denote the chromosomes that arrived to the spindle midplane between t = 2 min and t = 4.5 min, arrows denote the movement of DNA (left) or PRC1 bundles (right). Similar results were obtained in 5 independent experiments. c Time-lapse images (sum of 41 planes) of a horizontally oriented spindle in a HeLa-Kyoto cell expressing PRC1-GFP (green), and DNA stained with SiR-DNA (magenta) starting at early prometaphase. d Spindle width and fraction of DNA at the metaphase plate over time. Colored surfaces around the central lines (mean) represent standard deviation, n = 10 cells (thin lines) from 10 independent experiments. e Live-cell images (maximum-intensity projections of 41 planes) of metaphase spindles in HeLa-Kyoto cells expressing PRC1-GFP (green), and DNA stained with SiR-DNA (magenta), treated as indicated. f Spindle width for the indicated treatments. The black line shows the mean; the light and dark gray areas mark 95% confidence interval on the mean and standard deviation, respectively; p values for significant differences from control are shown, one-way ANOVA test. Number of cells was 15–31 per group. Data for Kif18A siRNA are replotted from ref. 77. All scale bars, 1 µm.