Fig. 6: Microtubules from the opposite side of the spindle facilitate the movement of polar chromosomes in the region close to the spindle surface.

a STED superresolution images of microtubules (MTs) immunostained for α-tubulin (white) in RPE1 cells stably expressing CENP-A-GFP (colorful, confocal). Images show maximum intensity projections of the whole cell. Kinetochores are color-coded for depth with the brgbcmyw LUT in ImageJ throughout 16 and 9 z-planes, corresponding to 4.8 and 2.7 µm, respectively. White boxes represent regions containing polar kinetochores attached to the microtubule from the opposite side of the spindle, which are enlarged to the right and represented schematically. White arrows denote the microtubule coming from the opposite side of the spindle. Enlargements are maximum intensity projections of 2 and 3 z-planes where the microtubule of interest is located, smoothed with 0.75-mm-sigma Gaussian blur. b Percentages of kinetochores that are unattached (blue) or attached to the microtubule from the opposite side of the spindle (yellow) measured in cells as in a. The shaded area represents kinetochores located at the angle above 90° with respect to the long spindle axis. N = 18 kinetochore pairs in 15 cells from 5 experiments. Statistical test: two-sided Fisher’s exact test. c Pie chart representing the percentage of different attachment types with the microtubule from the opposite side. N = 18 kinetochore pairs in 15 cells from 5 experiments. d Pie chart representing the percentage of polar kinetochores from LLS videos that initiate their movement towards the opposite pole after landing (blue), before landing at an angle below 90° (yellow) and before landing at an angle above 90° (orange), indicative of a force other than pivoting. N = 39 kinetochore pairs in 20 cells from 8 experiments. e Schematic representation of percentages of polar chromosomes that pivot without forming an attachment to the microtubule from the opposite side before landing (left), that pivot and attach to microtubule from the opposite side once they reach the angle below 90° (middle), and the ones that attach to microtubule from the opposite side at the angle above 90° (right). Scale bars, 2 µm.