Fig. 2: Polar chromosomes rely on spindle elongation-driven pivoting of astral microtubules to pass the polar region.

a Upper panel: time-lapse images of RPE1 cells stably expressing EYFP-tubulin (white, confocal) and H2B-mRFP (not imaged) with added HaloTag-CENP-A (blue, confocal). Each image represents a single central z-plane and was rotated and cropped to align the spindle horizontally and position it in the center of the frame. The orange arrow denotes the kinetochore pair attached to the pivoting astral microtubule. Lower panel: Schematic representation of pivoting (red arrow) based on the images from the upper panel, showing the current and previous microtubule positions. b Time-lapse images of RPE1 cells stably expressing EYFP-tubulin (white, confocal) and H2B-mRFP (not imaged) with added HaloTag-CENP-A (blue, confocal). Moving and stagnant spindle poles are denoted in the first image. The blue arrow represents the microtubule attached to the polar kinetochore (KC) tracked over time. Yellow and orange arrows represent microtubules without attached polar kinetochores on the moving and stagnant spindle pole, respectively. Insets and schematic representations of the previously described microtubule types are shown below the images of the whole spindle; spindle poles are depicted as white circles. Images are maximum intensity projections of 3 central z-planes, corresponding to 3 µm. c Images of RPE1 cells stably expressing EB3-GFP (colorful, confocal, upper image) and H2B-mCherry (colorful, confocal, lower image). Images are temporal maximum intensity projections of the centrosome (upper) and chromosome (lower) movements over 3 central z-planes (3 µm) and a time period of 3.3 min, color-coded for time with the Spectrum LUT in ImageJ. Spindle pole movement is depicted with white arrow. d Schematic representation of measured parameters for polar kinetochores along with the starting (end of centripetal movement) and last (landing) tracking point (t = 0 min). Spindle length was determined as the distance between the two spindle poles. The pivoting angle was determined as an angle that the midpoint of the kinetochore pair closes with the long spindle axis. e The pivoting angle of polar kinetochore pairs and f corresponding spindle length in time. The landing point of each kinetochore pair is set to zero. Values are shown as mean (dark line) and SEM (shaded areas). N = 39 kinetochore pairs in 20 cells from 8 experiments. g Anticorrelation of the pivoting angle and spindle length for the same polar kinetochores as in e. Each color represents one polar kinetochore pair and each dot represents its pivoting angle and the corresponding spindle length in time. Linear regression. N = 39 kinetochore pairs in 20 cells from 8 experiments. h The pivoting angle of the microtubule attached to the kinetochores on the moving pole (blue), microtubule without attached kinetochores on the moving pole (yellow) and microtubule without attached kinetochores on the stagnant pole (orange) in time. Values are shown as mean (dark line) and SEM (shaded areas). N = 16 microtubules with polar kinetochores attached to them on the moving pole in 10 cells from 4 experiments. N = 16 microtubules without polar kinetochores attached to them on the moving pole in 10 cells from 4 experiments. N = 14 microtubules without polar kinetochores attached to them on the stagnant pole in 6 cells from 4 experiments. The fourth group, microtubules with polar kinetochores attached to them on the stagnant pole, could not be found, as poles were stagnant only when they were already at the nuclear border at NEBD and, therefore, had no polar chromosomes to begin with. i Schematic representation of the spindle pole movement and pivoting angle for each microtubule marked in b in time (from red to blue). Note that only the microtubule attached to polar kinetochores pivots around the spindle pole. j Schematic representation of the centrosome, astral microtubule and chromosome movements during pivoting. Note that the centrosome moves significantly while microtubules pivot under the weight of a chromosome which moves only slightly. All scale bars, 2 µm.