Fig. 1: Polar chromosomes require an additional congression step to pass the polar region.

a STED superresolution images of microtubules 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 8–12 z-planes in prometaphase and 35 z-planes in metaphase, corresponding to 2.4–3.6 µm and 10.5 µm, respectively. The images represent different phases of mitosis: I – nuclear envelope breakdown (NEBD), II – a spindle starts forming and kinetochores in the polar region start their centripetal movement towards the spindle pole from the back, III – a prometaphase ring forms and centripetal movement of kinetochores ends, IV – a spindle rapidly elongates while kinetochores located at the back of the pole pass the polar region, V – spindle elongation ends and all kinetochores land onto the spindle surface, VI – a spindle is fully elongated and kinetochores travel towards the spindle equator. These stages were inferred from the arrangements of microtubules and kinetochores, see “Methods”. The polar region and the main spindle surface are denoted in image II. b Time-lapse images of RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP (colorful, lattice light-sheet (LLS)). Images show maximum intensity projections of the whole cell. Kinetochores are color-coded for depth with the brgbcmyw LUT in ImageJ throughout 32–40 z-planes, corresponding to 4.6–5.8 µm. Mitotic phases from I to VI correspond to the ones described in a. The polar region is denoted as a region behind two dashed lines perpendicular to the long spindle axes. Centrosomes are denoted with white circles. White arrows represent one polar kinetochore pair tracked from NEBD until alignment to the spindle equator. c Polar chromosome-centered (upper) and spindle-centered (lower) timelines of prometaphase events. Events are marked with roman numerals that correspond to the ones shown in a, b. The times are calculated based on 39 kinetochore pairs in 20 cells from 8 experiments. d Schematic representation of the first (end of centripetal movement) and the last (landing) known time points between which the time gap and possible passage across the polar region occur. e Three possible models for passing the spindle pole. In the first model, a motor bound to polar kinetochores brings them onto the spindle surface, representing diverse mechanisms based on kinetochore- and chromosome-bound motors. The second model proposes that spindle elongation drives pivoting of astral microtubules that consequently bring polar kinetochores in front of the spindle pole. In the last model, polar kinetochores attach to microtubules emanating from the opposite spindle half that bring them onto the spindle surface. All scale bars, 2 µm.