Extended Data Figure 7: Ki-67 has little secondary structure, is highly positively charged, and its absence can be partly compensated by overexpression of core histones.

a, Folding and charge prediction of full length Ki-67 based on FoldIndex and EMBOSS webtools using a sliding window of 100. Unfolded regions are depicted in green, folded regions in orange. Positive charge is marked in blue, negative charge in red. b, Quantification of overexpressed histone levels in individual cells related to the mitotic chromosome morphology phenotype, classified by visual inspection. Note that the mean fluorescence values are not comparable to Fig. 3c as different imaging settings had to be used. Cells are from 4–5 independent experiments. c, Radial localization of overexpressed H2B–mNeonGreen in live Ki-67 knockout cells (n = 20). Normalized fluorescence intensity along line profiles across a chromosome arm of live Ki-67 knockout cells transiently transfected with H2B–mNeonGreen (upper panel) or Ki-67–mNeonGreen (lower panel) indicate that overexpressed H2B binds to the surface as well as internal region within chromosomes. d, e, Stable association of H2B–mNeonGreen with mitotic chromosomes. d, Half of the mitotic chromosomes in Ki-67 knockout cells highly overexpressing H2B–mNeonGreen were photobleached and the recovery of fluorescence was followed by time-lapse recording in an image region (yellow box). Representative example of the quantification in e. e, Curves indicate mean ± s.d. of 20 photobleached and 19 unbleached control cells. f, Quantification of mitotic chromosome area relative to total cell area for cells shown in b and Fig. 3c. Boxes indicate median, quartiles and 1.5 × interquartile range (n = 30 for Ki-67, n = 64 for H2B). g, Live Ki-67 knockout cell transiently transfected with a H2B–mNeonGreen and stained with SiR-Hoechst. Although 25 of 64 rescued cells displayed fully separated chromosomes (Fig. 3e), a large fraction of cells (39 of 64 rescued cells) showed detectable chromosome individualization at a lower extent compared to wild-type cells. Representative single z-section of the latter is shown. h, Live Ki-67 knockout cells transiently transfected with plasmids for expression of the indicated histone fused to mNeonGreen and stained with SiR-Hoechst. Representative single z-sections of 12–18 cells from 2–3 independent experiments are depicted. Although chromosome individualization was restored, chromosomes were not separated to the same extent as in wild-type cells. Scale bars, 10 μm.