Extended Data Fig. 4: Chromosome clustering is independent of F-actin, precedes enwrapment by the nuclear envelope and suppresses chromosome mobility.
From: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly
a, b, Effect of actin depolymerization on chromosome clustering. a, Live HeLa cells stably expressing actin–eGFP and H2B–mCherry were imaged in the absence (control) or presence of latrunculin B. Nocodazole was present in both conditions. b, Quantification of chromosome convex hull area during a flavopiridol induced mitotic exit in the presence of nocodazole, in the absence (control) and presence of latrunculin B as shown in a, normalized to average area pre-flavopiridol addition. Cell numbers: n = 13 (control), n = 22 (latrunculin B). c, Timing of chromosome clustering relative to nuclear envelopment. Live mitotic HeLa cell expressing H2B–mCherry and the inner nuclear envelope protein AcGFP–LAP2β were imaged in presence of nocodazole; flavopiridol was added at t = 0 min to induce mitotic exit. d, Quantification of chromosomal area by convex hull (yellow line in H2B channel in c) and quantification of AcGFP–LAP2β accumulation at the surface of the chromatin region, within a rim of 1.6 μm width (yellow ROI in LAP2β channel in (c)). n = 21 cells. e–h, Kinetochore tracking in cells progressing through spindle-less mitotic exit. e, Live mitotic HeLa cell stably expressing H2B–mCherry and CENP-A–eGFP imaged in the presence of nocodazole before and after flavopiridol addition, time-lapse = 10 s; z-projection. f, Kinetochore trajectories (total length: 240 s) starting 240 s before and at 400 after flavopiridol addition. g, Representative MSD analyses of kinetochore tracks (CENP-A–eGFP) of nocodazole-treated mitotic HeLa cells before and after flavopiridol addition. h, Diffusion coefficients derived from mean square displacement (MSD) analyses of trajectories as in f. Significance was tested by a two-tailed ratio paired t-test (****P = 2.9 × 10−11). n = 13 cells. Bars and lines indicate mean, shaded areas indicate ± s.d., dashed vertical lines refer to flavopiridol addition. Scale bars, 10 μm.
