Extended Data Fig. 3: Chromosomes cluster and the nucleus reassembles in spindle-less cells after the induction of mitotic exit by different experimental procedures.
From: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly
a–d, Validation of mitotic exit in spindle-less cells by comparing H3(S10) dephosphorylation kinetics during unperturbed anaphase and spindle-less mitotic exit. a, Example images of wild-type HeLa cells during unperturbed anaphase fixed after time-lapse imaging, in metaphase, 6 min (maximally clustered) and 10 min after anaphase onset. Upper panel: chromatin labelled with H2B–mCherry, lower panel: H3(pS10) immunofluorescence. Single z-slice shown. b, Quantification of H3(pS10) mean fluorescence intensity and chromatin area in unperturbed mitosis, as shown in a. Cell numbers: n = 9 (metaphase), n = 14 (3-6 min after anaphase onset), n = 8 (≥8 min after anaphase onset). Normalization to average value of metaphase time point. c, Example images of wild-type HeLa cells during spindle-less mitosis fixed after time-lapse imaging, in prometaphase (no flavopiridol), 10 min (maximally clustered) and 20 min after flavopiridol addition. Imaging as in a. d, Quantification of H3(pS10) mean fluorescence intensity and chromatin area in spindle-less mitosis, as shown in (c), demonstrates that histone 3-serine 10 was efficiently dephosphorylated in flavopiridol-induced mitotic exit and chromosomes cluster to a degree comparable to that of normal late anaphase. Cell numbers: n = 11 (nocodazole arrested pro-metaphase), n = 12 (10 min after flavopiridol addition), n = 13 (20 min after flavopiridol addition). Normalization to average value of prometaphase time point. e, Time-lapse microscopy of HeLa cell expressing IBB–eGFP and H2B–mCherry incubated in nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. f, Quantification of IBB–eGFP mean fluorescence intensity within the chromosomal region, normalized to pre-flavopiridol time points, during spindle-less mitotic exit as in e. n = 20 cells. g, Time-lapse microscopy of a HeLa cell expressing H2B–mCherry, progressing through reversine-induced mitotic exit in the absence of spindle. Yellow line indicates convex hull around chromosomes, single z-slice shown. Time is relative to onset of clustering. h, Quantification of chromosome convex hull area of 11 cells as in g. Individual cell curves were aligned based on half-maximum value of convex hull area. Normalization to average of first 4 time points. i, Live HeLa cell undergoing mitosis upon RNAi-mediated depletion of the spindle checkpoint protein MAD2 in the absence of a spindle. The cell line stably expresses H2B–mCherry and membrane marker AcGFP–LAP2β. Time relative to nuclear envelope breakdown (NEBD), single z-slice shown. Representative example of 14 cells shown. Bars indicate mean in b, d; lines and shaded areas indicate mean ± s.d. in f, h. Scale bars, 10 μm.
