Extended Data Fig. 9: Characterization of peripherally localized missegregated chromosomes and the resulting MN: microtubule proximity, nuclear import, and chromosome number.
From: Nuclear envelope assembly defects link mitotic errors to chromothripsis
a, Restored assembly of NUP133 on MN derived from peripheral chromosomes as compared to central spindle lagging chromosomes in HeLa K cells. Left, quantification of the fraction of the lagging chromosome circumference with NUP133 (mean with 95% CI, n = 32, 40, from two experiments). ****P < 0.0001, two-tailed Mann–Whitney test. Right, western blots showing depletion of TTL by siRNA in HeLa K cells (three experiments; for gel source data see Supplementary Fig. 1). b, Fewer microtubules associated with peripheral missegregated chromosomes (red arrow) than with MN from central missegregated chromosomes (yellow arrow). Synchronization as in Fig. 4a. Representative images of RPE-1 cells after glutaraldehyde fixation (representative of 30 central or peripheral missegregated chromosome, from two technical replicates). Scale bars, 10 µm. The sparse microtubule density near the peripheral chromosome (bottom images) was never associated with more than three CHMP4B foci in 30 images. c–e, Normal nuclear import and DNA replication in MN from peripherally missegregated chromosomes (red arrows) as compared to MN from central missegregated chromosomes (yellow arrows). c, Top, scheme of experiment. RPE-1 cells were treated with p53 siRNA to facilitate cell-cycle progression. After mitotic exit, micronucleated cells were imaged at 20-min intervals. Bottom, images of fixed and labelled RPE-1 cells 4 h after RO release at the end of the live-cell experiment. Scale bars, 10 µm. See Fig. 4d for quantification. d, MN/PN FI ratio for lamin B1 in RPE-1 cells, 4 h after RO release (mean with 95% CI, n = 60, 32, from two experiments). ****P < 0.0001, two-tailed Mann–Whitney test. e, Restoration of DNA replication (EdU incorporation, shown as the MN/PN FI ratio) in MN from peripheral chromosomes in RPE-1 (left) and HeLa K (right) cells, 16–18 h after RO release (mean with 95% CI, n = 81, 60, from five experiments for RPE-1; n = 55, 86, from two experiments for HeLa K). In RPE-1 cells, as reported46, high dose MPS1 inhibition causes a fraction of cells to delay cell cycle progression. Therefore, we restricted analysis to cells that progressed into the S-phase of the cell cycle as evidenced by EdU labelling of the PN. **P < 0.0067, ****P < 0.0001, two-tailed Mann–Whitney test. f, MN derived from peripheral chromosomes were larger (DAPI area, top) and more decondensed (density of DAPI FI, bottom) than MN from central chromosomes. Data are from RPE-1 (left) and HeLa K (right) cells (mean with 95% CI, n = 145, 112, from three experiments for DAPI area for RPE-1, n = 145, 112, from three experiments for DAPI FI for RPE-1; n = 66, 21, from two experiments for DAPI area for HeLa K, n = 88, 58, from three experiments for DAPI FI for HeLa K). ****P < 0.0001, two-tailed Welch’s t-test for DAPI FI, two-tailed Mann–Whitney test for DAPI area. The extensive decondensation of MN from peripheral missegregated chromosomes may be because they initiate nuclear import slightly earlier than the assembling main daughter nuclei (Fig. 4c) and differ in surface area to volume ratio. g, h, Micronuclear chromosome number does not influence non-core NE assembly. Comparison of chromosome number in MN from central chromosomes and peripheral chromosomes. g, Confocal images of RPE-1 cells labelled for CENP-A (centromere marker, arrowheads), NUP133 and DNA. Yellow arrows, central chromosomes; red arrows, peripheral chromosomes. Synchronization as in Fig. 4a. Scale bars, 10 µm. Quantified in h. h, Top, distribution of chromosome number (CENP-A foci) in central (blue) and peripheral (red) MN. Data from RPE-1 (left) and HeLa K (right) cells. Bottom, fraction of lagging chromosome circumference with NUP133 for MN with the indicated chromosome number in RPE-1 (left) and HeLa K (right) cells (mean ± s.d., n = 40, 111, 20, 9, 3 each category for central chromosomes, n = 0, 6, 71, 0, 11 for peripheral chromosomes, from two experiments for RPE-1; mean ± s.d., n = 2, 35, 14, 2, 0 each category for central chromosomes, n = 0, 8, 6, 2, 0 for peripheral chromosomes, from two experiments for HeLa K). Although MN from peripheral chromosomes generally contain more chromosomes than MN from central chromosomes, non-core assembly is inhibited on MN from central chromosomes irrespective of their chromosome number. Furthermore, non-core NE assembly is restored to near-normal levels on MN from peripheral chromosomes, again irrespective of chromosome number.
