Extended Data Fig. 10: Peripheral localization of lagging chromosomes reduces MN NE disruption and DNA damage; actin-dependent peripheral MN disruption in RPE-1 cells.
From: Nuclear envelope assembly defects link mitotic errors to chromothripsis
a, Peripheral localization of lagging chromosomes reduces MN disruption and DNA damage. Representative images of HeLa K cells (C-MN from central chromosomes; P-MN from peripheral chromosomes). See Fig. 4e for quantification. Scale bars, 5 µm. b, Detection of MN disruption by GFP–BAF accumulation yields similar results as in a for NE disruption detection by the loss of RFP–NLS. Representative images from a live-cell/fixed cell imaging experiment (as in Fig. 4e) from HeLa K cells expressing GFP–BAF. C-MN show NE disruption detected by hyper-accumulation of GFP–BAF (top) whereas P-MN show infrequent disruption (bottom). NE disruption of C-MN is accompanied by the acquisition of DNA damage (γH2AX, see Fig. 4e). At the end of 16–18 h live imaging, cells were fixed and stained for γH2AX and lamin B1. See Fig. 4e for quantification. Scale bars, 5 µm. c, In RPE-1 cells, P-MN undergo actin-dependent NE disruption. Top, experimental scheme. Chromosome missegregation was induced in RPE-1 cells expressing RFP–H2B and GFP–BAF (or GFP–H2B and RFP–NLS). About 1 h after mitotic exit, cells were treated with DMSO or a low dose (150 nM) of the actin assembly inhibitor latrunculin A (LatA) and imaged for 16–18 h. Bottom, percentage of P-MN or C-MN that underwent NE disruption (hyperaccumulation of GFP–BAF, from four experiments for DMSO and LatA) or that displayed DNA damage (FI of γH2AX in MN >3 s.d. of background γH2AX in PN, from six experiments for DMSO and three experiments for LatA). NS, P = 0.0948, **P = 0.0044, ****P < 0.0001, two-tailed Fisher’s exact test. For NE disruption: n = 258 for C- MN (DMSO), n = 176 for P-MN (DMSO), n = 173 for C-MN (LatA), n = 125 for P-MN (LatA); for DNA damage: n = 306 for C-MN (DMSO), n = 182 for P-MN (DMSO), n = 128 for C-MN (LatA), n = 73 for P-MN (LatA). d, P-MN develop discontinuities in the lamin B1 nuclear rim. Left, representative images of RPE-1 cells ~18 h after mitotic exit, as in c. Yellow boxes indicate C-MN (yellow arrowheads) with reduced lamin B1. Right, quantification (mean with 95% CI, n = 61, 28, 38, 18, left to right, from two experiments). ****P < 0.0001, two-tailed Welch’s t-test for DMSO, two-tailed Mann–Whitney test for LatA. Red boxes indicate P-MN, one of which displays a prominent lamin B1 rim discontinuity. Red arrowhead in the enlarged image shows an NE herniation on a P-MN. Scale bars, 10 µm. Consistent with prior work, MN from lagging chromosomes undergo spontaneous disruption independent of actin13. By contrast, transient disruption of PN, as occurs during confined cell migration47, is mediated by actomyosin contractile forces13. We confirm that MN from central lagging chromosomes undergo disruption independent from actin. However, we noted a difference between RPE-1 and HeLa K cells in the behaviour of MN from peripheral chromosomes. Although NE assembly appeared to be restored in both HeLa K and RPE-1 cells, P-MN in RPE-1 cells underwent residual disruption (there is nevertheless a statistically significant reduction of NE disruption frequency when comparing P-MN and C-MN in RPE-1 cells). We hypothesized that in highly motile RPE-1 cells, large P-MN might be more likely to undergo actin-dependent NE breakage, essentially becoming more similar to the transient NE disruption of PN13,47. The data in c confirm that P-MN undergo actin-dependent breakage. Furthermore, P-MN in RPE-1 cells have lamin B1 gaps (d, red arrowhead)6. One mechanism that could generate these gaps is residual contact between peripheral missegregated chromosomes and astral microtubules (Extended Data Fig. 9b). In addition, P-MN are more decondensed, and have larger NE surface area, than PN, which may dilute lamins. The increased breakage of P-MN in RPE-1 cells relative to HeLa K cells may be due to higher contractile forces in RPE-1 cells.
