ATR and a Chk1-Aurora B pathway coordinate postmitotic genome surveillance with cytokinetic abscission

Abstract

Aurora B regulates cytokinesis timing and plays a central role in the abscission checkpoint. Cellular events monitored by this checkpoint are beginning to be elucidated, yet signaling pathways upstream of Aurora B in this context remain poorly understood. Here we reveal a new connection between postmitotic genome surveillance and cytokinetic abscission. Underreplicated DNA lesions are known to be transmitted through mitosis and protected in newly formed nuclei by recruitment of 53BP1 and other proteins until repair takes place. We find that this genome surveillance initiates before completion of cytokinesis. Elevating replication stress increases this postmitotic process and delays cytokinetic abscission by keeping the abscission checkpoint active. We further find that ATR activity in midbody-stage cells links postmitotic genome surveillance to abscission timing and that Chk1 integrates this and other signals upstream of Aurora B to regulate when the final step in the physical separation of daughter cells occurs.

FIGURE 1:

53BP1 foci are present in nuclei before abscission, and their presence corresponds to longer time in midbody stage. (A) HeLa and U2OS cells were cultured for 24 h in the presence of DMSO (control), HU, or APH and analyzed for the presence of nuclear foci containing 53BP1 (green). Midbody-stage cells were identified with an antibody directed against α-tubulin (magenta), and DNA was stained with DAPI (blue). Total percentage of midbody-stage cells with 53BP1 foci after each treatment is indicated (mean and SD from four experiments). Scale bar, 10 μm. (B) Quantification of 53BP1 foci per daughter cell nucleus in midbody-stage cells from each cell line after the indicated treatments. Data are combined results from four experiments. (C) Quantification of the number of midbody-stage cells (HeLa) after the indicated treatments. Error bars are mean and SD from three experiments. *p < 0.05 (Mann–Whitney test). (D) Montages of HeLa cells expressing GFP-tubulin and mCherry-53BP1-FFR subjected to time-lapse imaging. Time from midbody formation (0 min) to midbody disassembly (white arrowheads) was quantified by tracking GFP-tubulin for all cells that passed through this stage, with or without 53BP1 foci. (E) Quantification of abscission timing as defined in D. Boxplot represents the 25th, median, and 75th percentile of values from the indicated treatments (n = number of cells per treatment, combined from four experiments). Whiskers represent the 10th and 90th percentiles. ***p < 0.0001.

FIGURE 2:

Assay to assess abscission timing. (A) HeLa cells expressing GFP-tubulin (green) and H2B-mCherry (magenta) were subjected to time-lapse imaging every 5 min for 2 h. Time to abscission (midbody disassembly; white arrowheads) was quantified for all cells in midbody stage at the beginning of each movie. Either DMSO (Control) or Aurora B inhibitor (AurBi) was added to cells immediately before imaging. Cells that were in mitosis (prophase–metaphase) at the time of AurBi exposure were prone to cytokinesis failure as illustrated. (B) Time to abscission was quantified for all midbody-stage cells. Boxplot represents the 25th, median, and 75th percentile of values from the indicated treatments (n = number of cells per treatment, combined from three to five independent experiments). Whiskers represent the 10th and 90th percentiles. ***p < 0.0001 (Mann–Whitney test). (C) Cumulative frequency plots of progression through abscission. Each point represents the percentage of all midbody cells analyzed in C that had proceeded through abscission over the time course of the experiment. Error bars represent the mean and SD from three to five independent experiments per treatment. (D) Quantification of failed cytokinesis in mitotic (prophase–metaphase) cells and midbody-stage cells in the presence or absence of Aurora B inhibitor.

FIGURE 3:

Replication stress leads to prolonged time to abscission. (A) Timeline of experimental procedure and montages of HeLa cells stably expressing GFP-tubulin (green) and histone H2B-mCherry (magenta) cultured in HU or APH. Scale bar, 20 μm. (B) Quantification of abscission timing using the assay described in Figure 2. ***p < 0.0001. (C) Cumulative frequency plots of progression through abscission. Each point represents the percentage of all midbody cells analyzed in B that had proceeded through abscission over the time course of the experiment. Error bars are mean and SD from three to five experiments.

FIGURE 4:

Prolonged time to abscission after replication stress requires activity of Chk1 and ATR. (A) Western blot analysis of HeLa cells after release from a thymidine/nocodazole synchronization (see Supplemental Figure S5). (B, C) Quantification of abscission using the assay in Figure 2. APH-only data are the same as that in Figure 3, B and C, and are included here for comparison. ***p < 0.0001; n.s., not significant.

FIGURE 5:

ATR and a Chk1-Aurora B pathway coordinate postmitotic genome surveillance with abscission timing. (A, B) Quantification of abscission timing using the time-lapse imaging assay in Figure 2. Control and AurBi data from Figure 2 are included for comparison. ***p < 0.0001. (C) HeLa cells expressing GFP-tubulin and mCherry-53BP1-FFR were subjected to time-lapse imaging, and abscission timing was quantified for cells with and without 53BP1 foci in the presence or absence of the indicated inhibitors. The difference in median time to abscission compared with cells negative for 53BP1 foci (which take a median time of 50 min) is indicated. **p < 0.01; ***p < 0.0001. (D) HeLa cells were treated for 15 min with the indicated inhibitors and analyzed for localization of Aurora B-pT232 (green) at the midbody (α-tubulin, magenta). Graph represents quantification of Aurora B-pT232 fluorescence intensity after each treatment compared with control. Error bars indicate SEM. Scale bar, 10 μm.

FIGURE 6:

Model for abscission checkpoint regulation by ATR and Chk1. DNA lesions in postmitotic cells (depicted schematically in one nucleus; yellow hatches) trigger activation of a signaling pathway via ATR. This feeds into the abscission checkpoint via the central regulatory node of Chk1 and the downstream kinase Aurora B to delay abscission. Recruitment of 53BP1 (green circles), and likely other factors that protect DNA lesions during genome surveillance, would eventually lead to suppression of this signaling path when these lesions are properly poised for repair. Although the molecular network is not yet defined, other signals (e.g., those triggered by tension forces) feed independently into the Chk1-Aurora B pathway to further control abscission timing.