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• Research Paper Volume 11, Issue 23 pp 11686-11721

  Cytological and genetic consequences for the progeny of a mitotic catastrophe provoked by Topoisomerase II deficiency

  Relevance score: 7.3125873

  Cristina Ramos-Pérez, Margaret Dominska, Laura Anaissi-Afonso, Sara Cazorla-Rivero, Oliver Quevedo, Isabel Lorenzo-Castrillejo, Thomas D. Petes, Félix Machín

  Keywords: mitotic catastrophe, Top2, senescence, cell death, genomic instability

  Published in Aging on December 8, 2019

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  Topoisomerase II (Top2) removes topological linkages between replicated chromosomes. Top2 inhibition leads to mitotic catastrophe (MC) when cells unsuccessfully try to split their genetic material between the two daughter cells. Herein, we have characterized the fate of these daughter cells in the budding yeast. Clonogenic and microcolony experiments, in combination with vital and apoptotic stains, showed that 75% of daughter cells become senescent in the short term; they are unable to divide but remain alive. Decline in cell vitality then occurred, yet slowly, uncoordinatedly when comparing pairs of daughters, and independently of the cell death mediator Mca1/Yca1. Furthermore, we showed that senescence can be modulated by ploidy, suggesting that gross chromosome imbalances during segregation may account for this phenotype. Indeed, we found that diploid long-term survivors of the MC are prone to genomic imbalances such as trisomies, uniparental disomies and terminal loss of heterozygosity (LOH), the latter affecting the longest chromosome arms.

  

  Most progeny coming from a Top2-mediated mitotic catastrophe is inviable. (A) Haploid TOP2 (WT) or top2-5 cells were grown at 25 °C and spread on YPD agar plates. Unbudded cells (G₁/G₀) were identified and photographed again after 6 h at 37 °C. Number of cell bodies (buds) coming from these G₁/G₀ cells were then counted and plotted as indicated. (B) The same analysis as in panel A but including data coming from independent experiments as well as after 24 h incubation at 37 °C (mean ± s.e.m., n=3). (C) The principle of the solid medium-based clonogenic assay. Unlike the liquid medium-based clonogenic assay, cells are spread on the Petri dish before the condition that challenges survivability is transiently triggered (Top2 inactivation in our study). In the solid medium-based assay, the colony forming unit (CFU) reading after the challenge is binary, irrespective of how far cells keep on dividing during the challenge: “0” if all clonal cells are inviable (grey); “1” if at least one cell from the clone stays viable (yellowish orange). (D) Time course of clonogenic survivability. Asynchronous top2-5 cultures growing at 25 °C were spread onto several YPD plates. The plates were incubated at 37 °C for different periods before transferring them 25 °C. Four days after the initial plating, visible colonies (macrocolonies) were counted and normalized to a control plate which was never incubated at 37 °C (0h). (E) Analysis of the origin of macrocolonies after the 6 h x 37 °C regime as determined after microscanning plates at the time of seeding (N=33 macrocolonies; 2:1 unbudded:budded ratio at seeding).

  
  
  

  

  Most daughter cells coming from a Top2-mediated mitotic catastrophe are unable to divide again. (A) Haploid top2-5 cells were spread at high cell density on two Petri dishes. At the time of seeding, 0h (25 °C), several fields were photomicrographed before incubating the plates at 37 °C during either 6 h or 24 h. After the 37 °C incubations, the same fields were localized, photomicrographed again, and further incubated 16-24 h at 25 °C. An example of a microscope field of a 37 °C x 24 h experiment. Three representative unbudded cells at 0h (25 °C) are highlighted. In red, two cells that budded just once during the 37 °C x 24 h incubation (“2” cell bodies); one of them able to re-bud again a few times after the 25 °C downshift (“m”) and the second one that remained stuck as “2”. In green, a cell that reached “3” bodies at 37° C and remained so after the final 25 °C x 24 h incubation. Scale bar corresponds to 50 μm. (B) Analysis of how far the top2-5 MC progeny can go based on the microcolony approach shown in panel A. Only unbudded (G₁/G₀) cells at 0h (25 °C) were considered. The inner circle in the sunburst chart depicts proportions of cell bodies after the 37 °C incubation. The outer circle depicts the situation after the final 25 °C incubations (see supplemental information for a detailed description). On the left are results from a 37 °C x 6 h regime; on the right are results from a 37 °C x 24 h regime. Numbers point to the number of cell bodies; “m” means microcolonies of 5 or more bodies. (C) Capability of the top2-5 progeny to split apart and relationship with overall survivability. Unbudded cells were micromanipulated and arranged at defined plate positions before incubating them for 6 h at 37 °C. Then, those cells able to re-bud at least once were subjected to an attempt to physically separate the cell bodies. The inner circle in the sunburst depicts the number of cell bodies after the 37 °C incubation. The middle circle depicts the result of the separation attempt (“Y” or “N”, successful or unsuccessful, respectively). The outer circle indicates if any of the bodies was able to raise a macrocolony (Yes or No) after 4 d incubation at 25 °C. (D) Progression of the size (volume) of the original G₁/G₀ cells (mother) after the top2-5 mitotic catastrophe with and without the osmotic stabilizer Sorbitol (Sorb, 1.2 M). (E) Time course of clonogenic survivability in the presence of 1.2 M Sorbitol. The experiment was conducted as in Figure 1D. (F) Sunbursts of microcolony analyses in the presence of 1.2 M Sorbitol (Srb) at the 6 h and 24 h x 37 °C regimes. Interpretation as in panel B. In sunburst charts, N indicates number of original unbudded cells which were followed; blue sectors depict G₁/G₀ cells that remained unbudded during the 37 °C incubations; red sectors, cells that budded once at 37 °C; green sectors, cells that reached 3 bodies at 37 °C; orange sectors, cells that reached 4 bodies at 37 °C; cyan sectors, cells that reached 5 or more bodies at 37 °C.

  
  
  

  

  Cell vitality remain high for several hours after the top2 mitotic catastrophe and is not modulated by Yca1. (A) Morphological patterns of cell and nuclear sickness after the top2 MC. Haploid top2-5 HTA2-GFP cells were seeded onto agarose patches and the same fields visualized under the fluorescence microscope at 0 h, 6 h and 24 h after the 37 °C temperature upshift. White filled triangles point to darkened inclusion bodies, asterisks (*) swelled cells, open circles (○) cells that has lost their rounded shape, and hash (#) points to cells that have largely lost the H2A-GFP signal. BF, bright field. Scale bar corresponds to 20 μm. (B) Time course of cell vitality decline as reporter by methylene blue (MB) negative staining. Asynchronous cultures of the top2-5 and top2-5yca1Δ strains were grown at 25 °C before shifting the temperature to 37 °C. At the indicated time points (0, 2, 4, 6, 24 & 48 h), samples were taken and stained with the vital dye MB. (C) Cell vitality decline as reported by metabolic competence, intrinsic ROS generation, and loss of plasma membrane impermeability. Cells were treated as in B and stained at the indicated time points with the vital dye FUN1, the death marker propidium iodide (PI), and/or the ROS reporter DCFH-DA (mean ± s.e.m., n=3). (D) Clonogenic survival profile of top2-5 yca1Δ as determined on the low-density plates (mean ± s.e.m., n=3). The experimental procedure is described in Figure 1D. (E) Ability to re-bud of the top2-5 yca1Δ MC progeny as determined on the high-density plates. The experimental procedure is described in Figure 2.

  
  
  

  

  Mitotic catastrophe in top2-5 diploids leads to progeny with a greater capacity for cell division than observed in the haploid. Isogenic homozygous top2-5 diploid cells were grown and spread at either low or high cell density on Petri dishes. In addition, G₁/G₀ cells were micromanipulated, arrayed and treated as described in Figure 2C. (A) Ability to re-bud after transient (6 h or 24 h) incubations at 37 °C of the high-density plates. The experimental procedure is described in Figure 2. (B) Clonogenic survival profile as determined on the low-density plates (mean ± s.e.m., n=3). The experimental procedure is described in Figure 1D. (C) Capability of the progeny to split apart and relationship with overall survivability. The experimental procedure is described in Figure 2C.

  
  
  

  

  Mitotic catastrophe in top2-5 heterozygous diploids leads to survivors with specific genetic instability footprints. (A) Schematic of the engineered chromosome V (cV) from the hybrid highly heterozygous (~55,000 SNPs) diploids used in this study. As explained in the text, the genetic modifications applied in cV allowed for selection of chromosome rearrangements. (B) G₁/G₀ cells from the hybrid highly heterozygous top2-5 diploid FM1873 strain were micromanipulated, arrayed and treated as described in Figure 2C. The capability of the immediate progeny to split apart and its relationship with overall survivability is shown in the sunburst chart. (C) Clonogenic survival profile of FM1873 as determined on low-density plates (mean ± s.e.m., n=3). The experimental procedure is described in Figure 1D. (D) Percentage of red or sectored (either white:red or pink:red) colonies in the surviving clones. Both outcomes often reflect genetic alterations on cV as described in the text. (E) Results of SNP microarray analysis of colonies derived from FM1873 or MD684. Microarray patterns showing specific chromosome rearrangements are shown on the left side, and diagrams of the putative events producing these patterns are shown on the right side. The number of specific events out of 118 total events is indicated. For the microarray patterns, hybridization to SNPs specific to homologs derived from W303-1A are shown in red, and hybridizations to SNPs specific to YJM789 are shown in blue. The X-axis shows SGD coordinates for the chromosome, and the Y-axis shows the ratio of hybridization normalized to a heterozygous diploid strain. The representative examples correspond to (1) a T-LOH event on chromosome IV (MD684.1.1 (E1) in Table 1); (2) a I-LOH event (marked with a green arrow) plus T-LOH event on chromosome IV (FM1873-15c (C2) in Table 1); (3) a Trisomy for chromosome XIV (MD684.1.1 (E1) in Table 1); and (4) a UPD for chromosome V (This isolate has two copies of the W303-1A-derived and no copies of the YJM789-derived chromosome).

  
  
  

  

  Summary of the top2-mediated mitotic catastrophe and the fate of the immediate progeny. After inactivation of Top2, cells cannot resolve sister chromatids in anaphase, leading to an anaphase bridge between the mother (M) and its daughter (D1). These bridges are quickly severed (at least in the top2-5 mutant [25]). The immediate progeny coming from the top2 mitotic catastrophes (MCs) is largely unable to enter a new cell cycle (do not re-bud) despite remaining metabolically active for many hours; hence, these cells become senescent. Only ~25% of the original mothers re-bud once (D2) after the top2 MC. The long-term fate of most daughter cells is death. They will eventually die through accidental cell death (ACD), as deduced from both the asynchrony and asymmetry of death events and the lack of regulation by the death modulator Yca1(Mca1). The inability to enter a new cell cycle is likely a consequence of both the massive DNA damage as a consequence of bridge severing, and the misdistribution of essential genetic material coded on the chromosome arms between the daughter cells. A small proportion of the progeny, especially those cell that underwent a milder top2 MC (e.g., already in S/G₂ at the time of Top2 inactivation) survives to yield a population of cells with characteristic footprints of genomic instability. Two of these footprints, terminal loss of heterozygosity (T-LOH) and uniparental disomy (UPD) are expected outcomes from anaphase bridges.