Genome-scale genetic interactions and cell imaging confirm cytokinesis as deleterious to transient topoisomerase II deficiency in <i>Saccharomyces cerevisiae</i>

Topoisomerase II (Top2) is an essential protein that resolves DNA catenations. When Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumors become resistant to these drugs by acquiring hypomorphic mutations in the genes encoding Top2. Here, we have compared the cell cycle and nuclear segregation of two coisogenic Saccharomyces cerevisiae strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons: the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5. Furthermore, we have performed genome-scale synthetic genetic array (SGA) analyses for both alleles under permissive conditions, chronic sublethal Top2 downregulation, and acute, yet transient, Top2 inactivation. We find that slowing down mitotic progression, especially at the time of execution of the mitotic exit network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5; this correlated with cell biology experiments in this mutant, whereby we observed destabilization of both chromatin and ultrafine anaphase bridges by execution of MEN and cytokinesis. Interestingly, whereas transient inactivation of the critical MEN driver Cdc15 partly suppressed top2-5 lethality, this was not the case when earlier steps within anaphase were disrupted; i.e., top2-5 cdc14-1. We discuss the basis of this difference and suggest that accelerated progression through mitosis may be a therapeutic strategy to hypersensitize cancer cells carrying hypomorphic mutations in TOP2

Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction

Nucleolar and Ribosomal DNA Structure under Stress: Yeast Lessons for Aging and Cancer

Once thought a mere ribosome factory, the nucleolus has been viewed in recent years as an extremely sensitive gauge of diverse cellular stresses. Emerging concepts in nucleolar biology include the nucleolar stress response (NSR), whereby a series of cell insults have a special impact on the nucleolus. These insults include, among others, ultra-violet radiation (UV), nutrient deprivation, hypoxia and thermal stress. While these stresses might influence nucleolar biology directly or indirectly, other perturbances whose origin resides in the nucleolar biology also trigger nucleolar and systemic stress responses. Among the latter, we find mutations in nucleolar and ribosomal proteins, ribosomal RNA (rRNA) processing inhibitors and ribosomal DNA (rDNA) transcription inhibition. The p53 protein also mediates NSR, leading ultimately to cell cycle arrest, apoptosis, senescence or differentiation. Hence, NSR is gaining importance in cancer biology. The nucleolar size and ribosome biogenesis, and how they connect with the Target of Rapamycin (TOR) signalling pathway, are also becoming important in the biology of aging and cancer. Simple model organisms like the budding yeast Saccharomyces cerevisiae, easy to manipulate genetically, are useful in order to study nucleolar and rDNA structure and their relationship with stress. In this review, we summarize the most important findings related to this topic

rDNA structure under stress and chromosome remodelling

Chromosome structure in the yeast Saccharomyces cerevisiae is only visible at the microscopic level in the chromosome XII. The ribosomal DNA (rDNA) located in the long arm of this chromosome has been used to observed different phenomena of compaction, segregation and chromosomal structure at different phases of the cell cycle. The metaphase structure (“loop”) depends, among others, on the condensin complex; and its segregation during anaphase depends also in that complex and on the cell cycle phosphatase Cdc14. This study aims to elucidate chromosome remodelling under heatstress and other stresses and the influence of Cdc14 and the TOR pathway in this process. For this, I used different tagged proteins that bind to the rDNA locus for their monitoring and visualization with fluorescent microscopy under heat-stress conditions, besides, I applied several molecular and immunological techniques. I employed an auxin based degron system to degraded proteins by the proteasome. I found that the rDNA chromosome structure is condensed under heat-stress conditions and TORC1 inhibition, also I verified that the auxin mediated degradation system is a useful tool for this purpose. Finally, a role for TORC1 signalling pathway on the stress mediated condensation process, was established.La estructura cromosómica en Saccharomyces cerevisiae solo es visible a nivel microscópico en el cromosoma XII. El DNA ribosomal (rDNA) situado en el brazo largo de dicho cromosoma ha sido usado para observar fenómenos de compactación, segregación y estructuración cromosómica en diferentes fases del ciclo celular. La estructura tipo en metafase (“loop”) depende, entre otros, del complejo de la condensina; y para su segregación en anafase, ese mismo complejo y la fosfatasa de ciclo celular Cdc14 resultan esenciales. Este trabajo tiene como objetivo dilucidar la remodelación cromosómica bajo condiciones de estrés térmico y otros estreses, y la influencia de Cdc14 y la ruta TOR en dicho proceso. Para ello usé proteínas marcadas que se unen al rDNA para su seguimiento y visualización bajo microscopía de fluorescencia en condiciones de estrés térmico y otros estreses, además usé diferentes técnicas moleculares, e inmunológicas. Apliqué el uso de un sistema de degradación de proteínas por el proteasoma mediado por la auxina. Encontré que la estructura cromosómica del rDNA se condensa bajo estrés térmico e inhibición de TORC1, además verifiqué que el uso del sistema de degradación mediado por auxina es útil. Por último, se establece un papel de TORC1 sobre el proceso de condensación mediada por estrés

The ribosomal DNA metaphase loop of <i>Saccharomyces cerevisiae</i> gets condensed upon heat stress in a Cdc14-independent TORC1-dependent manner

<p>Chromosome morphology in <i>Saccharomyces cerevisiae</i> is only visible at the microscopic level in the ribosomal DNA array (rDNA). The rDNA has been thus used as a model to characterize condensation and segregation of sister chromatids in mitosis. It has been established that the metaphase structure (“loop”) depends, among others, on the condensin complex; whereas its segregation also depends on that complex, the Polo-like kinase Cdc5 and the cell cycle master phosphatase Cdc14. In addition, Cdc14 also drives rDNA hypercondensation in telophase. Remarkably, since all these components are essential for cell survival, their role on rDNA condensation and segregation was established by temperature-sensitive (ts) alleles. Here, we show that the heat stress (HS) used to inactivate ts alleles (25 ºC to 37 ºC shift) causes rDNA loop condensation in metaphase-arrested wild type cells, a result that can also be mimicked by other stresses that inhibit the TORC1 pathway. Because this condensation might challenge previous findings with ts alleles, we have repeated classical experiments of rDNA condensation and segregation, yet using instead auxin-driven degradation alleles (<i>aid</i> alleles). We have undertaken the protein degradation at lower temperatures (25 ºC) and concluded that the classical roles for condensin, Cdc5, Cdc14 and Cdc15 still prevailed. Thus, condensin degradation disrupts rDNA higher organization, Cdc14 and Cdc5 degradation precludes rDNA segregation and Cdc15 degradation still allows rDNA hypercompaction in telophase. Finally, we provide direct genetic evidence that this HS-mediated rDNA condensation is dependent on TORC1 but, unlike the one observed in anaphase, is independent of Cdc14.</p

A Yeast Mitotic Tale for the Nucleus and the Vacuoles to Embrace

The morphology of the nucleus is roughly spherical in most eukaryotic cells. However, this organelle shape needs to change as the cell travels through narrow intercellular spaces during cell migration and during cell division in organisms that undergo closed mitosis, i.e., without dismantling the nuclear envelope, such as yeast. In addition, the nuclear morphology is often modified under stress and in pathological conditions, being a hallmark of cancer and senescent cells. Thus, understanding nuclear morphological dynamics is of uttermost importance, as pathways and proteins involved in nuclear shaping can be targeted in anticancer, antiaging, and antifungal therapies. Here, we review how and why the nuclear shape changes during mitotic blocks in yeast, introducing novel data that associate these changes with both the nucleolus and the vacuole. Altogether, these findings suggest a close relationship between the nucleolar domain of the nucleus and the autophagic organelle, which we also discuss here. Encouragingly, recent evidence in tumor cell lines has linked aberrant nuclear morphology to defects in lysosomal function

Strains used in this work.

<p>Strains used in this work.</p

The G2/M arrest that follows a <i>cdc14-1</i> release is dependent on Rad9.

<p>(A) Strains FM459 (<i>cdc14-1 TUB1-GFP</i>) and FM576 (<i>cdc14-1 rad9Δ TUB1-GFP</i>) were arrested at 37°C for 3 h and then released. Samples were taken and micrographed 2 and 3 hours after the release. Upper panels show nuclei number after DAPI staining for the major foursome category. Lower panels indicate spindle morphologies for each nucleus in the foursomes (mean ± SEM, n = 3). (B) Strains FM515 (<i>cdc14-1 RAD52-YFP</i>) and FM883 (<i>cdc14-1 rad9Δ RAD52-YFP</i>) were arrested and released as in A. Two hours after the release, alpha-factor was added and cells were then incubated for 3 more hours before samples were taken and micrographed. Chart represents how many cells in each foursome responded to alpha-factor. Note how <i>cdc14-1 RAD9</i> was distributed in three major categories peaking at 0, 2 and 4 responsive cells; whereas most <i>cdc14-1 rad9Δ</i> foursomes had all cells responding to alpha-factor (i.e., all progeny passed the G2/M arrest) (mean ± SEM, n = 2). (C) Representative cells of a <i>cdc14-1 RAD9</i> foursome with two cells responding to alpha-factor and a <i>cdc14-1 rad9Δ</i> foursome with all its 4 cells responding (white arrows point to the shmoo). Note how there are 3 nuclei in the former (two of them in each of the responding cells) and 4 nuclei in the latter (see main text for more details).</p

Cells coming from a <i>cdc14-1</i> release frequently form Rad52 repair factories, which accumulate when rDNA missegregation had previously occurred.

<p>(A) Strains FM531 (<i>cdc15-2 RAD52-YFP</i>) and FM515 (<i>cdc14-1 RAD52-YFP</i>) were treated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002509#pgen-1002509-g002" target="_blank">Figure 2</a>. Cells from samples taken every 30′ were scored (>200 cells each) for number of Rad52-YFP foci (mean ± SEM, n = 3). (B) Time-lapse fluorescence microscopy (every 15–30′ for 6 h) of a FM515 (<i>cdc14-1 RAD52-YFP</i>) cell starting at the time of the telophase release. (C) Strain FM551 (<i>cdc14-1 RAD52-YFP tetO:rDNA tetR-mRFP</i>) was first arrested in the <i>cdc14-1</i> block and then released into a new cell cycle. After 2 hours, Rad52 foci were scored for those foursomes that have either segregated or missegregated the rDNA (mean ± SEM, n = 3). (D) A representative micrograph of two foursomes, one showing segregated <i>tetOs</i> (white triangles) and the other one with unresolved <i>tetOs</i> (the black triangle). Note the Rad52 focus near the unresolved <i>tetOs</i>. In the merged micrograph, mRFP is pseudocoloured in blue and DAPI in red.</p