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.

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. When cells divide they must segregate copies of their chromosomes to each of their daughters. A particular harmful situation arises when those copies are glued to each other (i.e., nondisjunction) at the moment of division. Previously, it has been possible to genetically favour this scenario, yet it has been difficult to limit the extent of nondisjunction to a single chromosome. We have developed and studied a yeast model where we control nondisjunction of one of its sixteen chromosomes. We show that dividing cells manage to complete nuclear and cell fission and therefore break that chromosome. We further show that new daughter cells then trigger a DNA damage response, yet only after they initiate a new round of replication. Remarkably, an uncommon repair strategy seems to be used to deal with this damage, which involves part of the homologous recombination machinery (i.e., RPA complex and Rad52) but lacks its primary sensor Mre11. Importantly though, both daughter cells arrest their cell cycle in G2 to prevent further damage from occurring. After a while, the cell that still carries an entire copy of the chromosome often survives, leading to aberrant forms of the chromosome in the progeny.

Chromosome morphology in Saccharomyces cerevisiae 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 (aid 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.

The ribosomal DNA (rDNA) array of Saccharomyces cerevisiae has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct presentations that arise as spatial adaptations to changes in the nuclear morphology triggered during mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of nuclear components yet colocalizes with the vacuole. We show that the rDNA-associated nuclear envelope (NE) often reshapes into a ladle to accommodate the vacuole in the SUL, with the nucleus becoming bilobed and doughnut-shaped. Finally, we demonstrate that the formation of the rDNA loop and the SUL require TORC1, membrane synthesis and functional vacuoles, yet is independent of nucleus–vacuole junctions and rDNA-NE tethering.

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.