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.

The Value of Klotho in Kidney Transplantation.

Kidney transplant recipients have better survival rates and improved quality of life than long-term dialysis patients. However, delayed graft function, immunosuppressive therapy nephrotoxicity, and rejection episodes may compromise graft and patient survival. The KL gene is highly expressed in kidney tubular cells and encodes the antiaging and kidney-protective protein Klotho, which has membrane-anchored and soluble forms and regulates mineral metabolism. Klotho expression decreases during acute kidney injury or chronic kidney disease, and human chronic kidney disease shares features of accelerated aging with murine Klotho deficiency. In this work, we review clinical studies on the relationship between Klotho and kidney transplantation. Specifically, we address the dynamics of serum and kidney Klotho levels in donors and kidney transplant recipients, the role of Klotho as a marker of current graft function and graft outcomes, and the potential impact of Klotho on kidney protection in the transplantation context. A better understanding of the potential biomarker and therapeutic utility of Klotho in kidney transplant recipients may provide new insights into the control of graft function and new therapeutic strategies to preserve allograft function.

The vacuole shapes the nucleus and the ribosomal DNA loop during mitotic delays.

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 deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae chromosomes.

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a chromosome segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and, probably, anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses, and GFP-tagged DNA damage markers suggest that neither recombination intermediates nor unfinished replication account for the postreplicative PFGE shift, which is further supported by the fact that the shift does not trigger the G2/M checkpoint. We propose that the absence of Top2 activity leads to a general chromosome structural/topological change in mitosis.

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.

Metal content in Mullus surmuletus in the Canary Islands (North-West African Atlantic).

The contents of 3 heavy metals (Al, Cd, and Pb) and 10 trace elements (B, Cr, Cu, Fe, Li, Mg, Ni, Sr, V, Zn) were determined by means of atomic emission spectrometry with inductively coupled plasma (ICP-OES) in 117 specimens of red mullet, Mullus surmuletus. The specimens were taken in two of the Canary Islands; two locations on the island of Tenerife: Candelaria on the northeast coast and Punta de Hidalgo on the north coast and Arguineguín, on the south coast of the island of Gran Canaria. No significant differences were found between the two sites in Tenerife regarding the metal content of the specimens studied, but differences were found between the specimens captured in Arguineguín and the two sites in Tenerife, the latter having higher concentrations of the analyzed metals.

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

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.

Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae.

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.

Yeast cytotoxic sensitivity to the antitumour agent ß-lapachone depends mainly on oxidative stress and is largely independent of microtubule- or topoisomerase-mediated DNA damage.

ß-Lapachone (ß-lap) is a promising antitumour drug currently undergoing clinical trials. Although it is known that ß-lap generates reactive oxygen species (ROS), its actual mechanism of action is still controversial. Especially important is to determine whether concomitant DNA or microtubule damage is the key target of its antitumour properties and whether DNA damage is mediated by topoisomerases as previously suggested. Here, we have searched for determinants of ß-lap cytotoxicity in the model organism Saccharomyces cerevisiae through a mechanism-driven approach whereby several pathways of the DNA and microtubule integrity responses, as well as the anti-oxidant response, were downregulated and the outcome of ß-lap treatment examined. We also included in the analysis several ß-lap derivatives expected to modify drug bioavailability and activity. We found that neither topoisomerase II nor microtubules contributed to yeast sensitivity to ß-lap and its equitoxic derivative 3-bromo-ß-lapachone. Instead, we found that oxidative and related environmental stresses were primarily responsible for toxicity. Accordingly, Yap1, the central transcription factor in the antioxidant response in yeast, together with several components involved in stress tolerance (i.e., Snf1 and Hog1) and chromatin remodelling (i.e., the SWR1 and RSC complexes), played major roles in protection against ß-lapachone. Critically, we show that dioxygen enhanced toxicity and that ROS scavengers protected cells from it. Furthermore, we show that both quinones resulted in cell death in a manner which cytologically resembled apoptosis/necrosis. We thus conclude that ß-lap is toxic to yeast through massive ROS production that either directly kills the cells or else triggers programmed cell death.

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.