Fernando Monje-Casas

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

Kinetochores of sister chromatids attach to microtubules emanating from the same pole (coorientation) during meiosis I and microtubules emanating from opposite poles (biorientation) during meiosis II. We find that the Aurora B kinase Ipl1 regulates kinetochore-microtubule attachment during both meiotic divisions and that a complex known as the monopolin complex ensures that the protein kinase coorients sister chromatids during meiosis I. Furthermore, the defining of conditions sufficient to induce sister kinetochore coorientation during mitosis provides insight into monopolin complex function. The monopolin complex joins sister kinetochores independently of cohesins, the proteins that hold sister chromatids together. We propose that this function of the monopolin complex helps Aurora B coorient sister chromatids during meiosis I.

Expression Analysis of the nrdHIEF Operon from Escherichia coli: conditions that trigger the transcript level in vivo

Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB andnrdHIEF operons. While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function. Here, we present evidence that nrdHIEF expression might be important under certain circumstances. Basal transcript levels were dramatically enhanced (25–75-fold), depending on the growth-phase and the growth-medium composition. Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants. Moreover, nrdHIEFexpression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium). The mechanism(s) that triggers nrdHIEFexpression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of thenrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. colimight optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors.

Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function

Alteration of the normal pattern and dynamics of Tem1 localization interferes with spindle checkpoint function and demonstrates that MEN signaling must initiate in the SPBs.

Absolute transcript levels of thioredoxin- and glutathione-dependent redox systems in Saccharomyces cerevisiae: response to stress and modulation with growth

We report the co-ordinated fine-tune of mRNA molecules that takes place in yeast (Saccharomyces cerevisiae) in response to diverse environmental stimuli. We performed a systematic and refined quantification of the absolute expression patterns of 16 genes coding for thioredoxin- and glutathione-dependent redox system components. Quantifications were performed to examine the response to oxidants, to sudden temperature upshifts and in association with metabolic changes accompanying culture growth and to explore the contribution of mRNA decay rates to the differences observed in basal expression levels. Collectively, these quantifications show (i) vast differences in the steady-state amounts of the investigated transcripts, cTPxI being largely overexpressed compared with GPX1 during the exponential phase and GPX2 beyond this growth stage; (ii) drastic changes in the relative abundance of the transcripts in response to oxidants and heat shock; and (iii) a unique temporal expression profile for each transcript as cells proceed from exponential to stationary growth phase, yet with some general trends such as maximal or near-maximal basal amounts of most mRNA species at early growth stages when glucose concentration is high and cells are actively growing. Moreover, the results indicate that (i) the half-lives of the investigated transcripts are longer and distributed within a narrower range than previously reported global mRNA half-lives and (ii) transcriptional initiation may play an important role in modulating the significant alterations that most mRNAs exhibit in their steady-state levels along with culture growth.

The Lrs4-Csm1 monopolin complex associates with kinetochores during anaphase and is required for accurate chromosome segregation

Lrs4 and Csm1, components of the monopolin complex, localize to the rDNA where they regulate rDNA maintenance and segregation. During meiosis, the complex also associates with kinetochores to bring about sister kinetochore co-orientation, an essential aspect of meiosis I chromosome segregation. We show here that the Lrs4-Csm1 complex associates with kinetochores during mitosis. This kinetochore localization is observed during anaphase and depends on the on the Mitotic Exit Network, a signaling cascade essential for the completion of mitosis. Furthermore, we find that Lrs4 and Csm1 are important for chromosome segregation fidelity. Our results reveal a previously unanticipated function for Lrs4-Csm1 in mitotic chromosome segregation.

Increased Aurora B activity causes continuous disruption of kinetochore-microtubule attachments and spindle instability

Significance During mitosis, Aurora B kinase plays a key role in ensuring that sister chromatids (each of the copies of a replicated chromosome) attach to different poles of the spindle to create a bipolar array of microtubules that allows correct distribution of the chromosomes. Aurora B deficiency leads to massive defects in chromosome segregation. Surprisingly, an increase in Aurora B activity also is deleterious for the cells and has been associated with various cancers. Here we demonstrate that in yeast an increase in Aurora B activity causes defects in chromosome segregation and spindle-assembly checkpoint activation by erroneously destabilizing even correct attachments of the chromosomes to the spindle, and also promotes premature collapse of the spindle midzone. Aurora B kinase regulates the proper biorientation of sister chromatids during mitosis. Lack of Aurora B kinase function results in the inability to correct erroneous kinetochore–microtubule attachments and gives rise to aneuploidy. Interestingly, increased Aurora B activity also leads to problems with chromosome segregation, and overexpression of this kinase has been observed in various types of cancer. However, little is known about the mechanisms by which an increase in Aurora B kinase activity can impair mitotic progression and cell viability. Here, using a yeast model, we demonstrate that increased Aurora B activity as a result of the overexpression of the Aurora B and inner centromere protein homologs triggers defects in chromosome segregation by promoting the continuous disruption of chromosome–microtubule attachments even when sister chromatids are correctly bioriented. This disruption leads to a constitutive activation of the spindle-assembly checkpoint, which therefore causes a lack of cytokinesis even though spindle elongation and chromosome segregation take place. Finally, we demonstrate that this increase in Aurora B activity causes premature collapse of the mitotic spindle by promoting instability of the spindle midzone.

Inhibition of the mitotic exit network in response to damaged telomeres.

When chromosomal DNA is damaged, progression through the cell cycle is halted to provide the cells with time to repair the genetic material before it is distributed between the mother and daughter cells. In Saccharomyces cerevisiae, this cell cycle arrest occurs at the G2/M transition. However, it is also necessary to restrain exit from mitosis by maintaining Bfa1-Bub2, the inhibitor of the Mitotic Exit Network (MEN), in an active state. While the role of Bfa1 and Bub2 in the inhibition of mitotic exit when the spindle is not properly aligned and the spindle position checkpoint is activated has been extensively studied, the mechanism by which these proteins prevent MEN function after DNA damage is still unclear. Here, we propose that the inhibition of the MEN is specifically required when telomeres are damaged but it is not necessary to face all types of chromosomal DNA damage, which is in agreement with previous data in mammals suggesting the existence of a putative telomere-specific DNA damage response that inhibits mitotic exit. Furthermore, we demonstrate that the mechanism of MEN inhibition when telomeres are damaged relies on the Rad53-dependent inhibition of Bfa1 phosphorylation by the Polo-like kinase Cdc5, establishing a new key role of this kinase in regulating cell cycle progression.

Impaired Manganese Metabolism Causes Mitotic Misregulation

Background: The P-type ATPase Pmr1 provides a major route for cellular detoxification of manganese. Results: Disregulation of Mn2+ homeostasis impairs genome replication and cell cycle progression. Conclusion: Genome instability and endomitosis can be triggered by alterations in cytosolic or Golgi Mn2+ levels. Significance: The Mn2+-dependent cell cycle defects might explain disease phenotypes observed in Hailey-Hailey patients having mutations in the human PMR1 orthologue ATP2C1. Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn2+ acts as a cofactor for many enzymes and excess of Mn2+ is toxic. Alterations in Mn2+ homeostasis affect metabolic functions and mutations in the human Mn2+/Ca2+ transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn2+ on cell cycle progression and show that an excess of cytosolic Mn2+ alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn2+ pool requires a functional morphology checkpoint to avoid the formation of polyploid cells.

Late rDNA Condensation Ensures Timely Cdc14 Release and Coordination of Mitotic Exit Signaling with Nucleolar Segregation

The nucleolus plays a pivotal role in multiple key cellular processes. An illustrative example is the regulation of mitotic exit in Saccharomyces cerevisiae through the nucleolar sequestration of the Cdc14 phosphatase. The peculiar structure of the nucleolus, however, has also its drawbacks. The repetitive nature of the rDNA gives rise to cohesion-independent linkages whose resolution in budding yeast requires the Cdc14-dependent inhibition of rRNA transcription, which facilitates condensin accessibility to this locus. Thus, the rDNA condenses and segregates later than most other yeast genomic regions. Here, we show that defective function of a small nucleolar ribonucleoprotein particle (snoRNP) assembly factor facilitates condensin accessibility to the rDNA and induces nucleolar hyper-condensation. Interestingly, this increased compaction of the nucleolus interferes with the proper release of Cdc14 from this organelle. This observation provides an explanation for the delayed rDNA condensation in budding yeast, which is necessary to efficiently coordinate timely Cdc14 release and mitotic exit with nucleolar compaction and segregation.

Defective histone supply causes condensin-dependent chromatin alterations, SAC activation and chromosome decatenation impairment

The structural organization of chromosomes is essential for their correct function and dynamics during the cell cycle. The assembly of DNA into chromatin provides the substrate for topoisomerases and condensins, which introduce the different levels of superhelical torsion required for DNA metabolism. In particular, Top2 and condensin are directly involved in both the resolution of precatenanes that form during replication and the formation of the intramolecular loop that detects tension at the centromeric chromatin during chromosome biorientation. Here we show that histone depletion activates the spindle assembly checkpoint (SAC) and impairs sister chromatid decatenation, leading to chromosome mis-segregation and lethality in the absence of the SAC. We demonstrate that histone depletion impairs chromosome biorientation and activates the Auroradependent pathway, which detects tension problems at the kinetochore. Interestingly, SAC activation is suppressed by the absence of Top2 and Smc2, an essential component of condensin. Indeed, smc2-8 suppresses catenanes accumulation, mitotic arrest and growth defects induced by histone depletion at semi-permissive temperature. Remarkably, SAC activation by histone depletion is associated with condensin-mediated alterations of the centromeric chromatin. Therefore, our results reveal the importance of a precise interplay between histone supply and condensin/Top2 for pericentric chromatin structure, precatenanes resolution and centromere biorientation.