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.

Are Anaphase Events Really Irreversible? The Endmost Stages of Cell Division and the Paradox of the DNA Double-Strand Break Repair.

It has been recently demonstrated that yeast cells are able to partially regress chromosome segregation in telophase as a response to DNA double-strand breaks (DSBs), likely to find a donor sequence for homology-directed repair (HDR). This regression challenges the traditional concept that establishes anaphase events as irreversible, hence opening a new field of research in cell biology. Here, the nature of this new behavior in yeast is summarized and the underlying mechanisms are speculated about. It is also discussed whether it can be reproduced in other eukaryotes. Overall, this work brings forwards the need of understanding how cells attempt to repair DSBs when transiting the latest stages of mitosis, i.e., anaphase and telophase.

Efficient Multicomponent Synthesis of Diverse Antibacterial Embelin-Privileged Structure Conjugates.

A library of embelin derivatives has been synthesized through a multicomponent reaction from embelin (1), aldehydes and privileged structures such as 4-hydroxycoumarin, 4-hydroxy-2H-pyran-2-one and 2-naphthol, in the presence of InCl3 as catalyst. This multicomponent reaction implies Knoevenagel condensation, Michael addition, intramolecular cyclization and dehydration. Many of the synthesized compounds were active and selective against Gram-positive bacteria, including one important multiresistant Staphylococcus aureus clinical isolate. It was found how the conjugation of diverse privileged substructure with embelin led to adducts having enhanced antibacterial activities.

Synthesis and biological evaluation of naphthoquinone-coumarin conjugates as topoisomerase II inhibitors.

Based on previous Topoisomerase II docking studies of naphthoquinone derivatives, a series of naphthoquinone-coumarin conjugates was synthesized through a multicomponent reaction from aromatic aldehydes, 4-hydroxycoumarin and 2-hydroxynaphthoquinone. The hybrid structures were evaluated against the α isoform of human topoisomerase II (hTopoIIα), Escherichia coli DNA Gyrase and E. coli Topoisomerase I. All tested compounds inhibited the hTopoIIα-mediated relaxation of negatively supercoiled circular DNA in the low micromolar range. This inhibition was specific since neither DNA Gyrase nor Topoisomerase I were affected. Cleavage assays pointed out that naphthoquinone-coumarins act by catalytically inhibiting hTopoIIα. ATPase assays and molecular docking studies further pointed out that the mode of action is related to the hTopoIIα ATP-binding site.

5-Ethynylarylnaphthalimides as antitumor agents: Synthesis and biological evaluation.

A set of 5-ethynylarylnaphthalimides was synthesized by Sonogashira cross-coupling reactions and evaluated for antiproliferative and antitopoisomerase II in vitro activities. Furthermore docking studies of these molecules as DNA-intercalators were carried out and the in vivo DNA-damaging activity was also determined with the model organism Saccharomyces cerevisiae. From the obtained results three naphthalimides 6, 13 and 14 showed strong topoisomerase II inhibitory activity. These three molecules also presented good docking scores as DNA-intercalators using a self-complementary oligodeoxynucleotide d(ATGCAT)2 as a model, and compounds 13 and 14 were among the most cytotoxic in the in vivo DNA-damaging activity.

Cdc14 phosphatase: warning, no delay allowed for chromosome segregation!

Cycling events in nature start and end to restart again and again. In the cell cycle, whose purpose is to become two where there was only one, cyclin-dependent kinases (CDKs) are the beginning and, therefore, phosphatases must play a role in the ending. Since CDKs are drivers of the cell cycle and cancer cells uncontrollably divide, much attention has been put into knocking down CDK activity. However, much less is known on the consequences of interfering with the phosphatases that put an end to the cell cycle. We have addressed in recent years the consequences of transiently inactivating the only master cell cycle phosphatase in the model yeast Saccharomyces cerevisiae, Cdc14. Transient inactivation is expected to better mimic the pharmacological action of drugs. Interestingly, we have found that yeast cells tolerate badly a relatively brief inactivation of Cdc14 when cells are already committed into anaphase, the first cell cycle stage where this phosphatase plays important roles. First, we noticed that the segregation of distal regions in the chromosome arm that carries the ribosomal DNA array was irreversibly impaired, leading to an anaphase bridge (AB). Next, we found that this AB could eventually be severed by cytokinesis and led to two different types of genetically compromised daughter cells. All these previous studies were done in haploid cells. We have now recently expanded this analysis to diploid cells and used the advantage of making hybrid diploids to study chromosome rearrangements and changes in the ploidy of the surviving progeny. We have found that the consequences for the genome integrity were far more dramatic than originally envisioned.

Domino Synthesis of Embelin Derivatives with Antibacterial Activity.

A series of dihydropyran embelin derivatives was synthesized through a direct and highly efficient approach based on a domino Knoevenagel intramolecular hetero-Diels-Alder reaction from natural embelin (1), using unsaturated aldehydes in the presence of organocatalysts such as ethylendiamine diacetate or l-proline. The aliphatic aldehydes yielded exclusively trans adducts, while mixtures of trans and cis isomers were found in reactions with aromatic aldehydes, with the cis form always predominating. Some of the compounds obtained were active and selective against Gram-positive bacteria, including multiresistant Staphylococcus aureus clinical isolates.

Structure and antimicrobial activity of phloroglucinol derivatives from Achyrocline satureioides.

The new prenylated phloroglucinol α-pyrones 1-3 and the new dibenzofuran 4, together with the known 23-methyl-6-O-demethylauricepyrone (5), achyrofuran (6), and 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphaliin A), were isolated from the aerial parts of Achyrocline satureioides. Their structures were determined by 1D and 2D NMR spectroscopic studies, while the absolute configuration of the sole stereogenic center of 1 was established by vibrational circular dichroism measurements in comparison to density functional theory calculated data. The same (S) absolute configuration of the α-methylbutyryl chain attached to the phloroglucinol nucleus was assumed for compounds 2-6 based on biogenetic considerations. Derivatives 7-16 were prepared from 1 and 5, and the antimicrobial activities of the isolated metabolites and some of the semisynthetic derivatives against a selected panel of Gram-positive and Gram-negative bacteria, as well as a set of yeast molds, were determined.

Cdc14 inhibits transcription by RNA polymerase I during anaphase.

Chromosome condensation and the global repression of gene transcription are features of mitosis in most eukaryotes. The logic behind this phenomenon is that chromosome condensation prevents the activity of RNA polymerases. In budding yeast, however, transcription was proposed to be continuous during mitosis. Here we show that Cdc14, a protein phosphatase required for nucleolar segregation and mitotic exit, inhibits transcription of yeast ribosomal genes (rDNA) during anaphase. The phosphatase activity of Cdc14 is required for RNA polymerase I (Pol I) inhibition in vitro and in vivo. Moreover Cdc14-dependent inhibition involves nucleolar exclusion of Pol I subunits. We demonstrate that transcription inhibition is necessary for complete chromosome disjunction, because ribosomal RNA (rRNA) transcripts block condensin binding to rDNA, and show that bypassing the role of Cdc14 in nucleolar segregation requires in vivo degradation of nascent transcripts. Our results show that transcription interferes with chromosome condensation, not the reverse. We conclude that budding yeast, like most eukaryotes, inhibit Pol I transcription before segregation as a prerequisite for chromosome condensation and faithful genome separation.

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.

The CRISPR/Cas9 system forms a condensate in the yeast nucleus.

CRISPR/Cas9 gene editing technology has revolutionized genetic engineering. However, the nuclear dynamics of Cas9 in eukaryotic cells, particularly in the model organism Saccharomyces cerevisiae , remains poorly understood. Here, we constructed yeast strains expressing fluorescently tagged Cas9 variants, revealing their accumulation in the nucleus over time. Notably, Cas9 was non-uniformly distributed in the nucleoplasm during the initial hours, suggesting the formation of a condensate. This condensate often co-localizes with the nucleolus and associates the target site to its periphery. Our findings provide insights into Cas9 nuclear dynamics in yeast, advancing our understanding of CRISPR/Cas9-based genetic manipulation.

Msc1 is a nuclear envelope protein that reinforces DNA repair in late mitosis.

Precise double-strand break (DSB) repair is a paramount for genome stability. Homologous recombination (HR) repairs DSBs when cyclin-dependent kinase (CDK) activity is high, which correlates with the availability of the sister chromatid as a template. However, anaphase and telophase are paradoxical scenarios since high CDK favors HR despite sister chromatids being no longer aligned. To identify factors specifically involved in DSB repair in late mitosis, we have undertaken comparative proteomics in Saccharomyces cerevisiae and found that meiotic sister chromatid 1 (Msc1), a poorly characterized nuclear envelope protein, is significantly enriched upon both random and guided DSBs. We further show that Δmsc1 is more sensitive to DSBs in late mitosis, and has a delayed repair of DBSs, as indicated by increased Rad53 hyperphosphorylation, a higher presence of RPA foci, fewer Rad52 repair factories, and slower HR completion. We propose that Msc1 favors the later stages of HR and the timely completion of DSB repair before cytokinesis.

Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination.

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.

Multicomponent synthesis of antibacterial dihydropyridin and dihydropyran embelin derivatives.

A series of dihydropyran and dihydropyridin embelin derivatives were synthesized through a novel and straightforward one-pot protocol based on a three-component reaction with embelin, aldehydes, and cyclic enaminones as synthetic imputs. The type of substituent on the nitrogen atom of the ß-enaminone is key to obtain nitrogenated or oxygenated rings. The obtained compounds were active against Gram-positive bacteria, including multiresistant Staphylococcus aureus clinical isolates.

Synthesis and study of antiproliferative, antitopoisomerase II, DNA-intercalating and DNA-damaging activities of arylnaphthalimides.

A series of arylnaphthalimides were designed and synthesized to overcome the dose-limiting cytotoxicity of N-acetylated metabolites arising from amonafide, the prototypical antitumour naphthalimide whose biomedical properties have been related to its ability to intercalate the DNA and poison the enzyme Topoisomerase II. Thus, these arylnaphthalimides were first evaluated for their antiproliferative activity against two tumour cell lines and for their antitopoisomerase II in vitro activities, together with their ability to intercalate the DNA in vitro and also through docking modelization. Then, the well-known DNA damage response in Saccharomyces cerevisiae was employed to critically evaluate whether these novel compounds can damage the DNA in vivo. By performing all these assays we conclude that the 5-arylsubstituted naphthalimides not only keep but also improve amonafide's biological activities.

Effect of carrier yeast RNAs in the detection of SARS-CoV-2 by RT-LAMP.

The COVID-19 pandemic caused by SARS-CoV-2 has underscored the need for rapid and accurate diagnostic methods. Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has emerged as a promising molecular tool in least developed countries due to its simplicity, speed, and sensitivity. Nevertheless, reliable SARS-CoV-2 detection can be challenged by the chain custody of the samples. In this context, carrier RNA can act as a preservative. In this study, we explored the potential of yeast total and transference RNA (tRNA) in the SARS-CoV-2 RT-LAMP. We have found that most optimal conditions are reached with 1 µg/µL tRNA in the RT-LAMP reaction.

Synthesis of Structurally Related Coumarin Derivatives as Antiproliferative Agents.

A library of structurally related coumarins was generated through synthesis reactions and chemical modification reactions to obtain derivatives with antiproliferative activity both in vivo and in vitro. Out of a total of 35 structurally related coumarin derivatives, seven of them showed inhibitory activity in in vitro tests against Taq DNA polymerase with IC50 values lower than 250 µM. The derivatives 4-(chloromethyl)-5,7-dihydroxy-2H-chromen-2-one (2d) and 4-((acetylthio)methyl)-2-oxo-2H-chromen-7-yl acetate (3c) showed the most promising anti-polymerase activity with IC50 values of 20.7 ± 2.10 and 48.25 ± 1.20 µM, respectively. Assays with tumor cell lines (HEK 293 and HCT-116) were carried out, and the derivative 4-(chloromethyl)-7,8-dihydroxy-2H-chromen-2-one (2c) was the most promising, with an IC50 value of 8.47 µM and a selectivity index of 1.87. In addition, the derivatives were evaluated against Saccharomyces cerevisiae strains that report about common modes of actions, including DNA damage, that are expected for agents that cause replicative stress. The coumarin derivatives 7-(2-(oxiran-2-yl)ethoxy)-2H-chromen-2-one (5b) and 7-(3-(oxiran-2-yl)propoxy)-2H-chromen-2-one (5c) caused DNA damage in S. cerevisiae. The O-alkenylepoxy group stands out as that with the most important functionality within this family of 35 derivatives, presenting a very good profile as an antiproliferative scaffold. Finally, the in vitro antiretroviral capacity was tested through RT-PCR assays. Derivative 5c showed inhibitory activity below 150 µM with an IC50 value of 134.22 ± 2.37 µM, highlighting the O-butylepoxy group as the functionalization responsible for the activity.

SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions.

Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5-Smc6 complexes. The Smc5-Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5-Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5-Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.

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.

A simplified and easy-to-use HIP HOP assay provides insights into chalcone antifungal mechanisms of action.

Elucidating the mechanism of action of an antifungal or cytotoxic compound is a time-consuming process. Yeast chemogenomic profiling provides a compelling solution to the problem but is experimentally complex. Here, we demonstrate the use of a highly simplified yeast chemical genetic assay comprising just 89 yeast deletion strains, each diagnostic for a specific mechanism of action. We use the assay to investigate the mechanism of action of two antifungal chalcone compounds, trans-chalcone and 4'-hydroxychalcone, and narrow down the mechanism to transcriptional stress. Crucially, the assay eliminates mechanisms of action such as topoisomerase I inhibition and membrane disruption that have been suggested for related chalcone compounds. We propose this simplified assay as a useful tool to rapidly identify common off-target mechanisms.