Anaphase is a key cell cycle transition that ensures faithful genome inheritance. At anaphase onset, sister chromatids separate abruptly and synchronously upon activation of the protease separase. Major cell cycle transitions often involve positive feedback, which contributes to their abruptness and irreversibility; however, whether such feedback is required for anaphase remains unclear. Here, we analyze sister chromatid separation dynamics in fission yeast using high-resolution live-cell imaging and computational modeling. We find that anaphase synchrony relies on fast degradation of the separase inhibitor securin but does not require separase-mediated positive feedback. Hence, sister chromatid separation, being inherently irreversible, may be one of the few major cell cycle transitions that can proceed without positive feedback. A stochastic model fitted to the data revealed that separation synchrony is limited by stochasticity resulting from small-number effects. Together, these results support a feedback-independent mechanism for anaphase onset and identify molecular noise as a fundamental constraint on its temporal precision.

Feedback loops have been described as universal, recurring motifs that regulate major cell cycle transitions. In this issue, Williams et al. (https://doi.org/10.1083/jcb.202602088) show, however, that chromosome segregation synchrony in fission yeast is feedback-independent, and instead, temporal precision of anaphase is dictated by molecular noise arising from small-number stochastic effects.

Ammonium is the preferred nitrogen source for microorganisms. We investigated the regulation of ammonium transporters Amt1, Amt2 and Amt3 in the fission yeast Schizosaccharomyces pombe. Expression of the amt1+ gene increases under nitrogen starvation as well as in the TORC1-deficient mutant tor2-287, suggesting negative regulation of amt1+ by TORC1. This regulation depends on the GATA transcription factor Gaf1, the phosphorylation of which is regulated by Ppe1 and PP2A phosphatases. Ppe1 is required for the nuclear accumulation of Gaf1, partly contributing to the controlled expression of amt1+. In contrast, PP2A phosphatase is essential for amt1+ induction upon starvation, indicating distinct roles for Ppe1 and PP2A in Gaf1 regulation. Nitrogen starvation also promotes the plasma membrane translocation of Amt1 and Amt2 in a manner dependent on the Tsc-Rhb1 pathway. Intriguingly, the amt1Δ amt2Δ tscΔ mutant is more sensitive to low-ammonium conditions than the amt1Δ amt2Δ amt3Δ triple mutant. Thus, there might be an unidentified ammonium transporter that is also dependent on the Tsc-Rhb1 pathway for its plasma membrane localization.

Optimized protocol for high-efficiency mitochondrial RNA isolation from fission yeast in log phase and stationary phase.

Class I cyclic nucleotide phosphodiesterases (PDEs) form a family of enzymes that hydrolyze the signaling molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Highly potent and selective inhibitors of mammalian PDEs have been developed, demonstrating that this enzyme family is eminently druggable. The genomes of the free-living nematode and model organism Caenorhabditis elegans and those of related parasitic nematodes possess six PDE genes representing six of the eleven PDE families found in mammals. Here, we expressed the C. elegans PDEs or their catalytic domains in the fission yeast Schizosaccharomyces pombe and screened a collection of small molecule inhibitors of mammalian PDEs obtained from our previous high throughput screens for ones with activity against one or more C. elegans PDEs. Consistent with an earlier study, the C. elegans PDE-4 enzyme is relatively insensitive to mammalian PDE4 inhibitors such as Rolipram, as are PDE-4 enzymes from three parasitic nematodes. Much of this is due to a single amino acid difference between mammalian PDE4s and nematode PDE4, as replacing arginine 580 with threonine in C. elegans PDE-4 restores substantial sensitivity to Rolipram. Finally, several of the most effective C. elegans PDE inhibitors were tested for their impact on C. elegans growth and fertility, two of which displayed toxic effects on C. elegans viability and fecundity using two different assessment methods, while a third showed a significant effect on fecundity. The strategy described herein offers an approach for discovery of novel anthelmintic and nematicidal compounds targeting parasitic nematode PDEs.

In Schizosaccharomyces pombe and Cryptococcus neoformans, the flavohemoglobins (flavoHbs) that protect these yeasts against nitrosative stress are Yhb1 and Fhb1, respectively. Although heme is an essential cofactor for flavoHb activity, the molecular circuitry governing the acquisition of exogenous heme by flavoHbs remains largely unknown. Because S. pombe is a powerful model organism for studying heme biology, Fhb1 was heterologously expressed in S. pombe mutant cells lacking Yhb1. Fhb1 expression functionally complements yhb1Δ cells that are defective in nitric oxide (˙NO) detoxification. Moreover, this Fhb1-mediated complementation requires the S. pombe heme transporter Str3 or the heterologous coexpression of two previously uncharacterized C. neoformans transporters, CnStr1 and CnStr3. Consistent with this finding, heterologous expression of CnStr1 and CnStr3 rescues the heme-dependent growth defect of S. pombe cells that are deficient in heme biosynthesis and lack endogenous heme uptake systems when hemin is provided as the sole heme source. Coimmunoprecipitation assays further demonstrate interactions between Fhb1 and S. pombe Str3 or C. neoformans Str3-like proteins when coexpressed in fission yeast cells. In C. neoformans, strains lacking CnStr1 and CnStr3 exhibit a heme-dependent growth defect in the absence of the cell-surface hemophore Cig1. Similar to the C. neoformans fhb1Δ mutant, a cig1Δ Cnstr1Δ Cnstr3Δ triple mutant fails to protect cells from nitrosative stress. Collectively, these findings reveal functional conservation between S. pombe Yhb1 and C. neoformans Fhb1 in their ability to acquire exogenous heme required for activation as defenders against nitrosative stress.

Cancer genomics, molecular pathology, targeted therapy, and immunotherapy have transformed how tumors are diagnosed and treated, yet several problems remain hard to resolve prospectively, including the interpretation of variants of uncertain significance, variable penetrance, recurrent drug resistance, and the early trajectories that predispose later therapeutic failure. Many of these are fundamentally evolutionary questions about which adaptive routes are accessible under selection and how reproducibly they arise. Here, we make the case that yeast experimental evolution can serve cancer biologists as a practical upstream discovery platform, because its control, scale, and temporal resolution expose recurrent adaptive routes, transient intermediates, compensatory mechanisms, and genotype-to-fitness relationships that mammalian systems resolve only slowly. We organize representative studies by evolutionary dynamic, set out design principles and an operational workflow, and mark where these models are strong, where they mislead, and where fission yeast adds complementary strengths. Together, these elements frame yeast evolution as a way to prioritize and sharpen downstream validation in cancer systems.

Rapid aging and disassembly of actin filaments from two evolutionary distant yeasts.

Protection and deprotection of the Rec8 cohesin complex during meiosis.

Ataxia telangiectasia and Rad3-related (ATR) initiates cell cycle checkpoints to maintain genome integrity in the presence of replication stress or various forms of DNA damage. However, how ATR is activated for checkpoint initiation remains incompletely understood. The canonical model suggests that binding of an ATR-activator protein relieves the autoinhibitory PIKK regulatory domain (PRD) within the kinase domain, thereby activating ATR by granting substrate access to the catalytic centre. To better understand the checkpoint initiation mechanism, we conducted a genetic screen in fission yeast that identified a charge-reversal mutation, E1369K, in the conserved FRAP-ATM-TRRAP (FAT) domain of Rad3, the ortholog of ATR. In vitro kinase assays show that the mutation converts Rad3 into a constitutively active form. This allows rescue of the Rad3 kinase signaling defect in cells lacking the phosphorylation of the Rad9-Rad1-Hus1 (9-1-1) complex specifically in the DNA replication checkpoint, not the damage checkpoint pathway. Since the mutation is not in the kinase domain and is away from the PRD, these findings show that, in addition to the canonical mechanism, Rad3 may also be activated allosterically via the FAT domain, a mechanism likely conserved in higher eukaryotes.

Removal of the 5' m⁷G cap by the Dcp1/Dcp2 complex commits mRNAs to degradation, yet the mechanisms regulating decapping remain incompletely understood. Here, we identify residue-level determinants within the extended C-terminus of fission yeast Dcp2 that repress activity. Mutations in conserved inhibitory motifs relieve autoinhibition, enhance RNA binding, and bypass the requirement for the activator Edc3. Strikingly, this activation persists within phase-separated condensates, demonstrating that conformational relief in solution is propagated to the dense phase. We further show that long-range interactions between the intrinsically disordered region of Dcp2 and the catalytic core restrict RNA engagement, providing a mechanistic basis for negative regulation. Together, these findings establish that sequence-encoded elements within the Dcp2 C-terminus control catalytic activity and functional output within biomolecular condensates. More broadly, our results reveal that competing interactions encoded within intrinsically disordered regions of proteins are balanced to allosterically tune enzyme activity, providing a general mechanism by which proteins modulate distinct enzymatic functions within biological condensates.

Mitotic chromosome dimensions differ between species, and they differ between developmental stages within an organism. The physiological determinants of chromosome size remain poorly understood. Here, we investigate chromosome size determinants in the fission yeast Schizosaccharomyces pombe. Super-resolution microscopy and semi-automated measurements reveal that cell and nuclear volume in interphase, and the time spent in mitosis (both previously proposed chromosome size determinants), have little influence on resultant chromosome dimensions. Instead, levels of the chromosomal condensin complex affect chromosome size, with increasing condensin levels resulting in more compact (thinner and shorter) chromosomes. Our observations inform the understanding of how chromosome dimensions are controlled in an organism. They suggest that a chromosome-intrinsic mechanism sets chromosome size, more so than the environment in which chromosomes find themselves in.

Living organisms have thermal limits above which they are unable to operate and survive. Our previous genetic screen identified proteins that impede the high-temperature growth of fission yeast, including the RNA-binding protein Dri1 and a fission yeast-specific protein termed Rhs1. Here, we show that Dri1 and Rhs1 form a complex and physically interact with the Ccr4-Not complex, a master regulator of mRNA metabolism. Gene expression analysis revealed that the Dri1-Rhs1 and Ccr4-Not complexes negatively regulate a set of genes implicated in ribosome biogenesis (Ribi genes). Loss of the Dri1-Rhs1 complex results in the augmented expression of Ribi genes, thereby suppressing the translation defects and the growth inhibition under high-temperature conditions. The thermosensitivity of the translational processes may be a determinant of the upper limit of the growth temperature in fission yeast.

The Tel2-Tti1-Tti2, or TTT complex, is the co-chaperone for co-translational maturation of all phosphatidylinositol 3-kinase-related kinases (PIKKs). The complex is highly conserved in eukaryotes and controls multiple cellular processes through PIKKs. Mutations of the TTT complex have recently been linked to disease syndromes and cancer. In Schizosaccharomyces pombe, six PIKKs are expressed: Rad3ATR, Tel1ATM, Tor1 and Tor2 (homologs of mTOR), and Tra1 and Tra2 (homologs of TRRAP). While Rad3ATR and Tel1ATM are the central cellcycle checkpoint kinases in response to DNA damage and replication stress, the other four PIKKs govern cell growth, nutrient sensing, and transcriptional regulation. Here, we report the identification of seven tti1 mutants in fission yeast that are sensitive to genotoxins. Characterization of one of the mutants, tti1-N18, reveals that the mutation selectively eliminates the kinase function of Rad3ATR, but not that of Tel1ATM. Further examination shows that, like Tel1ATM, the functions of the other four PIKKs are also largely uncompromised in the tti1-N18 mutant. These findings suggest a mechanism by which the TTT complex confers functional specificity towards Rad3ATR among the PIKKs. Since human Tel2 has been identified as a target of the antiparasitic drug Ivermectin, further investigation of the substrate specificity of the TTT complex may reveal a therapeutic vulnerability for treatment of cancer or other diseases.

The fungal pathogenCryptococcus neoformansis estimated to cause approximately 180,000 deaths annually, primarily among immunocompromised HIV patients in resource-limited countries in central and southern Africa. Economic constraints regularly restrict treatment to fluconazole monotherapy. Unstably resistant cells arise in any otherwise fluconazole-sensitiveC. neoformanspopulation, with such ‘heteroresistance’ often involving chromosome 1 disomy. Here we uncover a form of heteroresistance that occurs by a mechanism distinct from aneuploidy. Transient islands of histone H3 lysine 9 methylation-dependent heterochromatin occur at several locations across the genomes of clinical isolates from African patients. Some islands dissipate in the absence of fluconazole selection but are restored upon re-exposure. Deletion of specific heterochromatin island-located genes increased fluconazole resistance in an otherwise wild-type background, suggesting that heterochromatin-mediated alterations in gene expression elicit unstable resistance. Thus, in addition to aneuploidy, transient heterochromatin islands also appear to contribute toC. neoformansfluconazole heteroresistance. Heterochromatin islands have recently been shown to confer unstable antifungal resistance in the ascomycete fungusSchizosaccharomyces pombe(fission yeast) and fungal species of theMucor circinelloidescomplex. Thus, similar unstable resistant epimutations may be widespread in pathogenic fungi, however, their instability and inaccessibility to detection by direct sequencing poses challenges for monitoring this form of heteroresistance in clinical settings.

Septins are conserved polymer-forming proteins that scaffold the actomyosin cytoskeleton, its regulators, and other factors to cellular membranes. Septins also sense micron-scale curvature, bind microtubules, and establish membrane diffusion barriers.C. elegansis a powerful animal model to study septins’ roles because there are only two septin genes:unc-59andunc-61. In many fungal and animal cell types, septins are required for proper cytokinesis. In theC. eleganszygote, septins’ scaffolding roles in cytokinesis manifest during the chiral rotation of the cell cortex and the asymmetry of cytokinetic ring closure. Originally named for the uncoordinated movement exhibited by hypomorphic alleles, UNC-59 and UNC-61 are also required for normal postembryonic development, germline development, and fertility. To studyC. elegansseptins in these various contexts, we sought a fluorescent-protein tagging strategy that minimally perturbed septin function. We examined strains in which GFP, mKate2 or wrmScarlet had been inserted at theunc-59locus, or coupled tounc-61b/cat an exogenous locus, to encode fluorescently tagged fusion proteins. We compared these tagged septins to classical hypomorphic alleles, and to new null alleles. Null alleles phenocopied hypomorphic alleles in all our assays. Strains bearing fluorescently tagged septins exhibited defects in zygote cytokinesis, qualitatively phenocopying both hypomorphic and null alleles. These findings agreed with recent work with fission yeast, demonstrating the sensitivity of septin function to tagging. Interestingly, tagging septins did not perturb postembryonic development including animal mobility. This suggests that septins play distinct functions in the zygote versus later in development.

Pre-mRNA splicing is an essential step in gene expression regulation. It is mediated by the spliceosome, a large ribonucleoprotein complex that undergoes dynamic structural and compositional rearrangements during each splicing cycle. Although the mechanisms of splicing and the roles of main spliceosomal components are well defined, the identities and functions of transiently associated spliceosomal proteins remain incompletely understood. Here, we investigated the molecular function of the poorly characterized G-patch domain-containing protein SPAC6F6.19 (herein Sap34, for spliceosome-associated protein of 34kDa) in Schizosaccharomyces pombe, an ortholog of human GPATCH11. Using affinity purification and a yeast two-hybrid assay, we analyzed its interactome and identified its interaction partners. In addition, long-read sequencing was employed to assess Sap34-dependent changes in splicing efficiency. We found that Sap34 forms a complex with components of the U2 small nuclear ribonucleoprotein (snRNP) and the U4/U6 × U5 tri-snRNP, which are required for early spliceosome assembly and activation. Furthermore, we defined the interaction specificity of Sap34 with splicing proteins, demonstrating the importance of its C-terminal region for binding to Sap61 and Ini1, and of its G-patch domain for interaction with ATP-dependent RNA helicase Prp43, suggesting that G-patch domain of Sap34 may contribute to the regulation of Prp43 activity in early spliceosomes. Notably, we showed that deletion of sap34 leads to a global reduction in splicing efficiency, predominantly associated with increased intron retention. Together, these findings identify Sap34 as a previously unrecognized and important G-patch domain-containing protein regulating the early steps of pre-mRNA splicing in fission yeast.

The assembly of the Sm core, a ring-shaped protein complex essential for spliceosomal small nuclear ribonucleoprotein particle biogenesis, is chaperoned by the SMN complex in most eukaryotes. Intriguingly, the budding yeast Saccharomyces cerevisiae possesses a simplified version of this machinery-a single protein, Brr1-raising the question of whether it can functionally replace the entire multi-subunit SMN complex. Here, we engineered Brr1 (eBrr1) to bind Schizosaccharomyces pombe Sm proteins D1/D2/F/E/G (5Sm) and introduced it into fission yeast, which relies on a five-membered SMN complex. We demonstrate that eBrr1 functionally substitutes for the essential Gemin2 subunit. Surprisingly, in this context, the Gemin2-binding domain of SMN becomes dispensable, revealing that a stable SMN-Gemin2 interaction is not required for viability. Furthermore, we show that the SMN/Gemin6-8 subcomplex alone is sufficient to displace the assembly chaperone ICln from the ICln/5Sm/eBrr1 complex. Our findings provide key mechanistic insights into the stepwise action of the SMN complex and propose an evolutionary model in which the ICln-release machinery predated the stable incorporation of Gemin2.

Methods to Study Mitochondrial Metabolism and Homeostasis in Fission Yeast.

Tau pathology is increasingly recognized as a driver of metabolic dysfunction in neurodegenerative diseases, extending beyond protein aggregation to include impairments in glucose metabolism and redox homeostasis. However, how metabolic cofactors affect tau-associated metabolic stress remains incompletely understood. In this study, we employed a fission yeast (Schizosaccharomyces pombe) model expressing human tau to investigate the effects of vitamin B6 on glucose metabolism and cellular redox balance under glucose-limited conditions in tau-expressing cells. Cells were treated with vitamin B6 and analyzed for tau expression and phosphorylation, glucose consumption, NAD⁺/NADH ratio, and the expression of selected glucose metabolism-related genes under glucose starvation conditions. Vitamin B6 treatment was associated with a reduction in tau protein expression and phosphorylation at specific residues (S262, S396, S404). In addition, vitamin B6 affected glucose metabolism-related gene expression and was accompanied by modest changes in glucose consumption and redox balance. These effects were observed in both control and tau-expressing cells, although the patterns of response differed between the two conditions. While vitamin B6 generally enhanced the expression of glucose utilization-related genes in control cells, its effects in tau-expressing cells were more variable, indicating altered metabolic regulation under tau-associated stress. These findings suggest that vitamin B6 may contribute to cellular metabolic adaptation under tau-induced stress. This study demonstrates the usefulness of fission yeast as a tractable model for examining metabolic aspects of tau pathology and provides insight into how metabolic cofactors influence tau-associated cellular stress.