Fate of telomere entanglements is dictated by the timing of anaphase midregion nuclear envelope breakdown.

Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell's midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm.

Sororin is an evolutionary conserved antagonist of WAPL.

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

The SUN-family protein Sad1 mediates heterochromatin spatial organization through interaction with histone H2A-H2B.

Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing remains unclear. Here we found that Sad1, an inner nuclear membrane SUN-family protein in fission yeast, interacts with histone H2A-H2B but not H3-H4. We solved the crystal structure of the histone binding motif (HBM) of Sad1 in complex with H2A-H2B, revealing the intimate contacts between Sad1HBM and H2A-H2B. Structure-based mutagenesis studies revealed that the H2A-H2B-binding activity of Sad1 is required for the dynamic distribution of Sad1 throughout the nuclear envelope (NE). The Sad1-H2A-H2B complex mediates tethering telomeres and the mating-type locus to the NE. This complex is also important for heterochromatin silencing. Mechanistically, H2A-H2B enhances the interaction between Sad1 and HDACs, including Clr3 and Sir2, to maintain epigenetic identity of heterochromatin. Interestingly, our results suggest that Sad1 exhibits the histone-enhanced liquid-liquid phase separation property, which helps recruit heterochromatin factors to the NE. Our results uncover an unexpected role of SUN-family proteins in heterochromatin regulation and suggest a nucleosome-independent role of H2A-H2B in regulating Sad1's functionality.

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Pps1, phosphatidylserine synthase, regulates the salt stress response in Schizosaccharomyces pombe.

Phosphatidylserine (PS) is important for maintaining growth, cytoskeleton, and various functions in yeast; however, its role in stress responses is poorly understood. In Schizosaccharomyces pombe, the PS synthase deletion (pps1∆) mutant shows defects in growth, morphology, cytokinesis, actin cytoskeleton, and cell wall integrity, and these phenotypes are rescued by ethanolamine supplementation. Here, we evaluated the role of Pps1 in the salt stress response in S. pombe. We found that pps1∆ cells are sensitive to salt stresses such as KCl and CaCl2 even in the presence of ethanolamine. Loss of the functional cAMP-dependent protein kinase (git3∆ or pka1∆) or phospholipase B Plb1 (plb1∆) enhanced the salt stress-sensitive phenotype in pps1∆ cells. Green fluorescent protein (GFP)-Pps1 was localized at the plasma membrane and endoplasmic reticulum regardless of the stress conditions. In pka1∆ cells, GFP-Pps1 was accumulated around the nucleus under the KCl stress. Pka1 was localized in the nucleus and the cytoplasm under normal conditions and transferred from the nucleus to the cytoplasm under salt-stress conditions. Pka1 translocated from the nucleus to the cytoplasm during CaCl2 stress in the wild-type cells, while it remained localized in the nucleus in pps1∆ cells. Expression and phosphorylation of Pka1-GFP were not changed in pps1∆ cells. Our results demonstrate that Pps1 plays an important role in the salt stress response in S. pombe.

Mathematical model for the role of multiple pericentromeric repeats on heterochromatin assembly.

Although the length and constituting sequences for pericentromeric repeats are highly variable across eukaryotes, the presence of multiple pericentromeric repeats is one of the conserved features of the eukaryotic chromosomes. Pericentromeric heterochromatin is often misregulated in human diseases, with the expansion of pericentromeric repeats in human solid cancers. In this article, we have developed a mathematical model of the RNAi-dependent methylation of H3K9 in the pericentromeric region of fission yeast. Our model, which takes copy number as an explicit parameter, predicts that the pericentromere is silenced only if there are many copies of repeats. It becomes bistable or desilenced if the copy number of repeats is reduced. This suggests that the copy number of pericentromeric repeats alone can determine the fate of heterochromatin silencing in fission yeast. Through sensitivity analysis, we identified parameters that favor bistability and desilencing. Stochastic simulation shows that faster cell division and noise favor the desilenced state. These results show the unexpected role of pericentromeric repeat copy number in gene silencing and provide a quantitative basis for how the copy number allows or protects repetitive and unique parts of the genome from heterochromatin silencing, respectively.

Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.

Acetylation of Rec8 cohesin complexes regulates reductional chromosome segregation in meiosis.

For establishing sister chromatid cohesion and proper chromosome segregation in mitosis in fission yeast, the acetyltransferase Eso1 plays a key role. Eso1 acetylates cohesin complexes, at two conserved lysine residues K105 and K106 of the cohesin subunit Psm3. Although Eso1 also contributes to reductional chromosome segregation in meiosis, the underlying molecular mechanisms have remained elusive. Here, we purified meiosis-specific Rec8 cohesin complexes localized at centromeres and identified a new acetylation at Psm3-K1013, which largely depends on the meiotic kinetochore factor meikin (Moa1). Our molecular genetic analyses indicate that Psm3-K1013 acetylation cooperates with canonical acetylation at Psm3-K105 and K106, and plays a crucial role in establishing reductional chromosome segregation in meiosis.

CDK activity at the centrosome regulates the cell cycle.

In human cells and yeast, an intact "hydrophobic patch" substrate docking site is needed for mitotic cyclin centrosomal localization. A hydrophobic patch mutant (HPM) of the fission yeast mitotic cyclin Cdc13 cannot enter mitosis, but whether this is due to defective centrosomal localization or defective cyclin-substrate docking more widely is unknown. Here, we show that artificially restoring Cdc13-HPM centrosomal localization promotes mitotic entry and increases CDK (cyclin-dependent kinase) substrate phosphorylation at the centrosome and in the cytoplasm. We also show that the S-phase B-cyclin hydrophobic patch is required for centrosomal localization but not for S phase. We propose that the hydrophobic patch is essential for mitosis due to its requirement for the local concentration of cyclin-CDK with CDK substrates and regulators at the centrosome. Our findings emphasize the central importance of the centrosome as a hub coordinating cell-cycle control and explain why the cyclin hydrophobic patch is essential for mitosis.

The bistable mitotic switch in fission yeast.

In favorable conditions, eukaryotic cells proceed irreversibly through the cell division cycle (G1-S-G2-M) in order to produce two daughter cells with the same number and identity of chromosomes of their progenitor. The integrity of this process is maintained by "checkpoints" that hold a cell at particular transition points of the cycle until all requisite events are completed. The crucial functions of these checkpoints seem to depend on irreversible bistability of the underlying checkpoint control systems. Bistability of cell cycle transitions has been confirmed experimentally in frog egg extracts, budding yeast cells and mammalian cells. For fission yeast cells, a recent paper by Patterson et al. (2021) provides experimental evidence for an abrupt transition from G2 phase into mitosis, and we show that these data are consistent with a stochastic model of a bistable switch governing the G2/M checkpoint. Interestingly, our model suggests that their experimental data could also be explained by a reversible/sigmoidal switch, and stochastic simulations confirm this supposition. We propose a simple modification of their experimental protocol that could provide convincing evidence for (or against) bistability of the G2/M transition in fission yeast.

Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe.

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.

Quantifying redox transcription factor dynamics as a tool to investigate redox signalling.

A critical feature of the cellular antioxidant response is the induction of gene expression by redox-sensitive transcription factors. In many cells, activating these transcription factors is a dynamic process involving multiple redox steps, but it is unclear how these dynamics should be measured. Here, we show how the dynamic profile of the Schizosaccharomyces pombe Pap1 transcription factor is quantifiable by three parameters: signal amplitude, signal time and signal duration. In response to increasing hydrogen peroxide concentrations, the Pap1 amplitude decreased while the signal time and duration showed saturable increases. In co-response plots, these parameters showed a complex, non-linear relationship to the mRNA levels of four Pap1-regulated genes. We also demonstrate that hydrogen peroxide and tert-butyl hydroperoxide trigger quantifiably distinct Pap1 activation profiles and transcriptional responses. Based on these findings, we propose that different oxidants and oxidant concentrations modulate the Pap1 dynamic profile, leading to specific transcriptional responses. We further show how the effect of combination and pre-exposure stresses on Pap1 activation dynamics can be quantified using this approach. This method is therefore a valuable addition to the redox signalling toolbox that may illuminate the role of dynamics in determining appropriate responses to oxidative stress.

Bqt4 affects relative movement between SPB and nucleolus in fission yeast.

Movement dynamics in the nucleus involve various biological processes, including DNA repair, which is crucial for cancer prevention. Changes in the movement of the components of the nucleus indicate the changes in movement dynamics in the nucleus. In Schizosaccharomyces pombe, the inner nuclear membrane protein Bqt4 plays an essential role in attaching telomeres to the nuclear envelope. We observed that the deletion of bqt4+ caused a significant decrease in the mean square displacement (MSD) calculated from the distance between the nucleolar center and spindle pole body (SPB), hereafter referred to as MSD(SPB-Nucleolus). The MSD(SPB-Nucleolus) decrease in bqt4Δ was microtubule-dependent. The Rap1-binding ability loss mutant, bqt4F46A, and nonspecific DNA-binding ability mutants, bqt43E-A, did not exhibit an MSD(SPB-Nucleolus) decrease compared to the WT. Moreover, the bqt43E-Arap1Δ double mutant and 1-262 amino acids truncated mutant bqt4ΔN (263-432), which does not have either Rap1-binding or nonspecific DNA-binding abilities, did not exhibit the MSD(SPB-Nucleolus) decrease to the same extent as bqt4Δ. These results suggest that the unknown function of Bqt4 in the C-terminal domain is essential for the maintenance of the pattern of relative movement between SPB and the nucleolus.

The Schizosaccharomyces pombe DEAD-box protein Mss116 is required for mitoribosome assembly and mitochondrial translation.

DEAD-box helicases are important players in mitochondrial gene expression, which is necessary for mitochondrial respiration. In this study, we characterized Schizosaccharomyces pombe Mss116 (spMss116), a member of the family of DEAD-box RNA helicases. Deletion of spmss116 in a mitochondrial intron-containing background significantly reduced the levels of mitochondrial DNA (mtDNA)-encoded cox1 and cob1 mRNAs and impaired mitochondrial translation, leading to a severe respiratory defect and a loss of cell viability during stationary phase. Deletion of mitochondrial introns restored the levels of cox1 and cob1 mRNAs to wide-type (WT) levels but could not restore mitochondrial translation and respiration in Δspmss116 cells. Furthermore, deletion of spmss116 in both mitochondrial intron-containing and intronless backgrounds impaired mitoribosome assembly and destabilization of mitoribosomal proteins. Our findings suggest that defective mitochondrial translation caused by deletion of spmss116 is most likely due to impaired mitoribosome assembly.

Low-Molecular Weight Compounds that Extend the Chronological Lifespan of Yeasts, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

Yeast is an excellent model organism for research for regulating aging and lifespan, and the studies have made many contributions to date, including identifying various factors and signaling pathways related to aging and lifespan. More than 20 years have passed since molecular biological perspectives are adopted in this research field, and intracellular factors and signal pathways that control aging and lifespan have evolutionarily conserved from yeast to mammals. Furthermore, these findings have been applied to control the aging and lifespan of various model organisms by adjustment of the nutritional environment, genetic manipulation, and drug treatment using low-molecular weight compounds. Among these, drug treatment is easier than the other methods, and research into drugs that regulate aging and lifespan is consequently expected to become more active. Chronological lifespan, a definition of yeast lifespan, refers to the survival period of a cell population under nondividing conditions. Herein, low-molecular weight compounds are summarized that extend the chronological lifespan of Saccharomyces cerevisiae and Schizosaccharomyces pombe, along with their intracellular functions. The low-molecular weight compounds are also discussed that extend the lifespan of other model organisms. Compounds that have so far only been studied in yeast may soon extend lifespan in other organisms.

Dysfunction of Gpl1-Gih35-Wdr83 Complex in S. pombe Affects the Splicing of DNA Damage Repair Factors Resulting in Increased Sensitivity to DNA Damage.

Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1-Gih35-Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1-Gih35-Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1-Gih35-Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.

Identification and characterization of a novel antifungal compound tubeimoside I targeting cell wall.

Due to fungal diseases that threaten immunocompromised patients, along with the limited availability of antifungal agents, there is an urgent need for new antifungal compounds to treat fungal infections. Here, we aimed to identify potential antifungal drugs from natural products using the fission yeast Schizosaccharomyces pombe as a model organism since it shares many features with some pathogenic fungi. Here, we identified tubeimoside I (TBMS1), an extract from Chinese herbal medicine, that showed strong antifungal activity against S. pombe. To gain insight into the underlying mechanism, we performed transcriptomics analyses of S. pombe cells exposed to TBMS1. A significant proportion of the differential expressed genes were involved in cell wall organization or biogenesis. Additionally, TBMS1 treatment of S. pombe cells resulted in pleiotropic phenotypes, including increased sensitivity to ß-glucanase, enhanced calcineurin activity, translocation of GFP-Prz1 to the nucleus, as well as enhanced dephosphorylation of Prz1, suggesting that TBMS1 disrupted cell wall integrity of S. pombe cells. Notably, calcofluor staining showed that abnormal deposits of cell wall materials were observed in the septum and cell wall of the TBMS1-treated cells, which were further corroborated by electron microscopy analysis. We also found that oxidative stress might be involved in the antifungal action of TBMS1. Moreover, we confirmed the antifungal activities of TBMS1 against several clinical isolates of pathogenic fungi. Collectively, our findings suggest that TBMS1, a novel antifungal compound, exerts its antifungal activity by targeting cell walls, which may pave the way for the development of a new class of antifungals. IMPORTANCE: Fungal infections pose a serious threat to public health and have become an emerging crisis worldwide. The development of new antifungal agents is urgently needed. Here, we identified compound tubeimoside I (TBMS1) for the first time showing strong antifungal activity, and explored the underlying mechanisms of its antifungal action by using the model yeast Schizosaccharomyces pombe. Notably, we presented multiple evidence that TBMS1 exerts its antifungal activity through targeting fungal cell walls. Moreover, we verified the antifungal activities of TBMS1 against several pathogenic fungi. Our work indicated that TBMS1 may serve as a novel antifungal candidate, which provides an important foundation for designing and developing new cell wall-targeting agents for combating life-threatening fungal infections.

Chromodomain mutation in S. pombe Kat5/Mst1 affects centromere dynamics and DNA repair.

KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that is involved in multiple cellular activities. This family is characterized in part by containing a chromodomain, a motif associated with binding methylated histones. We show that a chromodomain mutation in the S. pombe Kat5, mst1-W66R, has defects in pericentromere silencing. mst1-W66R is sensitive to camptothecin (CPT) but only at an increased temperature of 36°C, although it is proficient for growth at this temperature. We also describe a de-silencing effect at the pericentromere by CPT that is independent of RNAi and methylation machinery. We also show that mst1-W66R disrupts recruitment of proteins to repair foci in response to camptothecin-induced DNA damage. Our data suggest a function of Mst1 chromodomain in centromere heterochromatin formation and a separate role in genome-wide damage repair in CPT.

Effects of ScRgt1-Like DNA-binding transcription factor SpRgt1 (SPCC320.03) on Hexose transporters gene expression in Schizosaccharomyces pombe.

Glucose, which plays an essential role in carbon and energy metabolism in eukaryotes, is vital in directing various energy-consuming cellular processes. In S. cerevisiae, transcription factors involved in regulating hexose transporters and their mechanisms of action under different carbon sources were revealed in detail. However, there is limited information on these processes in S. pombe. In this study, the effect of SPCC320.03 (named SpRgt1), the ortholog of ScRgt1 whose molecular mechanism is known in detail in S. cerevisiae, on the transcriptional regulation of hexose transporters (ght1-8) dependent on different carbon sources was investigated. We measured the transcript levels of ght1-8 using the qPCR technique and performed relative evaluation in S. pombe strains (parental, rgt1 deleted mutant, rgt1 overexpressed, and vectoral rgt1 carrying mutant). We aimed to investigate the transcriptional changes caused by the protein product of the rgt1 (SPCC320.03) gene in terms of ght1-8 genes in strains that are grown in different carbon sources (2% glucose, 2% glycerol + 0.1% glucose, and 2% gluconate). Here, we show that SpRgt1 is involved in the regulation of the ght3, ght4, ght6, and ght7 genes but that the ght1, ght2, ght5, and ght8 gene expression vary depending on carbon sources, independently of SpRgt1.