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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

TOR regulates variability of protein synthesis rates.

Cellular processes are subject to inherent variability, but the extent to which cells can regulate this variability has received little investigation. Here, we explore the characteristics of the rate of cellular protein synthesis in single cells of the eukaryote fission yeast. Strikingly, this rate is highly variable despite protein synthesis being dependent on hundreds of reactions which might be expected to average out at the overall cellular level. The rate is variable over short time scales, and exhibits homoeostatic behaviour at the population level. Cells can regulate the level of variability through processes involving the TOR pathway, suggesting there is an optimal level of variability conferring a selective advantage. While this could be an example of bet-hedging, but we propose an alternative explanation: regulated 'loose' control of complex processes of overall cellular metabolism such as protein synthesis, may lead to this variability. This could ensure cells are fluid in control and agile in response to changing conditions, and may constitute a novel organisational principle of complex metabolic cellular systems.

The deletion of ppr2 interferes iron sensing and leads to oxidative stress response in Schizosaccharomyces pombe.

Pentatricopeptide repeat proteins are involved in mitochondrial both transcriptional and posttranscriptional regulation. Schizosaccharomyces pombe Ppr2 is a general mitochondrial translation factor that plays a critical role in the synthesis of all mitochondrial DNA-encoded oxidative phosphorylation subunits, which are essential for mitochondrial respiration. Our previous analysis showed that ppr2 deletion resulted in increased expression of iron uptake genes and caused ferroptosis-like cell death in S. pombe. In the present work, we showed that deletion of ppr2 reduced viability on glycerol- and galactose-containing media.Php4 is a transcription repressor that regulates iron homeostasis in fission yeast. We found that in the ppr2 deletion strain, Php4 was constitutively active and accumulated in the nucleus in the stationary phase. We also found that deletion of ppr2 decreased the ferroptosis-related protein Gpx1 in the mitochondria. Overexpression of Gpx1 improves the viability of Δppr2 cells. We showed that the deletion of ppr2 increased the production of ROS, downregulated heme synthesis and iron-sulfur cluster proteins, and induced stress proteins. Finally, we observed the nuclear accumulation of Pap1-GFP and Sty1-GFP, suggesting that Sty1 and Pap1 in response to cellular stress in the ppr2 deletion strain. These results suggest thatppr2 deletion may cause mitochondrial dysfunction, which is likely to lead to iron-sensing defect and iron starvation response, resulting in perturbation of iron homeostasis and increased hydroxyl radical production. The increased hydroxyl radical production triggers cellular responses in theppr2 deletion strain.

The Rtf1/Prf1-dependent histone modification axis counteracts multi-drug resistance in fission yeast.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

MAPK-dependent control of mitotic progression in S. pombe.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress. RESULTS: We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit. CONCLUSIONS: Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.

RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.