Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast.

The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15's abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases' abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid-liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.

Shotgun knockdown of RNA by CRISPR-Cas13d in fission yeast.

The CRISPR-Cas13d system has a single small effector protein that targets RNA and does not require the presence of a protospacer flanking site in the targeted transcript. These features make CRISPR-Cas13d an attractive system for RNA manipulation. Here, we report the successful implementation of the CRISPR-Cas13d system in fission yeast for RNA knockdown. A high effectiveness of the CRISPR-Cas13d system was ensured by using an array of CRISPR RNAs (crRNAs) that are flanked by two self-cleaving ribozymes and are expressed from an RNA polymerase II promoter. Given the repressible nature of the promoter, RNA knockdown by the CRISPR-Cas13d system is reversible. Moreover, using the CRISPR-Cas13d system, we identified an effective crRNA array targeting the transcript of gfp and the effectiveness was demonstrated by successful knockdown of the transcripts of noc4-gfp, bub1-gfp and ade6-gfp. In principle, the effective GFP crRNA array allows knockdown of any transcript carrying the GFP sequences. This new CRISPR-Cas13d-based toolkit is expected to have a wide range of applications in many aspects of biology, including dissection of gene function and visualization of RNA.

Myosin II regulatory light chain phosphorylation and formin availability modulate cytokinesis upon changes in carbohydrate metabolism.

Cytokinesis, the separation of daughter cells at the end of mitosis, relies in animal cells on a contractile actomyosin ring (CAR) composed of actin and class II myosins, whose activity is strongly influenced by regulatory light chain (RLC) phosphorylation. However, in simple eukaryotes such as the fission yeast Schizosaccharomyces pombe, RLC phosphorylation appears dispensable for regulating CAR dynamics. We found that redundant phosphorylation at Ser35 of the S. pombe RLC homolog Rlc1 by the p21-activated kinases Pak1 and Pak2, modulates myosin II Myo2 activity and becomes essential for cytokinesis and cell growth during respiration. Previously, we showed that the stress-activated protein kinase pathway (SAPK) MAPK Sty1 controls fission yeast CAR integrity by downregulating formin For3 levels (Gómez-Gil et al., 2020). Here, we report that the reduced availability of formin For3-nucleated actin filaments for the CAR is the main reason for the required control of myosin II contractile activity by RLC phosphorylation during respiration-induced oxidative stress. Thus, the restoration of For3 levels by antioxidants overrides the control of myosin II function regulated by RLC phosphorylation, allowing cytokinesis and cell proliferation during respiration. Therefore, fine-tuned interplay between myosin II function through Rlc1 phosphorylation and environmentally controlled actin filament availability is critical for a successful cytokinesis in response to a switch to a respiratory carbohydrate metabolism.

The ecl family gene ecl3+ is induced by phosphate starvation and contributes to sexual differentiation in fission yeast.

In Schizosaccharomyces pombe, ecl family genes are induced by several signals, such as starvation of various nutrients, including sulfur, amino acids and Mg2+, and environmental stress, including heat or oxidative stress. These genes mediate appropriate cellular responses and contribute to the maintenance of cell viability and induction of sexual differentiation. Although this yeast has three ecl family genes with overlapping functions, any environmental conditions that induce ecl3+ remain unidentified. We demonstrate that ecl3+ is induced by phosphate starvation, similar to its chromosomally neighboring genes, pho1+ and pho84+, which respectively encode an extracellular acid phosphatase and an inorganic phosphate transporter. ecl3+ expression was induced by the transcription factor Pho7 and affected by the cyclin-dependent kinase (CDK)-activating kinase Csk1. Phosphate starvation induced G1 arrest and sexual differentiation via ecl family genes. Biochemical analyses suggested that this G1 arrest was mediated by the stabilization of the CDK inhibitor Rum1, which was dependent on ecl family genes. This study shows that ecl family genes are required for appropriate responses to phosphate starvation and provides novel insights into the diversity and similarity of starvation responses.

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex.

The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3'-end processing machineries.

Fission Yeast PUF Proteins Puf3 and Puf4 Are Novel Regulators of PI4P5K Signaling.

Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3+ gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3+ and puf4+, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3+ and puf4+ genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.

The SAGA histone acetyltransferase module targets SMC5/6 to specific genes.

BACKGROUND: Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Their physical binding to DNA requires accessible chromatin. RESULTS: We performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA-repair proteins, we first analysed the formation of DNA-damage-induced SMC5/6 foci in the Δgcn5 mutant. The SMC5/6 foci formed normally in Δgcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in Δgcn5 and Δada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant. CONCLUSION: Our data show genetic and physical interactions between SMC5/6 and SAGA complexes. The ChIP-seq analysis suggests that SAGA HAT module targets SMC5/6 to specific gene regions and facilitates their accessibility for SMC5/6 loading.

Interplays of AMPK and TOR in Autophagy Regulation in Yeast.

Cells survey their environment and need to balance growth and anabolism with stress programmes and catabolism towards maximum cellular bioenergetics economy and survival. Nutrient-responsive pathways, such as the mechanistic target of rapamycin (mTOR) interact and cross-talk, continuously, with stress-responsive hubs such as the AMP-activated protein kinase (AMPK) to regulate fundamental cellular processes such as transcription, protein translation, lipid and carbohydrate homeostasis. Especially in nutrient stresses or deprivations, cells tune their metabolism accordingly and, crucially, recycle materials through autophagy mechanisms. It has now become apparent that autophagy is pivotal in lifespan, health and cell survival as it is a gatekeeper of clearing damaged macromolecules and organelles and serving as quality assurance mechanism within cells. Autophagy is hard-wired with energy and nutrient levels as well as with damage-response, and yeasts have been instrumental in elucidating such connectivities. In this review, we briefly outline cross-talks and feedback loops that link growth and stress, mainly, in the fission yeast Schizosaccharomyces pombe, a favourite model in cell and molecular biology.

Comparative Research: Regulatory Mechanisms of Ribosomal Gene Transcription in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Restricting ribosome biosynthesis and assembly in response to nutrient starvation is a universal phenomenon that enables cells to survive with limited intracellular resources. When cells experience starvation, nutrient signaling pathways, such as the target of rapamycin (TOR) and protein kinase A (PKA), become quiescent, leading to several transcription factors and histone modification enzymes cooperatively and rapidly repressing ribosomal genes. Fission yeast has factors for heterochromatin formation similar to mammalian cells, such as H3K9 methyltransferase and HP1 protein, which are absent in budding yeast. However, limited studies on heterochromatinization in ribosomal genes have been conducted on fission yeast. Herein, we shed light on and compare the regulatory mechanisms of ribosomal gene transcription in two species with the latest insights.

Exploring Genetic Interactions with Telomere Protection Gene pot1 in Fission Yeast.

The regulation of telomere length has a significant impact on cancer risk and aging in humans. Circular chromosomes are found in humans and are often unstable during mitosis, resulting in genome instability. Some types of cancer have a high frequency of a circular chromosome. Fission yeast is a good model for studying the formation and stability of circular chromosomes as deletion of pot1 (encoding a telomere protection protein) results in rapid telomere degradation and chromosome fusion. Pot1 binds to single-stranded telomere DNA and is conserved from fission yeast to humans. Loss of pot1 leads to viable strains in which all three fission yeast chromosomes become circular. In this review, I will introduce pot1 genetic interactions as these inform on processes such as the degradation of uncapped telomeres, chromosome fusion, and maintenance of circular chromosomes. Therefore, exploring genes that genetically interact with pot1 contributes to finding new genes and/or new functions of genes related to the maintenance of telomeres and/or circular chromosomes.

Opposing Roles of FACT for Euchromatin and Heterochromatin in Yeast.

DNA is stored in the nucleus of a cell in a folded state; however, only the necessary genetic information is extracted from the required group of genes. The key to extracting genetic information is chromatin ambivalence. Depending on the chromosomal region, chromatin is characterized into low-density "euchromatin" and high-density "heterochromatin", with various factors being involved in its regulation. Here, we focus on chromatin regulation and gene expression by the yeast FACT complex, which functions in both euchromatin and heterochromatin. FACT is known as a histone H2A/H2B chaperone and was initially reported as an elongation factor associated with RNA polymerase II. In budding yeast, FACT activates promoter chromatin by interacting with the transcriptional activators SBF/MBF via the regulation of G1/S cell cycle genes. In fission yeast, FACT plays an important role in the formation of higher-order chromatin structures and transcriptional repression by binding to Swi6, an HP1 family protein, at heterochromatin. This FACT property, which refers to the alternate chromatin-regulation depending on the binding partner, is an interesting phenomenon. Further analysis of nucleosome regulation within heterochromatin is expected in future studies.

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color SMLM imaging.

The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.

A mechanism of salt bridge-mediated resistance to FtsZ inhibitor PC190723 revealed by a cell-based screen.

Bacterial cell division proteins, especially the tubulin homologue FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine, and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. On the basis of sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt bridge interactions between the central H7 helix and ß-strands S9 and S10 mediates resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.

The proline-rich domain of fission yeast WASp (Wsp1p) interacts with actin filaments and inhibits actin polymerization.

Members of the Wiskott-Aldrich Syndrome protein (WASp) family activate Arp2/3 complex (actin-related proteins 2 and 3 complex) to form actin filament branches. The proline-rich domain (PRD) of WASp contributes to branching nucleation, and the PRD of budding yeast Las17 binds actin filaments [Urbanek AN et al. (2013) Curr Biol 23, 196-203]. Biochemical assays showed the recombinant PRD of fission yeast Schizosaccharomyces pombe Wsp1p binds actin filaments with micromolar affinity. Recombinant PRDs of both Wsp1p and Las17p slowed the elongation of actin filaments by Mg-ATP-actin monomers by half and slowed the spontaneous polymerization of Mg-ATP-actin monomers modestly. The affinity of PRDs of WASp-family proteins for actin filaments is high enough to contribute to the reported stimulation of actin filament branching by Arp2/3 complex.

Protein S-palmitoylation regulates different stages of meiosis in Schizosaccharomyces pombe.

Posttranslational protein S-palmitoylation regulates the localization and function of its target proteins involved in diverse cellular processes including meiosis. In this study, we demonstrate that S-palmitoylation mediated by Erf2-Erf4 and Akr1 palmitoylacyltransferases is required at multiple meiotic stages in the fission yeast Schizosaccharomyces pombe We find that S-palmitoylation by Erf2-Erf4 is required for Ras1 localization at the cell periphery to enrich at the cell conjugation site for mating pheromone response. In the absence of Erf2 or Erf4, mutant cells are sterile. A role of Akr1 S-palmitoylating the nuclear fusion protein Tht1 to function in karyogamy is identified. We demonstrate that S-palmitoylation stabilizes and localizes Tht1 to ER, interacting with Sey1 ER fusion GTPase for proper meiotic nuclear fusion. In akr1, tht1, or sey1 mutant, meiotic cells, haploid nuclei are unfused with subsequent chromosome segregation defects. Erf2-Erf4 has an additional substrate of the spore coat protein Isp3. In the absence of Erf2, Isp3 is mislocalized from the spore coat. Together, these results highlight the versatility of the cellular processes in which protein S-palmitoylation participates.

Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe.

We constructed a panel of S. pombe strains expressing DNA polymerase ε variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES459F, and used these to compare mutation rates determined by canavanine resistance with other selective methods. Canavanine-resistance mutation rates are broadly similar to those seen with reversion of the ade-485 mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of ura4+ or ura5+ genes). Inactivation of several genes has been associated with canavanine resistance in S. pombe but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an R175C mutation in the any1/arn1 gene. This gene encodes an α-arrestin-like protein involved in mediating Pub1 ubiquitylation of target proteins, and the phenotypic resistance to canavanine by this single mutation is similar to that shown by the original "can1-1" strain, which also has the any1R175C mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The any1R175C strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.

A force balance model for a cell size-dependent meiotic nuclear oscillation in fission yeast.

Fission yeast undergoes premeiotic nuclear oscillation, which is dependent on microtubules and is driven by cytoplasmic dynein. Although the molecular mechanisms have been analyzed, how a robust oscillation is generated despite the dynamic behaviors of microtubules has yet to be elucidated. Here, we show that the oscillation exhibits cell length-dependent frequency and requires a balance between microtubule and viscous drag forces, as well as proper microtubule dynamics. Comparison of the oscillations observed in living cells with a simulation model based on microtubule dynamic instability reveals that the period of oscillation correlates with cell length. Genetic alterations that reduce cargo size suggest that the nuclear movement depends on viscous drag forces. Deletion of a gene encoding Kinesin-8 inhibits microtubule catastrophe at the cell cortex and results in perturbation of oscillation, indicating that nuclear movement also depends on microtubule dynamic instability. Our findings link numerical parameters from the simulation model with cellular functions required for generating the oscillation and provide a basis for understanding the physical properties of microtubule-dependent nuclear movements.

Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis.

Nuclear envelope (NE) expansion must be controlled to maintain nuclear shape and function. The nuclear membrane expands massively during closed mitosis, enabling chromosome segregation within an intact NE. Phosphatidic acid (PA) and diacylglycerol (DG) can both serve as biosynthetic precursors for membrane lipid synthesis. How they are regulated in time and space and what the implications are of changes in their flux for mitotic fidelity are largely unknown. Using genetically encoded PA and DG probes, we show that DG is depleted from the inner nuclear membrane during mitosis in the fission yeast Schizosaccharomyces pombe, but PA does not accumulate, indicating that it is rerouted to membrane synthesis. We demonstrate that DG-to-PA conversion catalyzed by the diacylglycerol kinase Dgk1 (also known as Ptp4) and direct glycerophospholipid synthesis from DG by diacylglycerol cholinephosphotransferase/ethanolaminephosphotransferase Ept1 reinforce NE expansion. We conclude that DG consumption through both the de novo pathway and the Kennedy pathway fuels a spike in glycerophospholipid biosynthesis, controlling NE expansion and, ultimately, mitotic fidelity.

A nuclear pore complex-associated regulation of SUMOylation in meiosis.

The nuclear pore complex (NPC) provides a permeable barrier between the nucleoplasm and cytoplasm. In a subset of NPC constituents that regulate meiosis in the fission yeast Schizosaccharomyces pombe, we found that nucleoporin Nup132 (homolog of human Nup133) deficiency resulted in transient leakage of nuclear proteins during meiosis I, as observed in the nup132 gene-deleted mutant. The nuclear protein leakage accompanied the liberation of the small ubiquitin-like modifier (SUMO)-specific ubiquitin-like protease 1 (Ulp1) from the NPC. Ulp1 retention at the nuclear pore prevented nuclear protein leakage and restored normal meiosis in a mutant lacking Nup132. Furthermore, using mass spectrometry analysis, we identified DNA topoisomerase 2 (Top2) and RCC1-related protein (Pim1) as the target proteins for SUMOylation. SUMOylation levels of Top2 and Pim1 were altered in meiotic cells lacking Nup132. HyperSUMOylated Top2 increased the binding affinity at the centromeres of nup132 gene-deleted meiotic cells. The Top2-12KR sumoylation mutant was less localized to the centromeric regions. Our results suggest that SUMOylation of chromatin-binding proteins is regulated by the NPC-bound SUMO-specific protease and is important for the progression of meiosis.

Critical role of Wat1/Pop3 in regulating the TORC1 signalling pathway in fission yeast S. pombe.

The target of rapamycin (TOR), a major pathway for the regulation of cell growth and proliferation is conserved from yeast to humans. Fission yeast contains two tor complexes, TORC1 is crucial for cell growth while TORC2 gets activated under stress conditions. Pop3/Wat1, a mammalian Lst8 ortholog is an important component of both TOR complexes and has been implicated in the oxidative stress response pathway. Here in this study, the genetic interaction analysis revealed a synthetic lethal interaction of wat1 with tor2-287 mutant cells. Co-immunoprecipitation analysis revealed Wat1 interacts with TORC1 components Tor2, Mip1, and Tco89 while wat1-17 mutant protein fails to interact with these proteins. In the absence of Wat1, the cells arrest at G1 phase with reduced cell size at non-permissive temperature reminiscent of tor2-287 mutant phenotype. Similarly, inactivation of Wat1 results in the failure of TORC1 mediated phosphorylation of Psk1 and Rps602, leading to dysregulation of amino acid permeases and delocalization of Gaf1, a DNA binding transcription factor. Overall, we have hypothesized that Wat1/Pop3 is required to execute the function of TORC1.