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International audience; The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

International audience; Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.

Heterochromatin protein‐1 (HP1) is a key component of heterochromatin. Reminiscent of the cohesin complex which mediates sister‐chromatid cohesion, most HP1 proteins in mammalian cells are displaced from chromosome arms during mitotic entry, whereas a pool remains at the heterochromatic centromere region. The function of HP1 at mitotic centromeres remains largely elusive. Here, we show that double knockout (DKO) of HP1α and HP1γ causes defective mitosis progression and weakened centromeric cohesion. While mutating the chromoshadow domain (CSD) prevents HP1α from protecting sister‐chromatid cohesion, centromeric targeting of HP1α CSD alone is sufficient to rescue the cohesion defects in HP1 DKO cells. Interestingly, HP1‐dependent cohesion protection requires Haspin, an antagonist of the cohesin‐releasing factor Wapl. Moreover, HP1α CSD directly binds the N‐terminal region of Haspin and facilitates its centromeric localization. The need for HP1 in cohesion protection can be bypassed by centromeric targeting of Haspin or inhibiting Wapl activity. Taken together, these results reveal a redundant role for HP1α and HP1γ in the protection of centromeric cohesion through promoting Haspin localization at mitotic centromeres in mammalian cells.

International audience; To maintain life across a fluctuating environment, cells alternate between phases of cell division and quiescence. During cell division, the spontaneous mutation rate is expressed as the probability of mutations per generation (Luria and Delbrü ck, 1943; Lea and Coulson, 1949), whereas during quiescence it will be expressed per unit of time. In this study, we report that during quiescence, the unicellular haploid fission yeast accumulates mutations as a linear function of time. The novel mutational landscape of quiescence is characterized by insertion/deletion (indels) accumulating as fast as single nucleotide variants (SNVs), and elevated amounts of deletions. When we extended the study to 3 months of quiescence, we confirmed the replication-independent mutational spectrum at the whole-genome level of a clonally aged population and uncovered phenotypic variations that subject the cells to natural selection. Thus, our results support the idea that genomes continuously evolve under two alternating phases that will impact on their size and composition.

International audience; Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1- and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.

International audience; Life is maintained through alternating phases of cell division and quiescence. The causes and consequences of spontaneous mutations have been extensively explored in proliferating cells, and the major sources include errors of DNA replication and DNA repair. The foremost consequences are genetic variations within a cell population that can lead to heritable diseases and drive evolution. While most of our knowledge on DNA damage response and repair has been gained through cells actively dividing, it remains essential to also understand how DNA damage is metabolized in cells which are not dividing. In this review, we summarize the current knowledge concerning the type of lesions that arise in non-dividing budding and fission yeast cells, as well as the pathways used to repair them. We discuss the contribution of these models to our current understanding of age-related pathologies.

International audience; Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage.

International audience; Defined as a chromatin structure that remains condensed throughout the cell cycle heterochromatin is generally transcriptionally silent and is characterized by a specific molecular signature. Constitutive heterochromatin at the pericentromere is a conserved feature throughout evolution, which impacts genome stability. Here, we will summarize recent advances in our understanding of the dynamics of mouse pericentric heterochromatin during the cell cycle and development. Comparison with heterochromatin maintenance in fission yeast will enable discussions of the common basic principles and various mechanisms exploited in the distinct organisms.

International audience; Quiescent cells play a predominant role in most organisms. Here we identify RNA interference (RNAi) as a major requirement for quiescence (G0 phase of the cell cycle) in Schizosaccharomyces pombe RNAi mutants lose viability at G0 entry and are unable to maintain long-term quiescence. We identified suppressors of G0 defects in cells lacking Dicer (dcr1Δ), which mapped to genes involved in chromosome segregation, RNA polymerase-associated factors, and heterochromatin formation. We propose a model in which RNAi promotes the release of RNA polymerase in cycling and quiescent cells: (i) RNA polymerase II release mediates heterochromatin formation at centromeres, allowing proper chromosome segregation during mitotic growth and G0 entry, and (ii) RNA polymerase I release prevents heterochromatin formation at ribosomal DNA during quiescence maintenance. Our model may account for the codependency of RNAi and histone H3 lysine 9 methylation throughout eukaryotic evolution.

International audience; Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways.

International audience; Cells normally grow to a certain size before they enter mitosis and divide. Entry into mitosis depends on the activity of Cdk1, which is inhibited by the Wee1 kinase and activated by the Cdc25 phosphatase. However, how cells sense their size for mitotic commitment remains unknown. Here we show that an intracellular gradient of the dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) Pom1, which emanates from the ends of rod-shaped Schizosaccharomyces pombe cells, serves to measure cell length and control mitotic entry. Pom1 provides positional information both for polarized growth and to inhibit cell division at cell ends. We discovered that Pom1 is also a dose-dependent G2-M inhibitor. Genetic analyses indicate that Pom1 negatively regulates Cdr1 and Cdr2, two previously described Wee1 inhibitors of the SAD kinase family. This inhibition may be direct, because in vivo and in vitro evidence suggest that Pom1 phosphorylates Cdr2. Whereas Cdr1 and Cdr2 localize to a medial cortical region, Pom1 forms concentration gradients from cell tips that overlap with Cdr1 and Cdr2 in short cells, but not in long cells. Disturbing these Pom1 gradients leads to Cdr2 phosphorylation and imposes a G2 delay. In short cells, Pom1 prevents precocious M-phase entry, suggesting that the higher medial Pom1 levels inhibit Cdr2 and promote a G2 delay. Thus, gradients of Pom1 from cell ends provide a measure of cell length to regulate M-phase entry.

International audience; BACKGROUND INFORMATION: In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. For faithful segregation of sister chromatids, each sister kinetochore must attach to microtubules that extend to opposite poles (chromosome bi-orientation). At the metaphase-anaphase transition, cohesion between sister chromatids is removed, and each sister chromatid is pulled to opposite poles of the cell by microtubule-dependent forces. RESULTS: We have studied the role of the minus-end-directed motor protein dynein by analysing kinetochore dynamics in fission yeast cells deleted for the dynein heavy chain (Dhc1) or the light chain (Dlc1). In these mutants, we found an increased frequency of cells showing defects in chromosome segregation, which leads to the appearance of lagging chromosomes and an increased rate of chromosome loss. By following simultaneously kinetochore dynamics and localization of the checkpoint protein Mad2, we provide evidence that dynein function is not necessary for spindle-assembly checkpoint inactivation. Instead, we have demonstrated that loss of dynein function alters chromosome segregation and activates the Mad2-dependent spindle-assembly checkpoint. CONCLUSIONS: These results show an unexpected role for dynein in the control of chromosome segregation in fission yeast, most probably operating during the process of bi-orientation during early mitosis.

International audience; LSD1 represses and activates transcription by demethylating histone H3K4me and H3K9me, respectively. Genetic ablation of the S. pombe homologs, splsd1 and splsd2, resulted in slow growth and lethality, respectively, underscoring their physiological importance. spLsd1 and spLsd2 form a stable protein complex, which exhibits demethylase activity toward methylated H3K9 in vitro. Both proteins were associated with the heterochromatin boundary regions and euchromatic gene promoters. Loss of spLsd1 resulted in increased H3K9 methylation accompanied by reduced euchromatic gene transcription and heterochromatin propagation. Removal of the H3K9 methylase Clr4 partially suppressed the slow growth phenotype of splsd1Delta. Conversely, catalytically inactivating point mutations in the splsd1 and splsd2 genes partially mimicked the growth and heterochromatin propagation phenotypes. Taken together, these findings suggest the importance of both enzymatic and nonenzymatic roles of spLsd1 in regulating heterochromatin propagation and euchromatic transcription and also suggest that misregulation of spLsd1/2 is likely to impact the epigenetic state of the cell.

International audience; Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G(1), and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.

We report here on the role of open reading frame (ORF) SPCC1183.04c of Schizosaccharomyces pombe in mitochondrial RNA metabolism. A mutant deleted for this ORF on chromosome III accumulates mitochondrial transcripts with the exception of the cob mRNA. A detailed Northern blot analysis showed that the effect results from a decrease in RNA degradation but not from RNA processing deficiencies. Overexpression of the SPCC1183.04c gene in a S. pombe wild-type strain is characterized by slow growth at 37 degrees C on non-fermentable carbon sources and a significant reduction of steady-state levels of mitochondrial transcripts. A NCBI BLASTP search with the amino acid sequence deduced from the S. pombe gene identified significant similarity to a number of proteins in fungi (e.g. Ascomycota, Basidiomycota) and in some non-fungal eukaryotes (e.g. ciliate, slime mold, red algae). By heterologous expression of SPCC1183.04c in a Saccharomyces cerevisiae pet127Delta strain, we demonstrate that the fission yeast protein and Pet127p from S. cerevisiae function similarly: The fission yeast gene complemented the respiratory defect associated with the pet127Delta allele and partially restored the RNA processing phenotype. Although it lacks any recognizable targeting signal, the S. pombe protein is imported into S. cerevisiae mitochondria in vivo. We conclude from our results that the fission yeast SPCC1183.04c gene is a member of a new protein family that functions to stimulate mitochondrial RNA degradation, a function that is conserved within the mitochondria of lower eukaryotes but seems to have been replaced by alternative pathways in metazoans and higher plants.

In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope., Author Summary In the genome of eukaryotic cells, domains of active and repressive chromatin alternate along the chromosome arms. Insulator elements are necessary to shield these different environments from each other. In the fission yeast Schizosaccharomyces pombe, the chromatin remodeler Fft3 is required to maintain the repressed subtelomeric chromatin. Here we show that Fft3 maintains nucleosome structure of insulator elements at the subtelomeric borders. We also observe that subtelomeres and insulator elements move away from the nuclear envelope in cells lacking Fft3. The nuclear periphery is known to harbor repressive chromatin in many eukaryotes and has been implied in insulator function. Our results suggest that chromatin remodeling through Fft3 is required to maintain proper chromatin structure and nuclear organization of insulator elements.

SUMMARY The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt α-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes.

International audience; In the fission yeast, Schizosaccharomyces pombe, uptake of the fluorescent styryl dye FM4-64 via the endocytic pathway to the vacuole was localised to the poles of growing, interphase cells and to the cell equator during cell division, regions of cell wall deposition that are rich in actin. When the pattern of growth or the plane of cytokinesis was altered, the relationship between the actin cytoskeleton and the site of endocytosis was maintained. Transfer of the label to the vacuolar membrane was dependent upon the Rab GTPase Ypt7 and, hence, vesicle fusion. Endocytic vesicles transiently colocalised with actin patches and endocytosis was inhibited in mutants that affected actin patch integrity and by the actin inhibitor latrunculin A. Concentrations of latrunculin that removed actin cables but left patches unaffected had no effect on endocytosis at the poles, but abolished endocytosis at the cell equator. Equatorial, but not polar, endocytosis was also inhibited in cells lacking the formin For3 (which have selectively destabilised actin cables), in mutants of the exocyst complex and in cells treated with brefeldin A. Differential effects on endocytosis at the cell poles and equator were also observed in the actin mutant cps8 and the Arp2/3 complex mutant arp2. The redirection of endocytosis from the cell poles to the cell equator in M phase coincided with the anaphase separation of sister chromatids and was abolished in the septation initiation network (SIN) mutants cdc7, sid1 and sid2, demonstrating that the spatial reorganisation of the endocytic pathway in the S. pombe cell cycle requires a functional SIN pathway. We conclude that endocytosis in fission yeast has two distinct components, both of which are actin-based, but which are mechanistically distinct, as well as being spatially and temporally separated in the S. pombe cell cycle.

International audience; RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.

International audience; In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.

International audience; UVA radiation, the most abundant solar UV radiation reaching Earth's surface, induces oxidative stress through formation of reactive oxygen species (ROS) that can damage different cell components. Because of the broad spectrum of the possible targets of ROS, the cellular response to this radiation is complex. While extensive studies have allowed dissecting the effects of UVB, UVC and gamma radiations on cell cycle progression, few studies have dealt with the effect of UVA so far. Here we use Schizosaccharomyces pombe as a model organism to study biological effects of UVA radiation in living organisms. Through analysis of cell cycle progression in different mutant backgrounds we demonstrate that UVA delays cell cycle progression in G(2) cells in a dose dependent manner. However, despite Chk1 phosphorylation and in contrast to treatments with others genotoxic agents, this cell cycle delay is only partially dependent on DNA integrity checkpoint pathway. We also demonstrate that UVA irradiation of S phase cells slows down DNA replication in a checkpoint independent manner, activates Chk1 to prevent entry into abnormal mitosis and induces formation of Rad22 (homologue to human Rad52) foci. This indicates that DNA structure integrity is challenged. Furthermore, the cell cycle delay observed in checkpoint mutants exposed to UVA is not abolished when stress response pathway is inactivated or when down regulation of protein synthesis is prevented. In conclusion, fission yeast is a useful model to dissect the fundamental molecular mechanisms involved in UVA response that may contribute to skin cancer and aging.

International audience; The morphogenesis of single cells depends on their ability to coordinate surface mechanics and polarity. During germination, spores of many species develop a polar tube that hatches out of a rigid outer spore wall (OSW) in a process termed outgrowth. However, how these awakening cells reorganize to stabilize this first growth axis remains unknown. Here, using quantitative experiments and modeling, we reveal the mechanisms underlying outgrowth in fission yeast. We find that, following an isotropic growth phase during which a single polarity cap wanders around the surface, outgrowth occurs when spores have doubled their volume, concomitantly with the stabilization of the cap and a singular rupture in the OSW. This rupture happens when OSW mechanical stress exceeds a threshold, releases the constraints of the OSW on growth, and stabilizes polarity. Thus, outgrowth exemplifies a self-organizing morphogenetic process in which reinforcements between growth and polarity coordinate mechanics and internal organization.

Summary The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome-wide modifications in replication program can modulate cellular physiology., Graphical Abstract, Highlights • Premeiotic S phase neither activates nor requires a specific origin usage program • Origin selection is a key regulator of the distribution of meiotic recombination • Organization of genome duplication has a functional impact on cellular physiology, Changes in the pattern of DNA replication are observed during development and in various pathologies, but it is unclear whether they contribute to these processes. Wu and Nurse show, by altering replication origin usage in fission yeast, that origin selection is critical for the organization of meiotic recombination.

International audience; Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.

Sap1 is a DNA-binding protein involved in controlling the mating type switch in fission yeast Schizosaccharomyces pombe. In the absence of any significant sequence similarity with any structurally known protein, a variety of biophysical techniques has been used to probe the solution low-resolution structure of the sap1 protein. First, sap1 is demonstrated to be an unusually elongated dimer in solution by measuring the translational diffusion coefficient with two independent techniques: dynamic light-scattering and ultracentrifugation. Second, sequence analysis revealed the existence of a long coiled-coil region, which is responsible for dimerization. The length of the predicted coiled-coil matches estimates drawn from the hydrodynamic experimental behaviour of the molecule. In addition, the same measurements done on a shorter construct with a coiled-coil region shortened by roughly one-half confirmed the localization of the long coiled-coil region. A crude T-shape model incorporating all these information was built. Third, small-angle X-ray scattering (SAXS) of the free molecule provided additional evidence for the model. In particular, the P(r) curve strikingly demonstrates the existence of long intramolecular distances. Using a novel 3D reconstruction algorithm, a low resolution 3D model of the protein has been independently constructed that matches the SAXS experimental data. It also fits the translation diffusion coefficients measurements and agrees with the first T-shaped model. This low-resolution model has clearly biologically relevant new functional implications, suggesting that sap1 is a bifunctional protein, with the two active sites being separated by as much as 120 A; a tetrapeptide repeated four times at the C terminus of the molecule is postulated to be of utmost functional importance.

International audience; Fission yeast centromeres are transcriptionally silent and form a heterochromatin-like structure essential for normal centromere function; this appears analogous to heterochromatin and position effect variegation in other eukaryotes. Conditional mutations in three genes designated cep (centromere enhancer of position effect) were found to enhance transcriptional silencing within centromeres. Cloning of the cep1(+) and cep2(+) genes by functional complementation revealed that they are identical to the previously described genes pad1(+) and mts2(+), respectively, which both encode subunits of the proteasome 19S cap. Like Mts2 and Mts4, epitope-tagged Cep1/Pad1 localizes to or near the nuclear envelope throughout the cell cycle. The cep mutants display a range of phenotypes depending on the temperature. Silencing within the central domain of centromeres is increased at 36 degrees C. This suggests that the proteasome is involved in regulating silencing and thus centromeric chromatin architecture, possibly by lowering the level of some chromatin-associated protein by ubiquitin-dependent degradation. This is the first report of defective proteasome function affecting heterochromatin-mediated transcriptional silencing. At 36 and 32 degrees C, the cep mutants lose chromosomes at an elevated rate, and at 18 degrees C, the mutants are cryosensitive for growth. Cytological analysis at 18 degrees C revealed a defect in sister chromatid separation while other mitotic events occurred normally, indicating that cep mutations might interfere specifically with the degradation of inhibitor(s) of sister chromatid separation. These observations suggest that 19S subunits confer a level of substrate specificity on the proteasome and raise the possibility of a link between components involved in centromere architecture and sister chromatid cohesion.

International audience; We report the cloning and characterization of a new S. pombe gene, efc25+, for 'exchange factor Cdc25-like'. The C-terminal region of the predicted product of this gene displays high sequence homology with a number of guanine nucleotide exchange factors for Ras. These include Cdc25 of Saccharomyces cerevisiae, Cdc25 of Saccharomyces kluyveri, Csc25 of Candida albicans, Sdc25 of S. cerevisiae and Ste6 of Schizosaccharomyces pombe. Disruption of efc25+ resulted in cells with a spherical shape reminiscent of the abnormal morphological phenotype of ras1 deletion mutants. However, unlike ras1 null mutants, strains deleted for efc25+ were proficient for mating and sporulation. This differs from the only other Ras1 exchange factor characterized so far in S. pombe, the Ste6 protein, whose deletion results in defects in mating and sporulation but not in cell shape. We hypothesize that Efc25 is an exchange factor for Ras1 and that it is involved in a signaling pathway different from that involving Ste6.

International audience; Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56(chk1) is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase delta strain at 37 degrees C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34(cdc2). In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of impaired DNA replication at 37 degrees C.

International audience; The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.

International audience; An interference assay has been devised in Schizosaccharomyces pombe to rapidly identify and clone genes involved in chromosome segregation. Random S.pombe cDNAs were overexpressed from an inducible promoter in a strain carrying an additional, non-essential minichromosome. Overexpression of cDNAs derived from four genes, two known (nda3+and ubc4+, encoding beta-tubulin and a ubiquitin conjugating enzyme, respectively) and two unknown, named mlo2+ and mlo3+ (missegregation & lethal when over expressed) caused phenotypes consistent with a failure to segregate chromosomes. Full overexpression of all four cDNAs was lethal. Cells overexpressing nda3+ and ubc4+ cDNAs arrested with condensed unsegregated chromosomes and cells overexpressing mlo2+ displayed an asymmetric distribution of nuclear chromatin. Sublethal levels of overexpression of nda3+, ubc4+ and mlo2+ cDNAs caused elevated rates of minichromosome loss. A third cDNA mlo3+, displayed no increase in the frequency of minichromosome loss at sublethal levels of overexpression but full overexpression caused a complete failure to segregate chromosomes. Our results confirm the assumption that beta-tubulin overexpression is lethal in S.pombe, implicate ubc4+ in the control of metaphase-anaphase transition in fission yeast and finally identify two new genes, mlo2+and mlo3+, likely to play an important role for chromosome transmission fidelity in mitosis.

International audience; The ura4+ gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4+ expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ura4+ gene adjacent to telomeres and at the three centromeric sites tested. The clr4-S5 and rik1-304 mutations cause the most dramatic derepression at two out of three sites within cen1. All six mutations had only slight or intermediate effects on a third site in the center of cen1 or on telomeric repression. Strains with lesions at the clr4, rik1, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.

International audience; In the fission yeast Schizosaccharomyces pombe, a chromosomal imprinting event controls the asym-metric pattern of mating-type switching. The orientation of DNA replication at the mating-type locus is instrumental in this process. However, the factors leading to imprinting are not fully identified and the mechanism is poorly understood. Here, we show that the replication fork pause at the mat1 locus (MPS1), essential for imprint formation, depends on the lysine-specific demethylase Lsd1. We demonstrate that either Lsd1 or Lsd2 amine oxidase activity is required for these processes, working upstream of the imprinting factors Swi1 and Swi3 (homologs of mammalian Timeless and Tipin, respectively). We also show that the Lsd1/2 complex controls the repli-cation fork terminators, within the rDNA repeats. These findings reveal a role for the Lsd1/2 demethy-lases in controlling polar replication fork progression, imprint formation, and subsequent asymmetric cell divisions.

International audience; Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.

International audience; The SAGA complex is a conserved, multifunctional co-activator that controls the transcription of many inducible genes in response to environmental changes. Recent studies have provided new insights into the functions of one of its subunits, Tra1/TRRAP, and suggest that it controls SAGA activity in response to external stimuli.

International audience; Pds5 and Wpl1 act as anti-establishment factors preventing sister-chromatid cohesion until counteracted in S-phase by the cohesin acetyl-transferase Eso1. However, Pds5 is also required to maintain sister-chromatid cohesion in G2. Here, we show that Pds5 is essential for cohesin acetylation by Eso1 and ensures the maintenance of cohesion by promoting a stable cohesin interaction with replicated chromosomes. The latter requires Eso1 only in the presence of Wapl, indicating that cohesin stabilization relies on Eso1 only to neutralize the anti-establishment activity. We suggest that Eso1 requires Pds5 to counteract anti-establishment. This allows both cohesion establishment and Pds5-dependent stable cohesin binding to chromosomes.

http://linkinghub.elsevier.com/; International audience; Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.

International audience; In budding yeast and humans, cohesion establishment during S phase requires the acetyltransferase Eco1/Esco1-2, which acetylates the cohesin subunit Smc3 on two conserved lysine residues. Whether Smc3 is the sole Eco1/Esco1-2 effector and how Smc3 acetylation promotes cohesion are unknown. In fission yeast (Schizosaccharomyces pombe), as in humans, cohesin binding to G(1) chromosomes is dynamic and the unloading reaction is stimulated by Wpl1 (human ortholog, Wapl). During S phase, a subpopulation of cohesin becomes stably bound to chromatin in an Eso1 (fission yeast Eco1/Esco1-2)-dependent manner. Cohesin stabilization occurs unevenly along chromosomes. Cohesin remains largely labile at the rDNA repeats but binds mostly in the stable mode to pericentromere regions. This pattern is largely unchanged in eso1Δ wpl1Δ cells, and cohesion is unaffected, indicating that the main Eso1 role is counteracting Wpl1. A mutant of Psm3 (fission yeast Smc3) that mimics its acetylated state renders cohesin less sensitive to Wpl1-dependent unloading and partially bypasses the Eso1 requirement but cannot generate the stable mode of cohesin binding in the absence of Eso1. Conversely, nonacetylatable Psm3 reduces the stable cohesin fraction and affects cohesion in a Wpl1-dependent manner, but cells are viable. We propose that Psm3 acetylation contributes to Eso1 counteracting of Wpl1 to secure stable cohesin interaction with postreplicative chromosomes but that it is not the sole molecular event by which this occurs.

International audience; Increasing evidence implicates cohesin in the control of gene expression. Here we report the first analysis of cohesin-dependent gene regulation in fission yeast. Global expression profiling of the mis4-367 cohesin loader mutant identified a small number of upregulated and downregulated genes within subtelomeric domains (SD). These 20- to 40-kb regions between chromosome arm euchromatin and telomere-proximal heterochromatin are characterized by a combination of euchromatin (methylated lysine 4 on histone H3/methylated Tysine 9 on histone H3 [H3K4me]) and heterochromatin (H3K9me) marks. We focused our analysis on the chromosome 1 right SD, which contains several upregulated genes and is bordered on the telomere-distal side by a pair of downregulated genes. We find that the expression changes in the SD also occur in a mutant of the cohesin core component Rad21. Remarkably, mutation of Rad21 results in the depletion of Swi6 binding in the SD. In fact, the Rad21 mutation phenocopied Swi6 loss of function: both mutations led to reduced cohesin binding, reduced H3K9me, and similar gene expression changes in the SD. In particular, expression of the gene pair bordering the SD was dependent both on cohesin and on Swi6. Our data indicate that cohesin participates in the setup of a subtelomeric heterochromatin domain and controls the expression of the genes residing in that domain.

The Pol I subunits Rpa34 and Rpa49, required for elongation and 35S rRNA production, promote clustering of neighboring Pol I complexes to enhance the rRNA gene transcription cycle., RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification. © 2011 Macmillan Publishers Limited. All rights reserved., This work was supported by grants BFU2008-01919 and Consolider-Ingenio CSD2007-00015 from the Spanish Ministry of Science and Innovation to F.A., and NIH R01 GM076396 to W.Z.C. and R.A.M.

International audience; Centromere-binding protein B (CENP-B) is a widely conserved DNA binding factor associated with heterochromatin and centromeric satellite repeats. In fission yeast, CENP-B homologues have been shown to silence long terminal repeat (LTR) retrotransposons by recruiting histone deacetylases. However, CENP-B factors also have unexplained roles in DNA replication. Here we show that a molecular function of CENP-B is to promote replication-fork progression through the LTR. Mutants have increased genomic instability caused by replication-fork blockage that depends on the DNA binding factor switch-activating protein 1 (Sap1), which is directly recruited by the LTR. The loss of Sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. We conclude that retrotransposons influence replication polarity through recruitment of Sap1 and transposition near replication-fork blocks, whereas CENP-B counteracts this activity and promotes fork stability. Our results may account for the role of LTR in fragile sites, and for the association of CENP-B with pericentromeric heterochromatin and tandem satellite repeats.

International audience; Multiple genome maintenance processes are coordinated at the replication fork to preserve genomic integrity. How eukaryotic cells accomplish such a coordination is unknown. Swi1 and Swi3 form the replication fork protection complex and are involved in various processes including stabilization of replication forks, activation of the Cds1 checkpoint kinase and establishment of sister chromatid cohesion in fission yeast. However, the mechanisms by which the Swi1-Swi3 complex achieves and coordinates these tasks are not well understood. Here, we describe the identification of separation-of-function mutants of Swi3, aimed at dissecting the molecular pathways that require Swi1-Swi3. Unlike swi3 deletion mutants, the separation-of-function mutants were not sensitive to agents that stall replication forks. However, they were highly sensitive to camptothecin that induces replication fork breakage. In addition, these mutants were defective in replication fork regeneration and sister chromatid cohesion. Interestingly, unlike swi3-deleted cell, the separation-of-functions mutants were proficient in the activation of the replication checkpoint, but their fork regeneration defects were more severe than those of checkpoint mutants including cds1Δ, chk1Δ and rad3Δ. These results suggest that, while Swi3 mediates full activation of the replication checkpoint in response to stalled replication forks, Swi3 activates a checkpoint-independent pathway to facilitate recovery of collapsed replication forks and the establishment of sister chromatid cohesion. Thus, our separation-of-function alleles provide new insight into understanding the multiple roles of Swi1-Swi3 in fork protection during DNA replication, and into understanding how replication forks are maintained in response to different genotoxic agents.

International audience; Cell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1-GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1Δ null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analogue-sensitive allele kin1-as1. Kin1 inhibition primarily promotes delocalization of plasma membrane-associated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen-activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen-activated protein kinase components reduces kin1Δ hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall.

Accurate chromosome segregation during eukaryotic cell division requires the timely release of cohesion between duplicated chromosomes so that they may separate into two daughter cells. Errors in this process have been linked to cancer progression, infertility, and debilitating genetic diseases such as Down syndrome. A surveillance mechanism known as the spindle assembly checkpoint delays this release until all chromosome pairs are properly attached to a structure called the mitotic spindle. The enzyme Bub1 is essential for this checkpoint, but the targets of its phosphorylation activity have been elusive. On page 172 of this issue, Kawashima et al. ( 1 ) report that histone H2A—a protein associated with DNA within chromosomes—is a substrate of Bub1, and that H2A phosphorylation directs subsequent events that control chromatid cohesion and the checkpoint mechanism during the early stages of cell division.

13 páginas, 7 figuras, 2 tablas., [Background]: The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of b-glucans. The b(1,3)-glucan synthase complex synthesizes linear b(1,3)- glucans, which remain unorganized until they are cross-linked to other b(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding b(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed. [Methodology/Principal Findings]: Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1D mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display b(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. [Conclusions/Significance]: We conclude that b(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth., This research was supported by grants from the Comision Interministerial de Ciencia y Tecnologia (BFU2004-00778) and Junta de Castilla y Leon (GR231) to C.R.V-A and from the European Community (LSHB-CT-2004-511952) to C.R.V-A. and J.P.L. M.M-R. held a fellowship from the Ministerio de Educacion y Ciencia.

Cytokinesis is the last step of the cell cycle, producing two daughter cells inheriting equal genetic information. This process involves the assembly of an actomyosin ring during mitosis. In the fission yeast Schizosaccharomyces pombe, cytokinesis occurs at the geometric cell centre, a position which is defined by the interphase nucleus and the anilin-related Mid1 protein. The pom1Delta, tea1Delta and tea4Delta mutants are defective in restricting Mid1 as a band around the nucleus and misplace the division site. We previously reported that inhibition of the protein kinase Kin1 promoted failure of cytokinesis in pom1Delta and tea1Delta cells but the mechanism involving Kin1 remained elusive. Here we investigated the contribution of Kin1 in cytokinesis. We show that Kin1-GFP has a dynamic cell cycle regulated distribution. Like pom1Delta and tea1Delta, tea4Delta exhibits a strong genetic interaction with kin1Delta. Using a conditional repressible kin1 allele that only alters interphase nuclear centering, we observed that Kin1 downregulation severely compromised actomyosin ring formation and septum synthesis in tea4Delta cells. In addition, nuclear displacement induced either by overexpression of a putative catalytically inactive Kin1 mutant, by chemically mediated microtubule depolymerization or by mutation in the par1Delta gene impaired cytokinesis in tea4Delta but not tea4(+) cells. We propose that nuclear mispositioning exacerbates the tea4Delta, pom1Delta and tea1Delta cell division phenotype. Our work reveal that nuclear centering becomes essential when Pom1/Tea1/Tea4 function is compromised and that Kin1 expression level is a key regulatory element in this situation. Our results suggest the existence of distinct overlapping control mechanisms to ensure efficient cell division.

International audience; Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34Delta weakened the binding of Rpa49 to RNA polymerase. rpa34Delta mutants were caffeine sensitive, and the rpa34Delta mutation was lethal in a top1Delta mutant and in rpa14Delta, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49Delta mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34Delta mutants, but strains carrying rpa49Delta and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30 degrees C, failed to grow at 25 degrees C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49Delta and rpa49-338::HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.

International audience; Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.

International audience; Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.

Background Microsporidia, parasitic fungi-related eukaryotes infecting many cell types in a wide range of animals (including humans), represent a serious health threat in immunocompromised patients. The 2.9 Mb genome of the microsporidium Encephalitozoon cuniculi is the smallest known of any eukaryote. Eukaryotic protein kinases are a large superfamily of enzymes with crucial roles in most cellular processes, and therefore represent potential drug targets. We report here an exhaustive analysis of the E. cuniculi genomic database aimed at identifying and classifying all protein kinases of this organism with reference to the kinomes of two highly-divergent yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. Results A database search with a multi-level protein kinase family hidden Markov model library led to the identification of 29 conventional protein kinase sequences in the E. cuniculi genome, as well as 3 genes encoding atypical protein kinases. The microsporidian kinome presents striking differences from those of other eukaryotes, and this minimal kinome underscores the importance of conserved protein kinases involved in essential cellular processes. ~30% of its kinases are predicted to regulate cell cycle progression while another ~28% have no identifiable homologues in model eukaryotes and are likely to reflect parasitic adaptations. E. cuniculi lacks MAP kinase cascades and almost all protein kinases that are involved in stress responses, ion homeostasis and nutrient signalling in the model fungi S. cerevisiae and S. pombe, including AMPactivated protein kinase (Snf1), previously thought to be ubiquitous in eukaryotes. A detailed database search and phylogenetic analysis of the kinomes of the two model fungi showed that the degree of homology between their kinomes of ~85% is much higher than that previously reported. Conclusion The E. cuniculi kinome is by far the smallest eukaryotic kinome characterised to date. The difficulty in assigning clear homology relationships for nine out of the twenty-nine microsporidian conventional protein kinases despite its compact genome reflects the phylogenetic distance between microsporidia and other eukaryotes. Indeed, the E. cuniculi genome presents a high proportion of genes in which evolution has been accelerated by up to four-fold. There are no orthologues of the protein kinases that constitute MAP kinase pathways and many other protein kinases with roles in nutrient signalling are absent from the E. cuniculi kinome. However, orthologous kinases can nonetheless be identified that correspond to members of the yeast kinomes with roles in some of the most fundamental cellular processes. For example, E. cuniculi has clear orthologues of virtually all the major conserved protein kinases that regulate the core cell cycle machinery (Aurora, Polo, DDK, CDK and Chk1). A comprehensive comparison of the homology relationships between the budding and fission yeast kinomes indicates that, despite an estimated 800 million years of independent evolution, the two model fungi share ~85% of their protein kinases. This will facilitate the annotation of many of the as yet uncharacterised fission yeast kinases, and also those of novel fungal genomes.