ABSTRACT
Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.
Subject(s)
Cell Membrane/chemistry , Schizosaccharomyces/chemistry , Schizosaccharomyces/cytology , Actins/metabolism , Cell Polarity , Cytoskeleton/metabolism , Membrane Microdomains , Microtubule-Associated Proteins/metabolism , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , cdc42 GTP-Binding Protein/metabolismABSTRACT
Cell growth is orchestrated by a number of interlinking cellular processes. Components of the TOR pathway have been proposed as potential regulators of cell growth, but little is known about their immediate effects on protein synthesis in response to TOR-dependent growth inhibition. Here, we present a resource providing an in-depth characterisation of Schizosaccharomyces pombe phosphoproteome in relation to changes observed in global cellular protein synthesis upon TOR inhibition. We find that after TOR inhibition, the rate of protein synthesis is rapidly reduced and that notable phosphorylation changes are observed in proteins involved in a range of cellular processes. We show that this reduction in protein synthesis rates upon TOR inhibition is not dependent on S6K activity, but is partially dependent on the S. pombe homologue of eIF4G, Tif471. Our study demonstrates the impact of TOR-dependent phospho-regulation on the rate of protein synthesis and establishes a foundational resource for further investigation of additional TOR-regulated targets both in fission yeast and other eukaryotes.
Subject(s)
Phosphoproteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , TOR Serine-Threonine Kinases/metabolism , Phosphorylation , Protein Biosynthesis , Proteome , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & developmentABSTRACT
Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis. Alternatively, dysfunctional forks are restarted by homologous recombination. Using fission yeast, we report that Rad52 and the DNA binding activity of Rad51, but not its strand-exchange activity, act to protect terminally arrested forks from unrestrained Exo1-nucleolytic activity. In the absence of recombination proteins, large ssDNA gaps, up to 3 kb long, occur behind terminally arrested forks, preventing efficient fork merging and leading to mitotic sister chromatid bridging. Thus, Rad52 and Rad51 prevent temporarily and terminally arrested forks from degrading and, despite the availability of converging forks, converting to anaphase bridges causing aneuploidy and cell death.
Subject(s)
DNA Replication , DNA, Fungal/biosynthesis , DNA, Single-Stranded/biosynthesis , Mitosis/physiology , Replication Origin , Schizosaccharomyces/metabolism , Sister Chromatid Exchange , Aneuploidy , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , DNA Breaks, Single-Stranded , DNA, Fungal/genetics , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Microbial Viability , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Time FactorsABSTRACT
Heterochromatin can be epigenetically inherited in cis, leading to stable gene silencing. However, the mechanisms underlying heterochromatin inheritance remain unclear. Here, we identify Fft3, a fission yeast homolog of the mammalian SMARCAD1 SNF2 chromatin remodeler, as a factor uniquely required for heterochromatin inheritance, rather than for de novo assembly. Importantly, we find that Fft3 suppresses turnover of histones at heterochromatic loci to facilitate epigenetic transmission of heterochromatin in cycling cells. Moreover, Fft3 also precludes nucleosome turnover at several euchromatic loci to prevent R-loop formation, ensuring proper replication progression. Our analyses show that overexpression of Clr4/Suv39h, which is also required for efficient replication through these loci, suppresses phenotypes associated with the loss of Fft3. This work uncovers a conserved factor critical for epigenetic inheritance of heterochromatin and describes a mechanism in which suppression of nucleosome turnover prevents formation of structural barriers that impede replication at fragile regions in the genome.
Subject(s)
Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , DNA, Fungal/biosynthesis , Epigenesis, Genetic , Heredity , Heterochromatin/metabolism , Nucleosomes/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , DNA, Fungal/genetics , Genotype , Heterochromatin/genetics , Histone-Lysine N-Methyltransferase , Histones/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Mutation , Nucleosomes/genetics , Phenotype , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/genetics , Time FactorsABSTRACT
Multiple lines of evidence implicate chromatin in the regulation of premessenger RNA (pre-mRNA) splicing. However, the influence of chromatin factors on cotranscriptional splice site usage remains unclear. Here we investigated the function of the highly conserved histone variant H2A.Z in pre-mRNA splicing using the intron-rich model yeast Schizosaccharomyces pombe Using epistatic miniarray profiles (EMAPs) to survey the genetic interaction landscape of the Swr1 nucleosome remodeling complex, which deposits H2A.Z, we uncovered evidence for functional interactions with components of the spliceosome. In support of these genetic connections, splicing-specific microarrays show that H2A.Z and the Swr1 ATPase are required during temperature stress for the efficient splicing of a subset of introns. Notably, affected introns are enriched for H2A.Z occupancy and more likely to contain nonconsensus splice sites. To test the significance of the latter correlation, we mutated the splice sites in an affected intron to consensus and found that this suppressed the requirement for H2A.Z in splicing of that intron. These data suggest that H2A.Z occupancy promotes cotranscriptional splicing of suboptimal introns that may otherwise be discarded via proofreading ATPases. Consistent with this model, we show that overexpression of splicing ATPase Prp16 suppresses both the growth and splicing defects seen in the absence of H2A.Z.
Subject(s)
Histones/genetics , Introns , RNA Splicing , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Adenosine Triphosphatases/metabolism , Gene Expression Regulation, Fungal , Nucleosomes/genetics , Promoter Regions, Genetic , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Spliceosomes/geneticsABSTRACT
Erh1, the fission yeast homolog of Enhancer of rudimentary, is implicated in meiotic mRNA elimination during vegetative growth, but its function is poorly understood. We show that Erh1 and the RNA-binding protein Mmi1 form a stoichiometric complex, called the Erh1-Mmi1 complex (EMC), to promote meiotic mRNA decay and facultative heterochromatin assembly. To perform these functions, EMC associates with two distinct complexes, Mtl1-Red1 core (MTREC) and CCR4-NOT. Whereas MTREC facilitates assembly of heterochromatin islands coating meiotic genes silenced by the nuclear exosome, CCR4-NOT promotes RNAi-dependent heterochromatin domain (HOOD) formation at EMC-target loci. CCR4-NOT also assembles HOODs at retrotransposons and regulated genes containing cryptic introns. We find that CCR4-NOT facilitates HOOD assembly through its association with the conserved Pir2/ARS2 protein, and also maintains rDNA integrity and silencing by promoting heterochromatin formation. Our results reveal connections among Erh1, CCR4-NOT, Pir2/ARS2, and RNAi, which target heterochromatin to regulate gene expression and protect genome integrity.
Subject(s)
Carrier Proteins/metabolism , Chromatin Assembly and Disassembly , Heterochromatin/metabolism , Meiosis , RNA Interference , RNA Stability , RNA, Fungal/metabolism , RNA, Messenger/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , mRNA Cleavage and Polyadenylation Factors/metabolism , Carrier Proteins/genetics , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Gene Expression Regulation, Fungal , Heterochromatin/genetics , Mutation , Nucleic Acid Conformation , Protein Binding , RNA, Fungal/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Retroelements , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , mRNA Cleavage and Polyadenylation Factors/geneticsABSTRACT
The fission yeast Schizosaccharomyces pombe is an excellent model organism to explore cellular events owing to rich tools in genetics, molecular biology, cellular biology, and biochemistry. Schizosaccharomyces pombe proliferates continuously when nutrients are abundant but arrests in G1 phase upon depletion of nutrients such as nitrogen and glucose. When cells of opposite mating types are present, cells conjugate, fuse, undergo meiosis, and finally form 4 spores. This sexual differentiation process in S. pombe has been studied extensively. To execute sexual differentiation, the glucose-sensing cAMP-PKA (cyclic adenosine monophosphate-protein kinase A) pathway, nitrogen-sensing TOR (target of rapamycin) pathway, and SAPK (stress-activating protein kinase) pathway are crucial, and the MAPK (mitogen-activating protein kinase) cascade is essential for pheromone sensing. These signals regulate ste11 at the transcriptional and translational levels, and Ste11 is modified in multiple ways. This review summarizes the initiation of sexual differentiation in S. pombe based on results I have helped to obtain, including the work of many excellent researchers.
Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Transcription Factors , Schizosaccharomyces/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Gene Expression Regulation, Fungal , Signal Transduction , Meiosis , Pheromones/metabolism , Sex Differentiation/genetics , Glucose/metabolism , Nitrogen/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Spores, Fungal/growth & development , Spores, Fungal/genetics , Spores, Fungal/physiologyABSTRACT
Alcohol fermentation comprises two phases: phase 1, alcohol fermentation occurs while yeast cells proliferate; phase 2, growth stops and alcohol fermentation continues. We categorized genes related to proliferation in low ethanol (phase 1) and viability in high ethanol (phase 2) as Alcohol Growth Ability (AGA) and Alcohol Viability (ALV), respectively. Although genes required for phase 1 are examined in budding yeast, those for phase 2 are unknown. We set conditions for ALV screening, searched for protein kinases (PKs) related to ALV in budding yeast, and expanded two screenings to fission yeast. Bub1 kinase was important for proliferation in low ethanol but not for viability in high ethanol, suggesting that the important PKs differ between the two phases. It was indeed the case. Further, 3 common PKs were identified as AGA in both yeasts, suggesting that the important cellular mechanism in phase 1 is conserved in both yeasts, at least partially.
Subject(s)
Ethanol , Schizosaccharomyces , Ethanol/metabolism , Ethanol/pharmacology , Schizosaccharomyces/genetics , Schizosaccharomyces/drug effects , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolism , Fermentation , Protein Kinases/metabolism , Protein Kinases/genetics , Cell Proliferation/drug effects , Saccharomycetales/genetics , Saccharomycetales/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/growth & developmentABSTRACT
Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.
Subject(s)
Fungal Proteins/metabolism , Intramolecular Transferases/metabolism , Schizosaccharomyces/physiology , Triterpenes/metabolism , Adaptation, Biological , Anaerobiosis , Bacterial Proteins/chemistry , Culture Media/chemistry , Culture Media/pharmacology , Ergosterol/pharmacology , Eukaryotic Cells/physiology , Fatty Acids, Unsaturated/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Intramolecular Transferases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/drug effects , Schizosaccharomyces/growth & development , Sterols/metabolismABSTRACT
R-loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of two fission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3-transcribed genes and produces long exosome-sensitive 3'-extended transcripts. Importantly, neither of these defects was affected by the removal of R-loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.
Subject(s)
DNA Helicases/metabolism , RNA Helicases/metabolism , RNA Polymerase III/genetics , Schizosaccharomyces/growth & development , Gene Expression Regulation, Fungal , RNA, Transfer/chemistry , RNA, Transfer/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Transcription Termination, GeneticABSTRACT
Unlike many single-celled organisms, the growth of fission yeast cells within a cell cycle is not exponential. It is rather characterized by three distinct phases (elongation, septation, and reshaping), each with a different growth rate. Experiments also showed that the distribution of cell size in a lineage can be bimodal, unlike the unimodal distributions measured for the bacterium Escherichia coli. Here we construct a detailed stochastic model of cell size dynamics in fission yeast. The theory leads to analytic expressions for the cell size and the birth size distributions, and explains the origin of bimodality seen in experiments. In particular, our theory shows that the left peak in the bimodal distribution is associated with cells in the elongation phase, while the right peak is due to cells in the septation and reshaping phases. We show that the size control strategy, the variability in the added size during a cell cycle, and the fraction of time spent in each of the three cell growth phases have a strong bearing on the shape of the cell size distribution. Furthermore, we infer all the parameters of our model by matching the theoretical cell size and birth size distributions to those from experimental single-cell time-course data for seven different growth conditions. Our method provides a much more accurate means of determining the size control strategy (timer, adder or sizer) than the standard method based on the slope of the best linear fit between the birth and division sizes. We also show that the variability in added size and the strength of size control in fission yeast depend weakly on the temperature but strongly on the culture medium. More importantly, we find that stronger size homeostasis and larger added size variability are required for fission yeast to adapt to unfavorable environmental conditions.
Subject(s)
Cell Cycle/physiology , Cell Size , Models, Biological , Schizosaccharomyces/cytology , Schizosaccharomyces/growth & development , Computational BiologyABSTRACT
Walled cells of plants, fungi, and bacteria come with a large range of shapes and sizes, which are ultimately dictated by the mechanics of their cell wall. This stiff and thin polymeric layer encases the plasma membrane and protects the cells mechanically by opposing large turgor pressure derived mechanical stresses. To date, however, we still lack a quantitative understanding for how local and/or global mechanical properties of the wall support cell morphogenesis. Here, we combine subresolution imaging and laser-mediated wall relaxation to quantitate subcellular values of wall thickness (h) and bulk elastic moduli (Y) in large populations of live mutant cells and in conditions affecting cell diameter in the rod-shaped model fission yeast. We find that lateral wall stiffness, defined by the surface modulus, σ = hY, robustly scales with cell diameter. This scaling is valid across tens of mutants spanning various functions-within the population of individual isogenic strains, along single misshaped cells, and even across the fission yeasts clade. Dynamic modulations of cell diameter by chemical and/or mechanical means suggest that the cell wall can rapidly adapt its surface mechanics, rendering stretched wall portions stiffer than unstretched ones. Size-dependent wall stiffening constrains diameter definition and limits size variations; it may also provide an efficient means to keep elastic strains in the wall below failure strains, potentially promoting cell survival. This quantitative set of data impacts our current understanding of the mechanics of cell walls and its contribution to morphogenesis.
Subject(s)
Cell Wall/chemistry , Morphogenesis , Schizosaccharomyces/chemistry , Stress, Mechanical , Actins/chemistry , Biomechanical Phenomena , Cell Membrane/chemistry , Elastic Modulus , Pressure , Schizosaccharomyces/growth & development , Surface PropertiesABSTRACT
The chromosomal position of each centromere is determined epigenetically and is highly stable, whereas incremental cases have supported the occurrence of centromere repositioning on an evolutionary time scale (evolutionary new centromeres, ENCs), which is thought to be important in speciation. The mechanisms underlying the high stability of centromeres and its functional significance largely remain an enigma. Here, in the fission yeast Schizosaccharomyces pombe, we identify a feedback mechanism: The kinetochore, whose assembly is guided by the centromere, in turn, enforces centromere stability. Upon going through meiosis, specific inner kinetochore mutations induce centromere repositioning-inactivation of the original centromere and formation of a new centromere elsewhere-in 1 of the 3 chromosomes at random. Repositioned centromeres reside asymmetrically in the pericentromeric regions and cells carrying them are competent in mitosis and homozygotic meiosis. However, when cells carrying a repositioned centromere are crossed with those carrying the original centromere, the progeny suffer severe lethality due to defects in meiotic chromosome segregation. Thus, repositioned centromeres constitute a reproductive barrier that could initiate genetic divergence between 2 populations with mismatched centromeres, documenting a functional role of ENCs in speciation. Surprisingly, homozygotic repositioned centromeres tend to undergo meiosis in an inverted order-that is, sister chromatids segregate first, and homologous chromosomes separate second-whereas the original centromeres on other chromosomes in the same cell undergo meiosis in the canonical order, revealing hidden flexibility in the perceived rigid process of meiosis.
Subject(s)
Centromere/genetics , Chromosome Segregation/genetics , Meiosis/physiology , Mitosis/physiology , Schizosaccharomyces/genetics , Kinetochores/metabolism , Meiosis/genetics , Mitosis/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
The septation initiation network (SIN), composed of a conserved SepH (Cdc7p) kinase cascade, plays an essential role in fungal cytokinesis/septation and conidiation for asexual reproduction, while the mitogen-activated protein kinase (MAPK) pathway depends on successive signaling cascade phosphorylation to sense and respond to stress and environmental factors. In this study, a SepH suppressor-PomA in the filamentous fungus A. nidulans is identified as a negative regulator of septation and conidiation such that the pomA mutant is able to cure defects of sepH1 in septation and conidiation and overexpression of pomA remarkably suppresses septation. Under the normal cultural condition, SepH positively regulates the phosphorylation of MAPK-HogA, while PomA reversely affects this process. In the absence of PbsB (MAPKK, a putative upstream member of HogA), PomA and SepH are unable to affect the phosphorylation level of HogA. Under the osmostress condition, the induced phosphorylated HogA is capable of bypassing the requirement of SepH, a key player for early events during cytokinesis but not for MobA/SidB, the last one in the core SIN protein kinase cascade, indicating the osmotic stimuli-induced septation is capable of bypassing requirement of SepH but unable to bypass the whole SIN requirement. Findings demonstrate that crosstalk exists between the SIN and MAPK pathways. PomA and SepH indirectly regulate HogA phosphorylation through affecting HogA-P upstream kinases.
Subject(s)
Aspergillus nidulans/genetics , Fungal Proteins/genetics , Mitogen-Activated Protein Kinases/genetics , Reproduction, Asexual/genetics , Aspergillus nidulans/growth & development , Cell Cycle Proteins/genetics , Cytokinesis/genetics , Mutation/genetics , Nuclear Proteins/genetics , Osmotic Pressure , Phosphorylation/genetics , Polymorphism, Single Nucleotide/genetics , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/genetics , Signal Transduction/geneticsABSTRACT
As part of a complex network of genome control, long regulatory RNAs exert significant influences on chromatin dynamics. Understanding how this occurs could illuminate new avenues for disease treatment and lead to new hypotheses that would advance gene regulatory research. Recent studies using the model fission yeast Schizosaccharomyces pombe (S. pombe) and powerful parallel sequencing technologies have provided many insights in this area. This review will give an overview of key findings in S. pombe that relate long RNAs to multiple levels of chromatin regulation: histone modifications, gene neighborhood regulation in cis and higher-order chromosomal ordering. Moreover, we discuss parallels recently found in mammals to help bridge the knowledge gap between the study systems.
Subject(s)
Chromatin/metabolism , RNA, Long Noncoding/genetics , Schizosaccharomyces/growth & development , Histone Code , Protein Processing, Post-Translational , RNA, Fungal/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
During the mitotic cycle, the rod-shaped fission yeast cells grow only at their tips. The newly born cells grow first unipolarly at their old end, but later in the cycle, the 'new end take-off' event occurs, resulting in bipolar growth. Photographs were taken of several steady-state and induction synchronous cultures of different cell cycle mutants of fission yeast, generally larger than wild type. Length measurements of many individual cells were performed from birth to division. For all the measured growth patterns, three different functions (linear, bilinear and exponential) were fitted, and the most adequate one was chosen by using specific statistical criteria, considering the altering parameter numbers. Although the growth patterns were heterogeneous in all the cultures studied, we could find some tendencies. In cultures with sufficiently wide size distribution, cells large enough at birth tend to grow linearly, whereas the other cells generally tend to grow bilinearly. We have found that among bilinearly growing cells, the larger they are at birth, the rate change point during their bilinear pattern occurs earlier in the cycle. This shifting near to the beginning of the cycle might finally cause a linear pattern, if the cells are even larger. In all of the steady-state cultures studied, a size control mechanism operates to maintain homeostasis. By contrast, strongly oversized cells of induction synchronous cultures lack any sizer, and their cycle rather behaves like an adder. We could determine the critical cell size for both the G1 and G2 size controls, where these mechanisms become cryptic. TAKE AWAY: Most individual fission yeast cells in steady-state cultures grow bilinearly. In strongly oversized fission yeast cells, linear growth dominates over bilinear. Above birth length thresholds, both the G1 and G2 size controls become cryptic.
Subject(s)
Cell Size , Mitosis , Schizosaccharomyces/cytology , Schizosaccharomyces/growth & development , Homeostasis , Mutation , Schizosaccharomyces/geneticsABSTRACT
Variations in cell wall composition and biomechanical properties can contribute to the cellular plasticity required during complex processes such as polarized growth and elongation in microbial cells. This study utilizes atomic force microscopy (AFM) to map the cell surface topography of fission yeast, Schizosaccharomyces pombe, at the pole regions and to characterize the biophysical properties within these regions under physiological, hydrated conditions. High-resolution images acquired from AFM topographic scanning reveal decreased surface roughness at the cell poles. Force extension curves acquired by nanoindentation probing with AFM cantilever tips under low applied force revealed increased cell wall deformation and decreased cellular stiffness (cellular spring constant) at cell poles (17 ± 4 mN/m) relative to the main body of the cell that is not undergoing growth and expansion (44 ± 10 mN/m). These findings suggest that the increased deformation and decreased stiffness at regions of polarized growth at fission yeast cell poles provide the plasticity necessary for cellular extension. This study provides a direct biophysical characterization of the S. pombe cell surface by AFM, and it provides a foundation for future investigation of how the surface topography and local nanomechanical properties vary during different cellular processes.
Subject(s)
Cell Membrane/physiology , Cell Wall/ultrastructure , Microscopy, Atomic Force/methods , Schizosaccharomyces/physiology , Schizosaccharomyces/ultrastructure , Cell Membrane/ultrastructure , Cell Wall/chemistry , Cell Wall/physiology , Schizosaccharomyces/growth & developmentABSTRACT
Cells balance glycolysis with respiration to support their metabolic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by aerobic glycolysis or fermentation. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentation-respiration balance. The laboratory and a few other strains depended more on respiration. This trait was associated with a single nucleotide polymorphism in a conserved region of Pyk1, the sole pyruvate kinase in fission yeast. This variant reduced Pyk1 activity and glycolytic flux. Replacing the "low-activity" pyk1 allele in the laboratory strain with the "high-activity" allele was sufficient to increase fermentation and decrease respiration. This metabolic rebalancing triggered systems-level adjustments in the transcriptome and proteome and in cellular traits, including increased growth and chronological lifespan but decreased resistance to oxidative stress. Thus, low Pyk1 activity does not lead to a growth advantage but to stress tolerance. The genetic tuning of glycolytic flux may reflect an adaptive trade-off in a species lacking pyruvate kinase isoforms.
Subject(s)
Carbon/metabolism , Mutation, Missense , Pyruvate Kinase/genetics , Schizosaccharomyces/growth & development , Fermentation , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Glycolysis , Oxidative Stress , Polymorphism, Single Nucleotide , Proteomics , Pyruvate Kinase/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365-920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371-920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe-S cluster in Asp1365-920 inside the cell. However, we show that the Fe-S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe-S cluster in vivo that is not involved in its pyrophosphatase activity.
Subject(s)
Cytoskeletal Proteins/chemistry , Iron-Sulfur Proteins/chemistry , Pyrophosphatases/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/enzymology , Biocatalysis , Cysteine/chemistry , Cytoskeletal Proteins/genetics , Iron-Sulfur Proteins/genetics , Multifunctional Enzymes/chemistry , Multifunctional Enzymes/genetics , Mutation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/genetics , Pyrophosphatases/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin.