RESUMO
Multiple researchers are reporting toxic agar, but the ultimate culprit remains unclear.
Assuntos
Ágar , Saccharomyces cerevisiae , Schizosaccharomyces , Ágar/provisão & distribuição , Ágar/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Laboratórios , Alga Marinha/química , Alga Marinha/metabolismoRESUMO
Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP8 toxicosis, whereby excessive 1,5-IP8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity. IMPORTANCE: Repression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.
Assuntos
Pirofosfatases , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Pirofosfatase Inorgânica/metabolismo , Pirofosfatase Inorgânica/genética , Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Regulação Fúngica da Expressão Gênica , Mutação , Nudix Hidrolases , Enzimas MultifuncionaisRESUMO
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.
Assuntos
Etanol , Schizosaccharomyces , Etanol/metabolismo , Etanol/farmacologia , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Fermentação , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Proliferação de Células/efeitos dos fármacos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Fatores de Transcrição , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Regulação Fúngica da Expressão Gênica , Transdução de Sinais , Meiose , Feromônios/metabolismo , Diferenciação Sexual/genética , Glucose/metabolismo , Nitrogênio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologiaRESUMO
The molecular mechanisms underlying the establishment of the monopolar growth of fission yeast spores have been less characterized. Here, we report that the Cdc42 GTPase-activating protein (GAP) Rga6 is required for promoting monopolar growth during spore germination. The absence of Rga6 increases the number of spores that grow in a bipolar fashion. Rga6 decorates the non-growing cortical region, binds phosphatidylinositol 4,5-bisphosphate, and colocalizes with the phosphatidylinositol 4,5-bisphosphate-binding protein Opy1. Overexpression of Opy1 diminishes the cortical localization of Rga6. The characteristic localization of Rga6 on the cell cortex depends on the C-terminal PBR region of Rga6. Moreover, engineered chimera composed of the Rga6 C-terminal PBR region fused to the GAP domain of Rga3 or Rga4 are sufficient to rescue the spore growth phenotype caused by the absence of Rga6. Hence, our work establishes a paradigm in which the lipid composition of the plasma membrane directs polarized cell growth by specifying the cortical localization of a GAP protein.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Esporos Fúngicos , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
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.
Assuntos
Ciclo Celular/fisiologia , Tamanho Celular , Modelos Biológicos , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Biologia ComputacionalRESUMO
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.
Assuntos
Cromatina/metabolismo , RNA Longo não Codificante/genética , Schizosaccharomyces/crescimento & desenvolvimento , Código das Histonas , Processamento de Proteína Pós-Traducional , RNA Fúngico/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Homeostatic maintenance of the physicochemical properties of cellular membranes is essential for life. In yeast, trehalose accumulation and lipid remodeling enable rapid adaptation to perturbations, but their crosstalk was not investigated. Here we report about the first in-depth, mass spectrometry-based lipidomic analysis on heat-stressed Schizosaccharomyces pombe mutants which are unable to synthesize (tps1Δ) or degrade (ntp1Δ) trehalose. Our experiments provide data about the role of trehalose as a membrane protectant in heat stress. We show that under conditions of trehalose deficiency, heat stress induced a comprehensive, distinctively high-degree lipidome reshaping in which structural, signaling and storage lipids acted in concert. In the absence of trehalose, membrane lipid remodeling was more pronounced and increased with increasing stress dose. It could be characterized by decreasing unsaturation and increasing acyl chain length, and required de novo synthesis of stearic acid (18:0) and very long-chain fatty acids to serve membrane rigidification. In addition, we detected enhanced and sustained signaling lipid generation to ensure transient cell cycle arrest as well as more intense triglyceride synthesis to accommodate membrane lipid-derived oleic acid (18:1) and newly synthesized but unused fatty acids. We also demonstrate that these changes were able to partially substitute for the missing role of trehalose and conferred measurable stress tolerance to fission yeast cells.
Assuntos
Glucosiltransferases/genética , Lipidômica/métodos , Monoéster Fosfórico Hidrolases/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/crescimento & desenvolvimento , Trealose/metabolismo , Glucosiltransferases/metabolismo , Temperatura Alta , Espectrometria de Massas , Mutação , Ácido Oleico/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Triglicerídeos/metabolismoRESUMO
In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryoelectron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.
Assuntos
Membrana Nuclear/fisiologia , Poro Nuclear/fisiologia , Poro Nuclear/ultraestrutura , Transporte Ativo do Núcleo Celular , Fenômenos Biomecânicos , Microscopia Crioeletrônica , Citoplasma/metabolismo , Metabolismo Energético , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Modelos Biológicos , Membrana Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Pressão Osmótica , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Estresse FisiológicoRESUMO
Cdc42 GTPase rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its downregulation. We describe here that oxidative stress causes Sty1-kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at tips and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles either by eliminating two Cdc42 GAPs or through the constitutive tethering of Gef1 to cell tips, and we biochemically demonstrate that the GAPs Rga3/6 and the GEF Gef1 are direct substrates of Sty1. We propose that phosphorylation of Rga3/6 and Gef1 mediates the Sty1-dependent inhibition of Cdc42 at cell tips, halting polarized growth during stress adaptation.
Assuntos
Polaridade Celular , Proliferação de Células , Proteínas Ativadoras de GTPase/metabolismo , Estresse Oxidativo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais , Fatores de Tempo , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Cytokinin (CK) is an important plant developmental regulator, having activities in many aspects of plant life and response to the environment. CKs are involved in diverse processes in the plant, including stem cell maintenance, vascular differentiation, growth and branching of roots and shoots, leaf senescence, nutrient balance, and stress tolerance. In some cases, phytopathogens secrete CKs. It has been suggested that to achieve pathogenesis in the host, CK-secreting biotrophs manipulate CK signaling to regulate the host cell cycle and nutrient allocation. CK is known to induce host plant resistance to several classes of phytopathogens from a few works, with induced host immunity via salicylic acid signaling suggested to be the prevalent mechanism for this host resistance. Here, we show that CK directly inhibits the growth, development, and virulence of fungal phytopathogens. Focusing on Botrytis cinerea (Bc), we demonstrate that various aspects of fungal development can be reversibly inhibited by CK. We also found that CK affects both budding and fission yeast in a similar manner. Investigating the mechanism by which CK influences fungal development, we conducted RNA next-generation sequencing (RNA-NGS) on mock- and CK-treated B. cinerea samples, finding that CK alters the cell cycle, cytoskeleton, and endocytosis. Cell biology experiments demonstrated that CK affects cytoskeleton components and cellular trafficking in Bc, lowering endocytic rates and endomembrane compartment sizes, likely leading to reduced growth rates and arrested developmental programs. Mutant analyses in yeast confirmed that the endocytic pathway is altered by CK. Our work uncovers a remarkably conserved role for a plant growth hormone in fungal biology, suggesting that pathogen-host interactions resulted in fascinating molecular adaptations on fundamental processes in eukaryotic biology. IMPORTANCE Cytokinins (CKs), important plant growth/developmental hormones, have previously been associated with host disease resistance. Here, we demonstrate that CK directly inhibits the growth, development, and virulence of B. cinerea (Bc) and many additional phytopathogenic fungi. Molecular and cellular analyses revealed that CK is not toxic to Bc, but rather, Bc likely recognizes CK and responds to it, resulting in cell cycle and individual cell growth retardation, via downregulation of cytoskeletal components and endocytic trafficking. Mutant analyses in yeast confirmed that the endocytic pathway is a CK target. Our work demonstrates a conserved role for CK in yeast and fungal biology, suggesting that pathogen-host interactions may cause molecular adaptations in fundamental processes in eukaryotic biology.
Assuntos
Citocininas/farmacologia , Citoesqueleto/efeitos dos fármacos , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Botrytis/efeitos dos fármacos , Botrytis/crescimento & desenvolvimento , Botrytis/patogenicidade , Ciclo Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resistência à Doença , Fungos/genética , Fungos/patogenicidade , Reguladores de Crescimento de Plantas , Patologia Vegetal , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , VirulênciaRESUMO
Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An in vitro assay revealed that five of these compounds are strong inhibitors of tubulin polymerization. These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by ßIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Microtúbulos/efeitos dos fármacos , Schizosaccharomyces/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colchicina/farmacologia , Descoberta de Drogas/métodos , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Schizosaccharomyces/crescimento & desenvolvimento , Células Tumorais CultivadasRESUMO
In our study we investigated the effect of different nickel (NiSO4·6H2O) (Ni) concentrations on cell division, cellular morphology and ionome homeostasis of the eukaryotic model organism Schizosaccharomyces pombe. Target of rapamycin (TOR) protein kinase is one of the key regulators of cell growth under different environmental stresses. We analyzed the effect of Ni on cell strains lacking the Tor1 signaling pathway utilizing light-absorbance spectroscopy, visualization, microscopy and inductively coupled plasma optical emission spectroscopy. Interestingly, our findings revealed that Ni mediated cell growth alterations are noticeably lower in Tor1 deficient cells. Greater size of Tor1 depleted cells reached similar quantitative parameters to wild type cells upon incubation with 400 µM Ni. Differences of ion levels among the two tested yeast strains were detected even before Ni addition. Addition of high concentration (1 mM) of the heavy metal, representing acute contamination, caused considerable changes in the ionome of both strains. Strikingly, Tor1 deficient cells displayed largely reduced Ni content after treatment compared to wild type controls (644.1 ± 49 vs. 2096.8 ± 75 µg/g), suggesting its significant role in Ni trafficking. Together our results predict yet undefined role for the Tor1 signaling in metal uptake and/or metabolism.
Assuntos
Regulação Enzimológica da Expressão Gênica , Níquel/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Regulação para Baixo , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Cinética , Níquel/química , Proteínas Quinases/química , Proteínas Quinases/genética , Schizosaccharomyces/química , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Nuclear movements during meiotic prophase, driven by cytoskeleton forces, are a broadly conserved mechanism in opisthokonts and plants to promote pairing between homologous chromosomes. These forces are transmitted to the chromosomes by specific associations between telomeres and the nuclear envelope during meiotic prophase. Defective chromosome movements (CMs) harm pairing and recombination dynamics between homologues, thereby affecting faithful gametogenesis. For this reason, modelling the behaviour of CMs and their possible microvariations as a result of mutations or physico-chemical stress is important to understand this crucial stage of meiosis. Current developments in high-throughput imaging and image processing are yielding large CM datasets that are suitable for data mining approaches. To facilitate adoption of data mining pipelines, we present ChroMo, an interactive, unsupervised cloud application specifically designed for exploring CM datasets from live imaging. ChroMo contains a wide selection of algorithms and visualizations for time-series segmentation, motif discovery, and assessment of causality networks. Using ChroMo to analyse meiotic CMs in fission yeast, we found previously undiscovered features of CMs and causality relationships between chromosome morphology and trajectory. ChroMo will be a useful tool for understanding the behaviour of meiotic CMs in yeast and other model organisms.
Assuntos
Algoritmos , Segregação de Cromossomos , Cromossomos Fúngicos , Interpretação de Imagem Assistida por Computador , Meiose , Microscopia de Fluorescência , Schizosaccharomyces/crescimento & desenvolvimento , Imagem com Lapso de Tempo , Automação Laboratorial , Computação em Nuvem , Ensaios de Triagem em Larga Escala , Schizosaccharomyces/genética , Fatores de TempoRESUMO
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.
Assuntos
Proteínas Fúngicas/metabolismo , Transferases Intramoleculares/metabolismo , Schizosaccharomyces/fisiologia , Triterpenos/metabolismo , Adaptação Biológica , Anaerobiose , Proteínas de Bactérias/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Ergosterol/farmacologia , Células Eucarióticas/fisiologia , Ácidos Graxos Insaturados/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Transferases Intramoleculares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Esteróis/metabolismoRESUMO
Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Forma Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Quinases Ativadas por p21/metabolismo , Polaridade Celular/fisiologia , Citoesqueleto/metabolismo , Fosforilação , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Fosfoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fosforilação , Biossíntese de Proteínas , Proteoma , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimentoRESUMO
Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in Schizosaccharomyces pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.
Assuntos
Cinesinas/metabolismo , Meiose , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fuso Acromático/metabolismo , Regulação Fúngica da Expressão Gênica , Cinesinas/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Proteínas Motores Moleculares/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais , Fuso Acromático/genética , Fatores de TempoRESUMO
The sla1+ gene of Schizosachharoymces pombe encodes La protein which promotes proper processing of precursor-tRNAs. Deletion of sla1 (sla1Δ) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4+ inhibits leucine uptake and growth of sla1Δ cells. Here, transcriptome analysis reveals that genes upregulated in sla1Δ cells exhibit highly significant overalp with general amino acid control (GAAC) genes in relevant transcriptomes from other studies. Growth in NH4+ media leads to additional induced genes that are part of a core environmental stress response (CESR). The sla1Δ GAAC response adds to evidence linking tRNA homeostasis and broad signaling in S. pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes in sla1Δ cells suggesting that nuclear surveillance-mediated signaling occurs in S. pombe. To study the NH4+-effects, we isolated sla1Δ spontaneous revertants (SSR) of the slow growth phenotype and found that GAAC gene expression and rapamycin hypersensitivity were also reversed. Genome sequencing identified a F32V substitution in Any1, a known negative regulator of NH4+-sensitive leucine uptake linked to TOR. We show that 3H-leucine uptake by SSR-any1-F32V cells in NH4+-media is more robust than by sla1Δ cells. Moreover, F32V may alter any1+ function in sla1Δ vs. sla1+ cells in a distinctive way. Thus deletion of La, a tRNA processing factor leads to a GAAC response involving reprogramming of amino acid metabolism, and isolation of the any1-F32V rescuing mutant provides an additional specific link.
Assuntos
Aminoácidos/metabolismo , Arrestinas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Arrestinas/metabolismo , Perfilação da Expressão Gênica , Genes Fúngicos/genética , Mutação/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density in fission yeast is maintained in a narrow distribution and exhibits homeostatic behavior. However, density varied during the cell cycle, decreasing during G2, increasing in mitosis and cytokinesis, and dropping rapidly at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages exacerbated density changes. Spatially heterogeneous patterns of density suggested links between density regulation, tip growth, and intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.