FY2015 Annual Report

G0 Cell Unit
Professor Mitsuhiro Yanagida

Abstract

The G0 Cell Unit studies how cells in the proliferative (dividing) or quiescent (non-dividing) phase respond to nutritional shifts (e.g., nitrogen source starvation and glucose limitation). We identify gene products (mostly proteins) and chemical factors (small molecules, metabolites) that affect adaptation mechanisms in order to understand the basis for longevity of long-term quiescent cells. We employ fission yeast as a eukaryotic cell model as the great majority of this organism’s genes are conserved in human. In addition, we are studying chromosomal regulatory mechanisms involving condensin, cohesin complexes (which lead to proper chromosome segregation in proliferating cells), and other nutrient adaptation-related nuclear chromatin proteins. This line of studies aims to understand dynamics of nuclear chromatin in response to nutritional cues. Study on human blood metabolomics initiated from comparative study with fission yeast metabolomics now directly aims to identify and understand the roles of metabolites intimately related to human aging.

Our current principal research projects may be summarized as below:

(1a) Methodological development for metabolomic analysis.

(1b) Comprehensive and quantitative human blood metabolomics.

(2) Understanding cell regulation in response to nitrogen deprivation.

(3) Understanding cell regulation in response to glucose starvation.

(4) Understanding chromosomal regulatory mechanisms involving condensin, cohesin complexes, and other nutrient adaptation-related nuclear proteins.

In FY2015, we published five original articles on human blood metabolome identifying age-related differences, oxidative and metal stress responsive genes in fission yeast, two glucose related topics in fission yeast (transient G2 cell arrest at glucose restriction, and critical glucose concentration for respiration-independent proliferation) and condensin enriched sites on chromosomes in fission yeast, as introduced in 3. Activities and Findings and listed in 4. Publications.

1. Staff

  • Dr. Takeshi Hayashi, Group leader
  • Dr. Kazuki Kumada, Group leader (until September)
  • Dr. Romanas Chaleckis, Researcher (until February)
  • Dr. Norihiko Nakazawa, Researcher
  • Dr. Tomas Pluskal, Researcher (until July)
  • Dr. Paul-Emile Poleni, Researcher (from November)
  • Dr. Kenichi Sajiki, Researcher
  • Dr. Takayuki Teruya, Researcher
  • Dr. Emily Tsang, Researcher
  • Dr. Xingya Xu, Researcher
  • Dr. Haifeng Zhang, Researcher (from December)
  • Ms. Orie Arakawa, Technical staff
  • Mr. Masahiro Ebe, Technical staff (until September)
  • Ms. Wendy Gutierrez, Technical staff (from September)
  • Ms. Ayaka Mori, Technical staff
  • Ms. Yuria Tahara, Technical staff
  • Ms. Junko Takada, Technical staff
  • Ms. Risa Uehara, Technical staff
  • Ms. Li Wang, Technical staff
  • Ms. Chikako Sugiyama, Research administrator
  • Ms. Caroline Starzynski, OIST Student
  • Mr. Subarna Sharma, Research Intern (from June, until August)
  • Mr. Zenan Wang, Research Intern (until June)

2. Collaborations

  • Theme: Metabolite structure determination using NMR
    • Type of collaboration: Joint research
    • Researchers:
      • Dr. Masaru Ueno, Department of Molecular Biotechnology, Graduate school of Advanced Science of Matter, Hiroshima University
  • Theme: Screening for low-glucose sensitive mutants in fission yeast
    • Type of collaboration: Joint research
    • Researchers:
      • Professor Shigeaki Saitoh, Institute of Life Science, Kurume University
  • Theme: Identification of the molecular mechanism, required for the maintenance of cell viability in low glucose condition
    • Type of collaboration: Joint research
    • Researchers:
      • Professor Kunihiro Ohta, Department of Life Science, Graduate School of Arts and Science, The University of Tokyo
  • Theme: Analysis of biomarkers, discovered by research using S.pombe, in human blood
    • Type of collaboration: Joint research
    • Researchers:
      • Dr. Hiroshi Kondoh, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University
      • Dr. Takumi Mikawa, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University
  • Theme: Functional analysis of condensin complex in the regulation of chromosome segregation and gene expression
    • Type of collaboration: Joint research
    • Researchers:
      • Dr. Takashi Sutani, Institute of Molecular Cellular Biosciences, The University of Tokyo
  • Theme: Physiological/genetic analysis of cellular adaptation strategy to environmental changes
    • Type of collaboration: Joint research
    • Researchers:
      • Dr. Kojiro Takeda, Department of Biology, Faculty of Science and Engineering, Konan University
  • Theme: Comparative analysis of blood metabolome between healthy people and metabolic abnormality patients
    • Type of collaboration: Joint research
    • Researchers:
      • Professor Hiroaki Masuzaki, Division of Endocrinology, Diabetes and Metabolism, Hematology, Rheumatology (Second Department of Internal Medicine), Graduate School of Medicine, University of the Ryukyus
  • Theme: Analysis of biomarkers, discovered by research using S. pombe, in human blood
    • Type of collaboration: Joint research
    • Researchers:
      • Dr. Rasa Liutkevičienė, Lithuanian University of Health Sciences
      • Virginija Ašmonienė, Lithuanian University of Health Sciences

3. Activities and Findings

3.1 Individual variability in human blood metabolites identifies age-related differences

Metabolites present in human blood document individual physiological states influenced by genetic, epigenetic, and lifestyle factors. Using high-resolution liquid chromatography-mass spectrometry (LC-MS), we performed nontargeted, quantitative metabolomics analysis in blood of 15 young (29 ± 4 y of age) and 15 elderly (81 ± 7 y of age) individuals. Immediately after taken from volunteers, all blood samples were quenched in methanol/water at −40 °C. This quick-quenching step ensured accurate measurement of many labile metabolites.

Coefficients of variation (CV = SD/mean) were obtained for 126 blood metabolites of all 30 donors. We found previously unidentified CVs of 51 blood compounds, while others are mostly consistent with those CVs previously published. Compounds having low CV values, such as ATP and glutathione, may be vitally essential, and related to various diseases because their concentrations are strictly controlled. Compounds having moderate to high CV values (0.4–2.5) are often modified.

We found 14 compounds that differed significantly between the two age groups; citrulline, pantothenate, dimethyl-guanosine, N-acetyl-arginine, and N6-acetyl-lysine are higher in elderly group, while 1,5-anhydroglucitol, acetyl-carnosine, carnosine, ophthalmic acid, UDP-acetyl-glucosamine, leucine, isoleucine, NAD+, and NADP+ are higher in young population (Figure 1). Six of them are RBC-enriched, suggesting that RBC metabolomics is highly valuable for human aging research. Such age differences are partly explained by a decrease in antioxidant production, inefficiency of urea metabolism, or impairment in kidney, muscle, brain, and liver among the elderly. Moreover, Pearson’s coefficients demonstrated that some age-related compounds are strongly correlated, suggesting that aging affects them concomitantly. This study was performed in collaboration with Dr. Hiroshi Kondoh (Kyoto University) and published in PNAS (Chaleckis et al., 2016) (Kondoh & Yanagida, 2016).

Figure 1: Characterization of 14 age-related metabolites we identified.

3.2 Diverse fission yeast genes required for responding to oxidative and metal stress: Comparative analysis of glutathione-related and other defense gene deletions

We constructed a collection of twelve single-gene deletion strains of the fission yeast Schizosaccharomyces pombe (S. pombe) designed for the study of oxidative and heavy metal stress responses. This collection contains deletions of biosynthetic enzymes of glutathione (∆gcs1 and ∆gsa1), phytochelatin (∆pcs2), ubiquinone (∆abc1) and ergothioneine (∆egt1), as well as catalase (∆ctt1), thioredoxins (∆trx1 and ∆trx2), Cu/Zn- and Mn-superoxide dismutases (SODs; ∆sod1and ∆sod2), sulfiredoxin (∆srx1) and sulfide-quinone oxidoreductase (∆hmt2). First, we employed metabolomic analysis to examine the mutants of the glutathione biosynthetic pathway. We found that ophthalmic acid, an age related metabolite found in human blood, was produced by the same enzymes as glutathione in S. pombe (Figure 2A, B). Too high peaks of ophthalmic acid and its precursor in ∆gsa1 suggest they are not merely byproducts of glutathione, but have yet undiscovered function.

The identical genetic background of the strains allowed us to assess the severity of the individual gene knockouts by treating the deletion strains with oxidative agents. The results show the astonishing diversity in cellular adaptation mechanisms to various types of oxidative and metal stress. Among other results, we found that glutathione deletion strains were not particularly sensitive to peroxide or superoxide, but highly sensitive to cadmium stress (Figure 2C). Our collection provides a useful tool for further research into stress responses. This study was published in Genes to Cells (Pluskal et al., 2016).

Figure 2: (A) Biosynthetic pathways for ergothioneine, selenoneine, glutathione, ophthalmic acid, and phytochelatin, in S. pombe. Colors indicate the flow of each pathway. (B) Results of metabolomic analysis of three deletion strains of the glutathione/phytochelatin pathway in comparison with wild-type cells. Raw peak areas of [M+H]+ ions of four key metabolites are shown. (C) Spot test assays on YE plates containing CdSO4 in aerobic (+O2) and anaerobic (-O2) conditions. Glutathione deletion strains showed high sensitivity.

3.3 Glucose restriction induces transient G2 cell cycle arrest extending cellular chronological lifespan

While glucose is the fundamental source of energy in most eukaryotes, it is not always abundantly available in natural environments, including within the human body. Eukaryotic cells are therefore thought to possess adaptive mechanism to survive glucose-limited conditions, which remains unclear. We explored the mechanism underlying the regulation of cell cycle progression under glucose-limited conditions, and showed that glucose restriction causes transient G2 arrest in S. pombe. This G2 arrest due to glucose restriction is thought to be determined genetically, as deletion of the wee1+ gene, which encodes an evolutionarily conserved tyrosine kinase regulating CDK (cyclin-dependent kinase) activities, bypasses this G2 block and causes the accumulation of cells with unreplicated DNA during proliferation under glucose-limited conditions. S. pombe cells lacking Wee1 lost cell viability faster than wild-type cells in glucose-depleted medium.

Our findings indicate the presence of a novel cell cycle checkpoint monitoring glucose availability, which allows cells to adapt to glucose-limited environments (Figure 3). During the period of G2 arrest in response to glucose restriction, cells may enhance their capability for glucose transport and mitochondrial ATP generation, and gain energy and carbon source sufficient for completion of mitosis and cytokinesis. This glucose-monitoring checkpoint may, at least in part, extend the cellular chronological lifespan in the absence of glucose, as mutant cells lacking the wee1+ gene have a shorter lifespan than WT cells. Although it remains to be determined whether the “glucose checkpoint” described above exists in higher eukaryotes, if present, inhibition of Wee1 activity in these organisms may selectively kill glucose-starved cells, such as cells in a tumor. This study was published in Scientific reports (Masuda et al., 2016).

Figure 3 

3.4 The critical glucose concentration for respiration-independent proliferation of fission yeast

Glucose is the fundamental energy source for life. Therefore, an appropriate response to fluctuations in glucose concentration within the media is deterministic for cell growth and survival. We uncovered the relationship between media glucose concentration and respiration-dependency during proliferation in S. pombe. In our study respiratory complex III impairment was induced by drug treatment. Cell growth was measured for 10 hours under varying concentration of glucose, which ranged from ample amount (2%) to low glucose (0.08%). In parallel, treatment with antimycin A (AA), an inhibitor of Complex III, was performed in a separate batch (Figure 4A-C). Cells cultured in low glucose are extremely susceptible to AA treatment, whereas cells grown in sufficient glucose are resistant. Moreover, AA treatment revealed a threshold of respiratory dependency (Figure 4E).