Research ArticleMOLECULAR BIOLOGY

Genetic regulation of mitotic competence in G0 quiescent cells

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Science Advances  15 Aug 2018:
Vol. 4, no. 8, eaat5685
DOI: 10.1126/sciadv.aat5685
  • Fig. 1 Nitrogen-deprived cells maintained high MC with elevated cellular activity, apparently related to recycling nitrogen.

    (A) Time course of MC in WT cells under nitrogen deprivation (−N). 4′,6-Diamidino-2-phenylindole (DAPI) images of VE and −N 4-week cells are shown as insets. (B) Differential interference contrast (DIC) images were taken at 5-min intervals of cells incubated 24 hours in EMM2−N (upper panels) and in EMM2–glc (lower panels). In both conditions, high MC was maintained (93 and 80%, respectively), but white particles observed inside cells showed position changes only in EMM2−N cells. (C) Fluorescence images of nitric oxide (green, DAF-FM DA), vacuoles (red, FM4-64), and merged images of WT cells and Δaut12, a deletion strain of the vacuolar fusion protein. The upper row shows VE cells, and the lower row shows cells 24 hours after −N. Signal intensity of DAF-FM DA was quantified and shown in the images, proportional to WT VE cells. (D) Time course changes of normalized peak areas of amino acids detected by quantitative metabolomics analysis, using liquid chromatography–mass spectrometry.

  • Fig. 2 85 genes proved essential to sustain MC during the G0 phase, and these were categorized into three classes.

    (A) Schematized identification process for GZE genes. (B) Genes belonging to each class and diagram of class 1 genes.

  • Fig. 3 nem1 showed the most severe MC loss and manifested deformed nuclei that resembled those of the ned1-264 mutant.

    Western blot analysis also showed that Nem1 was required for Ned1 dephosphorylation after −N. (A) MC graph of class 1 genes related to phosphorylation signaling. (B) Fluorescence images of Nem1-GFP (green) and Cut11-mCherry (nuclear membrane, red) in WT cells in the VE phase and 24 hours after −N. (C) Fluorescence images of nuclei (DAPI) and vacuoles (FM4-64) in WT and Δnem1 cells. (D) Diagram of Ned1 protein. The mutation site of ned1-264 is indicated. (E) DAPI images of cell shape and nuclei in ned1-264 24 hours after −N. (F) MC graphs of the indicated strains. (G) Western blot analysis of Ned1-FLAG in WT, Δnem1, and ned1-264 in a 6% Phos-tag gel. Phos-tag traps phosphorylated proteins, reducing electrophoretic mobility. Samples were prepared from VE cells and 2, 6, and 12 hours after −N. Red and blue arrowheads indicate low and high electrophoretic mobility bands, respectively. (H) Fluorescence images of lipid droplets (Nile red) in WT, Δnem1, and ned1-264 cells in the VE phase and 24 hours after −N. Numbers of lipid droplets counted from midsection images of 20 cells for each strain were averaged and shown in right bar graphs with SD.

  • Fig. 4 MC loss and nuclear defects of Δnem1 were rescued by deletions of autophagy genes.

    (A) Electron micrographs of WT and Δnem1 24 hours after −N. N, nuclei. (B) DNA contents of WT and Δnem1 in the VE phase, 24 hours and 3 days after −N, by FACS. (C) Fluorescence images of nuclear chromatin region–localized protein Arp8/Eat1-GFP (green), vacuoles (FM4-64, red), and merged images of WT and Δnem1 in the VE phase and 2 days after −N. (D) MC graphs of the indicated strains. A diagram of autophagy processes associated with the strains is shown. (E) Percentages of DAPI-stained nuclear shapes found in the indicated strain and DAPI images 24 hours after −N. Two hundred cells for each strain were counted. (F) Schematized diagram of the GZE (nem1-SPBC902.03) and SHK (ned1) genes.

  • Table 1 Class 1 genes with functional group, human and budding yeast orthologs, MC (%) at the indicated time points, average cell length 24 hours after −N, cell number increment 24 hours after −N, and major DNA peaks in VE and 24 hours after −N, measured by FACS.
    Functional groupStrain nameOrthologMC (%)Cell
    length
    Cell
    number
    Major DNA
    peak
    HumanBudding
    yeast
    VE24
    hours
    1
    week
    2
    weeks
    3
    weeks
    4
    weeks
    −N 24
    hours
    (μm)
    Fold
    change
    VE-N 24
    hours
    WT104.497.887.8103.390.690.04.93.62C1C
    Protein
    phosphatase–
    related
    Nem1-
    Spo7
    Δnem1CTDNEP1NEM196.780.05.03.32.02.85.43.02C1C
    ΔSPBC902.03CNEP1R1SPO7108.380.04.80.00.00.06.22.42C1C/2C
    CWIΔrnc1HNRNPKPBP2103.3100.096.77.03.30.85.03.82C1C
    PP1Δctf1CSTF2TRNA1566.778.320.00.00.00.04.84.02C1C
    PP2AΔmug134ENSAIGO1110.0106.750.08.35.05.010.52.42C2C
    Δpar1PPP2R5BRTS193.388.348.30.00.00.05.02.22C2C
    Δypa1PPP2R4RRD186.775.070.05.84.34.75.82.22C2C
    Protein kinaseΔkin1MARKKIN1101.765.023.70.00.00.05.82.52C2C
    RNA/protein
    process
    Δexo2XRN1XRN141.761.78.80.00.00.010.11.72C2C
    Δrpl2002RPL18ARPL20B105.0106.746.71.25.80.04.43.52C1C
    Δsbh1SEC61BSBH161.763.35.01.70.01.24.64.12C1C
    Vesicle
    transport
    Δvps3TGFBRAP1VPS3115.045.01.00.00.00.05.12.42C1C
    Δvps26VPS26BPEP8113.363.325.01.73.31.76.61.62C2C
    Δvps35VPS35VPS3578.330.05.00.70.70.37.21.92C2C
    TransporterΔvht1SLC17VHT170.090.010.00.00.00.06.21.81C1C
    VacuoleΔaut12MON1BMON1100.071.70.00.00.00.08.21.42C1C/2C
    Δvac7VAC7108.371.70.00.00.00.08.52.12C2C
    ΔSPBC83.16cTTC39BYKR018C103.390.07.50.00.00.04.93.62C1C
    Metabolism
    (antioxidant)
    Δsod2SOD2SOD276.748.33.70.00.00.05.12.02C1C
    Δspe1ODCC1SPE190.083.375.04.21.01.85.03.11C/2C1C/2C

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaat5685/DC1

    Fig. S1. Fluorescence images of nitric oxide (green, DAF-FM DA) and vacuoles (red, FM4-64) and merged images of indicated strains.

    Fig. S2. A low-mobility band of Ned1-FLAG is phosphorylated.

    Fig. S3. Electron micrograph of ΔSPBC902.03 at 24 hours after −N.

    Fig. S4. ned1-264 showed MC loss and deformed nuclei, which were rescued by deletions of autophagy genes.

    Fig. S5. Scheme of two screening procedures.

    Fig. S6. PCR confirmation of gene deletion.

    Table S1. Table of 85 GZE genes.

    Table S2. List of cancer-related references for 85 GZE genes.

    Table S3. Strains that failed to backcross or grow.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Fluorescence images of nitric oxide (green, DAF-FM DA) and vacuoles (red, FM4-64) and merged images of indicated strains.
    • Fig. S2. A low-mobility band of Ned1-FLAG is phosphorylated.
    • Fig. S3. Electron micrograph of ΔSPBC902.03 at 24 hours after −N.
    • Fig. S4. ned1-264 showed MC loss and deformed nuclei, which were rescued by deletions of autophagy genes.
    • Fig. S5. Scheme of two screening procedures.
    • Fig. S6. PCR confirmation of gene deletion.
    • Legends for tables S1 to S3

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Table of 85 GZE genes.
    • Table S2 (Microsoft Excel format). List of cancer-related references for 85 GZE genes.
    • Table S3 (Microsoft Excel format). Strains that failed to backcross or grow.

    Download Tables S1 to S3

    Files in this Data Supplement:

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