TORC1 activity is required for sporulation (original) (raw)

2016

Abstract

(A) Cells (FW1762) were treated with different concentrations of rapamycin, and doubling times (left panel) as well as the fraction of cells that underwent meiosis (right panel) were quantified. Left panel, cells were grown in YPD, shifted to YPD plus 0, 5, 20, or 1000 ng/ml rapamycin and doubling times were measured during exponential growth. Right panel, cells were diluted into YPD plus PKA inhibitors and treated with different concentrations of rapamycin as indicated. DAPI masses were counted after 48 hours of treatment. (B) Control (FW1762) and KOG-AID/pTEF1-osTIR1 (FW1904) cells harbouring tpk1-as were grown in YPD overnight, diluted into fresh YPD and treated with 1NM-PP1, rapamycin or IAA. The nuclei number in cells was counted after 48 hours of treatment by DAPI staining, and percentage of cells that underwent meiosis (MI+MII) was quantified. (C) Quantification of IME1 mRNA levels in control (FW1762) and KOG1-AID/pTEF1-osTIR1 (FW1904) cells harbouring tpk1-as and treated with 1NM-PP1. KOG1-AID/pTEF1-osTIR1 cells were also treated with IAA. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and IME1 mRNA levels were measured by quantitative PCR. Signals were normalized to ACT1 levels. The standard error of the mean of at least two biological experiments is shown. (D) Percentage of cells that underwent meiotic divisions (MI+MII) was determined in gene deletion strains, all harbouring tpk1-as and pIME1-LacZ (FW1976, control). The following gene deletion mutants were used for the analyses: control (FW1976), tco89Δ (FW2154), gtr1Δ (FW2164) or tor1Δ (FW2162). Samples were grown in YPD medium, fixed, and DAPI masses were counted at 48 hours after treatment with 1NM-PP1 or with 1NM-PP1 and rapamycin. (E) IME1 promoter activity was measured in strains described in D. Cells were grown in YPD overnight, diluted into YPD plus 1NMPP1 and/or rapamycin, and samples were taken after 0, 4, 8, and 12 hours. β-galactosidase activity was measured using a quantitative liquid ortho-Nitrophenyl-β-galactoside (ONPG) assay (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). The promoter activities are displayed in Miller Units, and the standard error of the mean of at least two biological experiments is shown. (F) IME1 promoter activity was measured as described in E for control (FW1976) and tco89Δ (FW2154) strains. Cells were grown in YPD overnight, diluted into YPD plus 1NMPP1 and/or rapamycin, and samples were taken after 0, 2, 4, 6, 8, 10, 12 and 24 hours. (G) Kinetics of meiotic division (MI+MII) of strains and treatments described in F. Samples were taken at the indicated time points, fixed, and DAPI masses were counted.</p

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