Measurement of the e+e-→D(*)+D(*)- cross sections

journal articl

Inactive TORC1 represses <i>IME1</i>.

(A) IME1 mRNA quantification in control (FW1976) and tco89Δ (FW2154) cells shifted from YPD to YPD plus rapamycin and 1NM-PP1. 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. (B) IME1 mRNA and Ime1 protein quantification in tco89Δ cells expressing Ime1-V5 (FW2403). Cells were grown in YPD overnight, diluted into YPD and treated with rapamycin, 1NM-PP1, or both compounds. Samples were taken at the indicated time points, and IME1 mRNA levels were measured as described in A. Ime1 protein levels were quantified by western blot with antibodies direct against V5 and Hxk1 (control).</p

Inhibition of PKA and TORC1 induces meiosis.

(A) Kinetics of meiotic divisions (MI + MII) of cells harbouring the tpk1-as allele (FW1762) shifted from YPD to fresh YPD with no treatment (NT) (closed circles), rapamycin (open circles), 1NM-PP1 (closed triangles), or rapamycin/1NM-PP1 (open triangles). Samples were taken at the indicated time point, fixed, and DAPI masses were counted. Cells that contain 2, 3, or 4 DAPI masses were classified as cells that underwent meiosis. For each time point 100 cells were counted. (B) Quantification of sporulation efficiencies in control (FW1511) and tpk1-as (FW1762) diploid cells. Cell were grown overnight in YPD, shifted to pre-sporulation medium, and then transferred to sporulation medium (SPO). Percentages of sporulated cells were determined after 48 hours in SPO. (C-D) Quantification of number of nuclei and spores. Cells harbouring the tpk1-as allele were grown in YPD, shifted to pre-sporulation medium, and subsequently shifted to SPO medium or to YPD plus 1NM-PP1 and rapamycin. The percentage of cells harbouring three or four spores was determined at 24 hours after treatment (C). DAPI labelled nuclei were counted at 10 hours (D, left panel) and 24 hours (D, right panel) after treatment. For each time point, 200 cells were counted.</p

TORC1 activity is required for sporulation.

<p>(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 <i>KOG-AID</i>/<i>pTEF1-osTIR1</i> (FW1904) cells harbouring <i>tpk1-as</i> 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 <i>IME1</i> mRNA levels in control (FW1762) and <i>KOG1-AID</i>/<i>pTEF1-osTIR1</i> (FW1904) cells harbouring <i>tpk1-as</i> and treated with 1NM-PP1. <i>KOG1-AID</i>/<i>pTEF1-osTIR1</i> cells were also treated with IAA. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and <i>IME1</i> mRNA levels were measured by quantitative PCR. Signals were normalized to <i>ACT1</i> 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 <i>tpk1-as</i> and <i>pIME1-LacZ</i> (FW1976, control). The following gene deletion mutants were used for the analyses: control (FW1976), <i>tco89</i>Δ (FW2154), <i>gtr1</i>Δ (FW2164) or <i>tor1</i>Δ (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) <i>IME1</i> 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) <i>IME1</i> promoter activity was measured as described in E for control (FW1976) and <i>tco89</i>Δ (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

Single cell quantification of <i>IME1</i>.

<p>(A) Representative images used for the analyses of <i>IME1</i> and <i>ACT1</i> transcript levels in single cells. Cells harbouring <i>tpk1-as</i> (FW1762) were grown in YPD overnight and were either shifted to SPO, or diluted into fresh YPD plus rapamycin, 1NM-PP1 or 1NM-PP1/rapamycin. Cells were fixed, hybridized with probes directed against <i>IME1</i> (AF594) and <i>ACT1</i> (Cy5), and were imaged (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). <i>ACT1</i> was used as an internal control and only <i>ACT1</i> positive cells were selected for the analysis. (B) Mean of <i>IME1</i> and <i>ACT1</i> transcripts’ number among single cells as described in A. Cells harbouring <i>tpk1-as</i> (FW1762) were grown in YPD overnight, diluted into fresh YPD plus 1NM-PP1 or 1NM-PP1/ rapamycin and samples were taken at the indicated time points. Cells were imaged and quantified (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). At least, 60 cells (n = 60) were quantified per time point. The standard error of the mean of at least two biological experiments is shown. (C) Distribution of <i>IME1</i> and <i>ACT1</i> transcripts among single cells grown in YPD as described in A and B. Cells were binned in five classes of transcript levels (0 to 5; 6 to 10; 11 to 15; 16 to 20 and 21 or more transcripts per cell) and the percentages of each class contributing to the total population are indicated. At least, 60 cells (n = 60) were quantified per time point. The standard error of the mean of at least two biological experiments is shown. (D-G) Similar to C except that cells were grown in YPD overnight and transferred to YPD plus rapamycin (D), YPD plus 1NM-PP1 (E), YPD plus 1NMPP and rapamycin (F), or SPO (G). Samples were taken at the indicated time points. Cells were binned in five classes of transcript levels (0 to 5; 6 to 10; 11 to 15; 16 to 20 and 21 or more transcripts per cell) and the percentages of each class contributing to the total population are indicated.</p

Tup1 binds, represses, and mediates nutrient control of the <i>IME1</i> promoter.

<p>(A) Data taken from <i>Rizzo et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#pgen.1006075.ref039" target="_blank">39</a>] showing the nucleosome distribution at the <i>IME1</i> locus in control (closed circles) and <i>tup1</i>Δ mutant (open squares) cells. The x-axis shows the coordinates of the <i>IME1</i> locus at chromosome X in kilobases (kb), and y-axis shows the nucleosome occupancy score as described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#pgen.1006075.ref039" target="_blank">39</a>]. The position of each dot or point on the graph represents the coordinate of the nucleosome dyad center at the <i>IME1</i> locus. Regions lacking dots are depleted for nucleosomes. (B) Binding of Tup1 to the <i>IME1</i> promoter measured by chromatin immunoprecipitation. Diploid cells harbouring <i>tpk1-as</i> (control, FW1762) and <i>tpk1-as</i> plus Tup1 tagged at the C-terminus with 3xV5 (FW3078) were grown in rich medium (YPD) to mid-log and cross-linked with formaldehyde. Tup1 was immunoprecipitated from chromatin extracts. The recovered DNA was quantified by real-time PCR with 9 different primer sets across the <i>IME1</i> promoter and gene. Signals were normalized to the silent mating type locus (<i>HMR</i>), which does not bind Tup1. The error bars represent the standard error of the mean of two biological experiments. (C) Tup1 binding to the <i>IME1</i> promoter was measured by chromatin immunoprecipitation in control (FW3078) and <i>tco89</i>Δ (FW3096) cells. Cells were grown in YPD and shifted to YPD and were either untreated or treated with rapamycin, 1NM-PP1 or both compounds. Tup1 tagged with 3xV5 epitope was immunoprecipitated from chromatin extracts. The recovered DNA was quantified by real-time PCR with primer set five corresponding to middle of the <i>IME1</i> promoter. Signals were normalized to the silent mating type locus (<i>HMR</i>), which does not bind Tup1. The error bars represent the standard error of the mean of two biological experiments. (D) <i>IME1</i> promoter activity upon depletion of Tup1. Cells harbouring <i>IME1</i> promoter fused to LacZ (<i>pIME1-LacZ</i>) and expressing either Tup1 fused to the auxin induced degron (<i>TUP1-AID</i>) (FW3188) or <i>TUP1-AID</i> together with <i>pTEF1-osTIR1</i> (FW3184) were grown in YPD overnight. Cells were diluted to fresh YPD, either untreated or treated with indole-3-acetic acid (<i>IAA</i>) (500 μM), and samples were taken at the indicated time points. 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. (E) Comparison of <i>IME1</i> promoter activity during different treatments and growth conditions. Diploid cells harbouring <i>tpk1-as</i> and <i>pIME1-LacZ</i> (FW1976) were grown overnight in YPD, and diluted to YPD with 1NM-PP1 and rapamycin or cells were washes with water before transferred to sporulation medium. Diploid cells harbouring <i>TUP1-AID</i> and <i>pTEF1-osTIR1</i> (FW3188) were grown and treated as described D. Samples were taken at the indicated time points, and β-galactosidase activity was measured as described in D.</p

Respiration is not essential to <i>IME1</i> induction.

<p>(A) Northern blot analysis of <i>IME1</i> expression in control (FW1762, lanes 1–4) and <i>pet100</i>Δ mutant (FW1770, lanes 5–8). Cells were grown overnight in YPD medium and shifted to sporulation medium (SPO), and samples were taken at the indicated time points. (B) Similar to A except that cells (FW1762) were grown in pre-sporulation medium before shifted to SPO. Subsequently, cells were either not treated (lanes 1–4), treated with antimycin A (50 μM, lanes 5–8), oligomycin (10 μM, lanes 9–12), or with CCCP (20 μM, lanes 13–16). (C) Similar to A, except that cells were diluted into 1NM-PP1 and rapamycin containing YPD (lanes 1–16), and treated with antimycin A (lanes 5–8) or oligomycin (lanes 9–12). The <i>pet100</i>Δ mutant (lanes 13–16) was also included in the analyses. (D) <i>IME1</i> promoter activity was measured using a diploid strain harbouring <i>tpk1-as</i> and the <i>IME1</i> promoter fused to <i>LacZ</i> reporter (FW1976). Cells were grown in YPD overnight, diluted into YPD plus 1NM-PP1/rapamycin or shifted to sporulation medium (SPO) in the presence or absence of oligomycin (10 μM). Samples were taken after 0, 4, 6, 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. (E) Quantification of <i>IME1</i> mRNA levels in a strain harbouring the <i>GAL1</i> promoter fused to <i>IME1</i> and <i>GAL4-ER</i> (FW3243). Cells were grown in YPD, shifted to pre-sporulation medium, and transferred to SPO in the absence or presence of antimycin A. The <i>GAL1</i> promoter was activated using estradiol. Total RNA was isolated, reverse transcribed, and <i>IME1</i> mRNA levels were measured by quantitative PCR. Signals were normalized to <i>ACT1</i> levels. The standard error of the mean of at least two biological experiments is shown. (F) Quantification of sporulation efficiency of strains and treatments described in C. At least n = 200 cells were counted at 48 hours after treatment. Untreated diploid cells were included as a negative control.</p

Inhibition of PKA and TORC1 induces <i>IME1</i> and meiosis in rich medium.

<p>(A) Scheme of the signalling pathways controlling <i>IME1</i> expression. 1NM-PP1 and rapamycin compounds inhibit <i>tpk1-as</i> and TORC1 respectively. (B) Wild-type (FW1511) cells or cells harbouring the <i>tpk1M164G</i>, <i>tpk2</i>Δ, <i>tpk3</i>Δ alleles (<i>tpk1-as</i>, FW1762) were grown in YPD overnight, diluted to 0.2 (OD600), and subsequently cells were treated with 5μM 1NM-PP1or untreated. Cell density (OD600) was measured over time at the indicated time points. (C) <i>IME1</i> promoter activity was measured in a diploid <i>tpk1-as</i> strains and the <i>IME1</i> promoter fused to <i>LacZ</i> reporter (FW1976). Cells were grown in YPD overnight, diluted into YPD or shifted to sporulation medium (SPO), and samples were taken after 0, 4, 6, 8, 10, 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. (D) Similar to C except that cells were shifted to SPO, SPO plus 2% glucose or SPO with 2% glucose and 5μM 1NM-PP1. Samples were taken at the indicated time points. (E) Similar to C, except that samples shifted to YPD or YP-acetate (YPA), YPA plus rapamycin or YPA plus 1NM-PP1. (F) Similar to C except that cells were shifted to SPO or YPD and treated with rapamycin, 1NM-PP1 or both compounds. (G) <i>IME1</i> mRNA quantification of cells shifted from YPD to SPO or YPD plus rapamycin, 1NM-PP1, or both compounds. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and <i>IME1</i> mRNA levels were measured by quantitative PCR. Signals were normalized to <i>ACT1</i> levels. The standard error of the mean of at least two biological experiments is shown.</p

Sch9 controls <i>IME1</i> and entry into sporulation.

<p>(A) <i>IME1</i> promoter activity was measured in strains harbouring <i>tpk1-as</i> and <i>pIME1-LacZ</i> (control, FW1976) and <i>sch9</i>Δ (FW2498). Cells were grown in YPD overnight shifted to SPO and samples were taken at the indicated time points. 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. (B) Similar to A except that cells were diluted into YPD with 1NM-PP1 or 1NM-PP1 plus rapamycin. (C) <i>IME1</i> transcript distribution among single cells in control (FW1762), <i>tco89</i>Δ (FW1966) and <i>sch9</i>Δ (FW2437) strains (all in <i>tpk1-as</i> background). After 8 hours treatment with 1NM-PP1, cells were fixed, hybridized with probes directed against <i>IME1</i> (AF594) and <i>ACT1</i> (Cy5). Finally, cells were imaged and transcript levels were quantified (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). <i>ACT1</i> was used as an internal control and only cells positive for <i>ACT1</i> were selected for the analysis. At least 120 cells (n = 120) were quantified. The error bars represent the standard error of the mean of two biological experiments. (D) Same treatments and strains as A and B, but here the percentage of cells that underwent meiotic divisions (MI+MII) was determined after 48 hours of treatment.</p

Scheme of how PKA and TORC1 signalling controls <i>IME1</i> and entry into sporulation.

<p>Scheme of how PKA and TORC1 signalling controls <i>IME1</i> and entry into sporulation.</p