Figure 1.
Defining the stages of meiosis with time-lapse microscopy.
A) Cell with Spc42-mCherry, Zip1-GFP, and GFP-TUB1 were imaged using time-lapse microscopy. Still pictures from the time-lapse fluorescence microscopy representing the different stages of meiosis are shown. Fluorescent images are overlaid with a DIC image (Scale bar - 3 µm). The white arrowhead marks the SC, shown with Zip1-GFP. B) A plot of spindle length in µm (Y-axis) at 10-minute timepoints after SC disassembly (X-axis) of 5 cells undergoing meiosis. The distances between SPBs were measured to obtain the spindle length as the cells progressed from prophase I to anaphase I. C) Cell with Pds1-GFP and Spc42-mCherry shows the spindle length at the metaphase I to anaphase I transition when Pds1-GFP is degraded (Scale bar - 3 µm). D) Diagram depicting the timing of the stages of prometaphase I and metaphase I based on spindle length measurements.
Table 1.
The duration of the meiotic stages (time in minutes ± standard error. n = 75 cells analyzed for wildtype and 80 cells analyzed for NDT80/ndt80Δ).
Figure 2.
The meiotic commitment point occurs in prometaphase I.
A) Graph of the percent of cells with each cell-cycle outcome (y-axis) upon complete medium addition to cells with different spindle lengths (x-axis). Cells that returned to mitosis are shown in blue, finished meiosis are shown in red, and arrested in meiosis I are shown in green. B) Graph of the percent of cells with each cell-cycle outcome (y-axis) at the meiotic stage in which complete medium was added. Cells that returned to mitosis are shown in blue, finished meiosis are shown in red, and arrested in meiosis I are shown in green. C-F) Cells with Spc42-mCherry, Zip1-GFP, and GFP-TUB1 initiated meiosis in microfluidic chambers. Complete medium was flowed into the chambers (at timepoint with grey arrowhead) and time-lapse microscopy was used to monitor the cell-cycle outcome. Fluorescent images are overlaid with a DIC image (Scale bar – 3 µm). C) Cell in prophase I at the time of complete medium addition returned to mitosis. D) Cell in prometaphase I at the time of complete medium addition returned to mitosis. E) Cell in prometaphase I at the time of complete medium addition finished meiosis. F) Cell in metaphase I at the time of complete medium addition finished meiosis. G) Diagram of the meiotic commitment point. SPBs are shown in red, chromosomes in blue, SC in green, and spindle microtubules in grey. Cells commit to meiosis as the SPBs are separating in prometaphase I.
Figure 3.
Cdc5-sfGFP is present prior to meiotic commitment.
Fluorescence microscopy of cells with Cdc5-sfGFP, Spc42-mCherry, and DAPI staining. Scale bar 2 µm. A) Cdc5-sfGFP is not present in prophase I. B) Cdc5-sfGFP is present in early prometaphase I, prior to the meiotic commitment point (spindle length of 0.73 µm). C) Cdc5-sfGFP is present in prometaphase I (spindle length of 2.38 µm).
Figure 4.
Altering NDT80 expression results in inappropriately uncommitted cells.
A) Diagram of the experimental method. Cells in microfluidic chambers are first exposed to sporulation medium. The cells enter meiosis and arrest at pachytene (due to the lack of NDT80 expression). Beta-estradiol is added to induce NDT80 and the cells exit prophase I. Complete medium is added to cells in different chambers at one of the 10-minute timepoints shown after the addition of estradiol. Cell Cycle outcomes are recorded. B) Graph of the percent of cells at each cell-cycle outcome (y-axis) upon complete medium addition at timepoints after SC disassembly (in minutes) (x-axis). Cells that returned to mitosis are in blue. Cells that finished meiosis are in red. Cells that were inappropriately uncommitted are in dark grey. Cells that arrested are in brown and green. C) Time-lapse images from a cell that is inappropriately uncommitted. The cell expresses GFP-Tub1. Complete medium was added to the cell in metaphase I (timepoint with grey arrowhead). The cell underwent meiosis I, assembled two spindles, budded and then underwent nuclear division, in which two nuclei divided (Scale bar - 3 µm).
Figure 5.
Decreasing the levels of Ndt80 results in fewer cells that commit to meiosis.
A) Analysis of Ndt80 protein levels in PGAL1,10NDT80/PGAL1,10NDT80 cells. Beta-estradiol was added to cells in prophase I to induce the expression of NDT80. After 110 minutes of Ndt80 expression (t = 0 minutes), complete medium without beta-estradiol (complete medium) or sporulation medium with beta-estradiol (sporulation medium) was added to the cells and protein was collected every 30 minutes for 150 minutes. Western blot was performed using anti-Ndt80 and anti-Nop1 (loading control) antibodies. B) Analysis of Ndt80 protein levels in PNDT80NDT80/PNDT80NDT80 and PGAL1,10NDT80/PGAL1,10NDT80 cells. Beta-estradiol was added to cells in prophase I to induce the expression of NDT80. After 110 minutes of Ndt80 expression, complete medium without beta-estradiol (complete medium) was added to the cells (t = 0 minutes) and protein was collected every 15 minutes for 30 minutes. Western blot was performed using anti-Ndt80 and anti-Nop1 (loading control) antibodies. C) Comparison plots of PGAL1,10NDT80/PGAL1,10NDT80 and PGAL1,10NDT80/ndt80Δ strains. Graphs show the percent of cells with each cell cycle outcome (y-axis) upon nutrient addition at different meiotic stages (x-axis). Cells that returned to meiosis are in blue. Those that finish meiosis are in red. Cells that were inappropriately uncommitted are in dark grey. Cells that arrested are in brown or green. D) Analysis of Ndt80 protein levels in different strain backgrounds. Western blot was performed using anti-Ndt80 and anti-Nop1 (loading control) antibodies. E) Plot of the PNDT80-MSE1Δ,MSE2Δ-NDT80 strain, in which positive feedback in the NDT80 promoter was deleted. Graphs show the percent of cells with each cell cycle outcome (y-axis) upon nutrient addition at different meiotic stages (x-axis). Cells that returned to meiosis are in blue. Cells that were inappropriately uncommitted are in dark grey. Cells that arrested in meiosis I are in green.
Figure 6.
Disruption of positive feedback in NDT80 expression results in inappropriately uncommitted cells.
A-C) Time-lapse images of PNDT80-MSE1ΔMSE2ΔNDT80/PNDT80-MSE1ΔMSE2ΔNDT80 cell expressing Spc42-mCherry, Zip1-GFP, and GFP-Tub1 (Scale bar – 3 µm). A) A PNDT80-MSE1ΔMSE2ΔNDT80/PNDT80-MSE1ΔMSE2ΔNDT80 cell that buds and returns to mitosis upon complete medium addition (grey arrowhead) in metaphase I. B) A PNDT80-MSE1ΔMSE2ΔNDT80/PNDT80-MSE1ΔMSE2ΔNDT80 cell that is inappropriately uncommitted. Complete medium was added (grey arrowhead) to the cell at anaphase I spindle breakdown. Cell finished meiosis I, assembled two spindles, budded, and underwent nuclear division, dividing one nucleus in the mother cell and one nucleus across the bud neck. C) A PNDT80-MSE1ΔMSE2ΔNDT80/PNDT80-MSE1ΔMSE2ΔNDT80 cell initiated meiosis and arrested at metaphase II.
Figure 7.
Deletion of one copy of NDT80 shifts the meiotic commitment point.
Graph showing the percent of NDT80/ndt80Δ cells at each cell cycle outcome (y-axis) with complete medium addition at the different stages of meiosis (x-axis). Cells that returned to mitosis are in blue. Cells that finished meiosis are in red. Cells that were inappropriately uncommitted are shown in dark grey. Cells that arrest are in green or brown.
Figure 8.
Cells that were inappropriately uncommitted are multinucleate and have an increase in ploidy.
A-C) Immunostaining of inappropriately uncommitted cells. The spindle is shown in red with anti-tubulin antibody. The LacO array on chromosome III is marked in green with LacI-GFP and anti-GFP antibody. And, the nucleus is shown in blue with DAPI (Scale bar – 2 µm). There are three nuclear segregation phenotypes. A) Both nuclei divided across the bud neck, resulting in two nuclei in the mother cell and two nuclei in the bud. B) One nucleus divided across the bud neck and one nucleus divided in the mother cell, resulting in three nuclei in the mother cell and one nucleus in the bud. C) One nucleus divided in the mother cell and one nucleus divided in the bud, resulting in two nuclei in the mother cell and two nuclei in the bud. D) Fluorescence microscopy of an inappropriately uncommitted cell that underwent a first division. Mcm7-GFP is shown in green. The spindle is shown in red with mCherry-Tub1 (Scale bar – 3 µm).