Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

Abstract

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/C(Cdc20)), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.

Figure 1.

Dynamics of the spindle, the chromosome, and Cdc13 at meiosis in the wild type. (A and B) Spindle behavior at MI and MII. Graphs show changes in spindle length. In the graph in B, lengths of the two MII spindles are shown as blue and red lines. (C) An approximate map of GFP-visualized loci on three chromosomes used in this study. (D and E) Behavior of homologous centromeres (cen2; white, arrowheads) and the SPB (yellow) at MI. The graph shows changes in distance between two SPBs (D3) and between one of the SPBs and each centromere (D1 and D2). (F) Behavior of the arm locus (ade8; bottom, white, arrowheads, and graph) and the SPB (yellow) at MI. The graph shows the mean time of sister locus separation. Time 0 is PIII onset. (G) Behavior of sister centromeres (arrowheads) at MII. Arrowheads in enlarged images highlight transient sister centromere separation before anaphase. The graph shows changes in SPB-cen (D1 and D2) and SPB-SPB (D3) distances at MII. (H) Centromeres were visualized by Mis6-GFP and the spindle was visualized by mDsRed-α2-tubulin at MII (8 min before anaphase). (right) Images are enlarged views of Mis6 dots (arrows). (I) Dynamics of Cdc13-GFP at meiosis in the wild type. White lines in images indicate cell shapes. Dotted lines in graphs show boundaries of the spindle phases. Error bars indicate standard deviation. PI, phase I; PII, phase II; PIII, phase III. Bars: (A, B, and D–H) 5 μm; (G, inset) 2 μm.

Figure 2.

Dynamics of the chromosome, the spindle, and Cdc13 at MI in rec12 mutant. (A) Behavior of homologous centromeres and the SPB at MI. Arrowheads indicate the homologous centromeres (cen2). Bottom panels highlight independent oscillations of the homologous centromeres (white and red arrowheads) at phase II. The graph shows changes in the SPB-cen (D1 and D2) and SPB-SPB (D3) distances. (B) Behavior of the MI spindle in rec12. The arrow in the top graph shows the sudden regression of the spindle. Arrowheads indicate the thin spindle midzone. Dotted lines in graphs show boundaries of the spindle phases. PI, phase I; PII, phase II; PIII, phase III. (C) Duration of spindle phases. (D) Dynamics of Cdc13-GFP at MI. (E) Timing of arm locus separation at MI. Bars show time of separation of the ade6, ade8, and sod2 loci after spindle formation. At least seven cells were examined for each locus. Wt, wild type. Error bars indicate standard deviation. Bars: (A, top, B, and D) 5 μm; (A, bottom) 2 μm.

Figure 3.

Dynamics of the chromosome and the spindle at MII in rec8 and clr4 mutants. (A) Behavior of sister centromeres (white and red arrowheads) and the SPB (yellow) at MII in rec8 (top left and right) and clr4 (bottom left and right) mutants. The graph shows changes in the SPB-cen (D1 and D2) and SPB-SPB (D3) distances. PI, phase I; PII, phase II; PIII, phase III. Bar, 2 μm. (B) Mean distances of centromeres at phase II. More than eight pairs of centromeres were examined for each analysis. (C) Changes in spindle length at MII in the rec8 (left) and clr4 (right) mutants. Graphs show kinetics of two MII spindles in the same cell. Dotted lines in graphs show boundaries of the spindle phases. (D) Duration of the spindle phases at MII. (E) Duration of MI, MII, and the MI–MII interval. Wt, wild type. Error bars indicate standard deviation.

Figure 4.

Changes in the amount of nuclear Cdc13. (A) Changes in Cdc13-GFP amount in the nucleus at meiosis. After first nuclear division, the amount is shown as the total amount in the two nuclei. (B) Effect of Fzr1 depletion on changes in nuclear Cdc13-GFP amount at MI. Arrows show disappearance of Cdc13-GFP from the spindle. Gray areas show anaphase determined by Cdc13 disappearance from the spindle and spindle behavior. Time 0 is the start of MI spindle formation. Dotted lines in graphs show boundaries of different meiotic stages.

Figure 5.

Functional relationship of Mes1 with Fzr1 and Slp1. (A) The population of cells that underwent MI and MII. MI and MII were judged by the formation of the MI and MII spindles, respectively. 21, 18, and 17 cells were examined for the wild type, mes1, and other mutant strains, respectively. (B) Duration of the spindle phases in the mes1 mutants. Wt, wild type. *, results of 4 spindles; **, results of 10 spindles. Error bars indicate standard deviation.