Polo kinase Cdc5 is a central regulator of meiosis I

Abstract

During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome segregation. CDC5 also establishes conditions for centromeric cohesin removal during meiosis II by promoting the degradation of Spo13, a protein that protects centromeric cohesin during meiosis I. Despite CDC5's central role in meiosis I, the protein kinase is dispensable during meiosis II and does not even phosphorylate its meiosis I targets during the second meiotic division. We conclude that Cdc5 has evolved into a master regulator of the unique meiosis I chromosome segregation pattern.

Fig. 1.

CDC5 is required for Rec8 cleavage. (A) Cells depleted for CDC20 (A27808) or CDC5 (A27809) were induced to sporulate. Rec8-S179 phosphorylation and total Rec8-HA levels were determined at the indicated times. (B) Wild-type (A33368) and CUP-CDC5 (A32851) cells were arrested in prophase I by depleting Ndt80. CDC5 expression was induced by 50 μM CuSO₄ addition 4 h after transfer into sporulation medium to examine Rec8–S179 phosphorylation and total Rec8-V5 protein levels. A vegetative no-tag control (A4962) is shown. (C) HA-tagged REC8 phosphomimetic mutants (A30411, A30407, A30408, A32252, A30409, A32254, A32256, A32250, A32258, A30410, and A30406) were induced to sporulate and GFP dot distribution was analyzed. One hundred cells were counted for three experiments. Statistical analyses are shown in Table S1. (D) spo11∆ CDC20-mn (A33493, filled squares) or spo11∆ CDC20-mn rec8-S136D S179D S197D-HA (A33491, filled circles) were sporulated. The percentage of cells in metaphase II was quantified (n = 100 cells per time point). (E) Wild-type (A33293), ama1Δ (A33119), CDC5-mn (A33292), or ama1Δ CDC5-mn (A33118) cells were sporulated and Rec8-HA and Pgk1 (loading control) protein levels analyzed.

Fig. 2.

Cells overexpressing CDC5 undergo a mixed meiosis I. (A) Wild-type (A31020) or CUP-CDC5 (A32746) cells were sporulated. The percentage of wild-type or CUP–CDC5 cells with stretched nuclei (squares), two nuclei (circles), and four nuclei (triangles) is shown (n = 100 cells per time point). (B) Wild-type (A31020) and CUP-CDC5 (A32746) cells were sporulated and followed by live cell microscopy (n = 75 cells). A representative montage for wild type (Upper) and CUP-CDC5 (Lower) is shown.

Fig. 3.

Cdc5 regulates Spo13 stability. (A) Wild-type (A31020), CUP-CDC5 (A32746), or spo13∆ (A30960) cells were sporulated and the percentage of metaphase I cells in wild type, spo13∆, and CUP–CDC5 was determined (n = 100 cells per time point). (B) Wild-type (A33501) or CUP-CDC5 (A33497) cells were sporulated using the Ndt80 block-release system and Spo13 levels examined. The peaks of meiosis I (MI) and meiosis II (MII) are indicated. Meiotic progression is shown in Fig. S 4 A and B. (C) Wild-type (A23405), CDC5-mn (A23757), and CDC20-mn (A23664) cells were sporulated using the Ndt80 block-release system. Spo13 levels were analyzed. Meiotic progression is shown in Fig. S4C.

Fig. 4.

CDC5 is dispensable during meiosis II. Wild-type (A22132) or cdc5-as1 (A33513) cells were sporulated using the Ndt80 block-release system and meiotic progression was assessed (n = 100 cells per time point). The cdc5-as1 inhibitor CMK (chloromethylketone), 5 μM, was added at the following times: wild type: 1 h after release into the meiotic divisions and cdc5-as1: 1.5 h after release into the meiotic divisions. Percentage of metaphase II (A) and anaphase II (B) cells is shown (n = 100 cells per time point). Detailed meiotic progressions are shown in Fig. S5 A–D.

Fig. 5.

Cdc5 targets are phosphorylated in meiosis I but not meiosis II. (A) Rec8-S136 and Rec8-S179 phosphorylation was analyzed in A21230 cells that were sporulated using the Ndt80 block-release system. A rec8-17A-HA strain (17A; A21235) arrested in metaphase I was used as a control for the phospho-antibodies. (B and C) A twofold dilution series of samples from the 6.25-h time point (lanes 1–4), 7-h time point (lanes 5–8) from A and rec8-17A-HA metaphase I (lanes 9 and 10) and metaphase II (lanes 11 and 12) were analyzed for total Rec8 levels and Rec8-S136 and Rec8-S179 phosphorylation. Quantifications of lanes 1 and 5 are shown in C. (D) A23650 cells were sporulated using the Ndt80 block-release system to examine Clb1-V5 protein. (E and F) REC8-HA (A22804) or rec8-29A-HA (A22803) mutants were sporulated using the Ndt80 block-release system to examine meiotic progression. (G) spo11∆ REC8-HA (A33469) and spo11∆ rec8-29A-HA (A33453) mutants were sporulated using the Ndt80 block-release system to examine meiotic progression.