Synchronized fission yeast meiosis using an ATP analog-sensitive Pat1 protein kinase

Abstract

Synchronous cultures are often indispensable for studying meiosis. Here we present an optimized protocol for induction of synchronous meiosis in the fission yeast Schizosaccharomyces pombe. Chemical inactivation of an ATP analog-sensitive form of the Pat1 kinase (pat1-as2) by adding the ATP analog 1-NM-PP1 in G1-arrested cells allows the induction of synchronous meiosis at optimal temperature (25°C). Importantly, this protocol eliminates detrimental effects of elevated temperature (34°C), which is required to inactivate the commonly used temperature-sensitive Pat1 kinase mutant (pat1-114). The addition of the mat-Pc gene to a mat1-M strain further improves chromosome segregation and spore viability. Thus, our protocol offers highly synchronous meiosis at optimal temperature, with most characteristics similar to those of wild-type meiosis. The synchronization protocol can be completed in 5 d (not including strain production, which may take as long as 2 or 3 months).

Figure 1. Zygotic and azygotic meiosis in the fission yeast S. pombe

(a) Scheme of zygotic meiosis. Haploid cells of the opposite mating type (h⁺ and h⁻) mate to form diploid zygotes, which undergo two meiotic divisions (this is called zygotic meiosis) and generate asci, typically curved, with four haploid spores. (b) Scheme of azygotic meiosis. Diploid cells heterozygous at the mating type locus (h⁺/h⁻) enter meiosis and generate azygotic asci, typically straight, with four haploid spores. (c) DIC (differential interference contrast) images and DAPI staining of azygotic asci from wild-type diploid h⁺ /h⁻ strain (JG16539) and zygotic asci from wild-type h⁹⁰ strain (JG11355). (d) Regulation of initiation of meiosis in the fission yeast S. pombe. Mei3 is expressed upon activation of the mating pheromone signaling cascade (MAPK) and starvation for nitrogen. Mei3 inhibits Pat1 kinase, and hence liberates Mei2 from its inhibitory phosphorylation. Additional factors, including the Ste11 transcription factor, are also important.

Figure 1. Zygotic and azygotic meiosis in the fission yeast S. pombe

(a) Scheme of zygotic meiosis. Haploid cells of the opposite mating type (h⁺ and h⁻) mate to form diploid zygotes, which undergo two meiotic divisions (this is called zygotic meiosis) and generate asci, typically curved, with four haploid spores. (b) Scheme of azygotic meiosis. Diploid cells heterozygous at the mating type locus (h⁺/h⁻) enter meiosis and generate azygotic asci, typically straight, with four haploid spores. (c) DIC (differential interference contrast) images and DAPI staining of azygotic asci from wild-type diploid h⁺ /h⁻ strain (JG16539) and zygotic asci from wild-type h⁹⁰ strain (JG11355). (d) Regulation of initiation of meiosis in the fission yeast S. pombe. Mei3 is expressed upon activation of the mating pheromone signaling cascade (MAPK) and starvation for nitrogen. Mei3 inhibits Pat1 kinase, and hence liberates Mei2 from its inhibitory phosphorylation. Additional factors, including the Ste11 transcription factor, are also important.

Figure 1. Zygotic and azygotic meiosis in the fission yeast S. pombe

(a) Scheme of zygotic meiosis. Haploid cells of the opposite mating type (h⁺ and h⁻) mate to form diploid zygotes, which undergo two meiotic divisions (this is called zygotic meiosis) and generate asci, typically curved, with four haploid spores. (b) Scheme of azygotic meiosis. Diploid cells heterozygous at the mating type locus (h⁺/h⁻) enter meiosis and generate azygotic asci, typically straight, with four haploid spores. (c) DIC (differential interference contrast) images and DAPI staining of azygotic asci from wild-type diploid h⁺ /h⁻ strain (JG16539) and zygotic asci from wild-type h⁹⁰ strain (JG11355). (d) Regulation of initiation of meiosis in the fission yeast S. pombe. Mei3 is expressed upon activation of the mating pheromone signaling cascade (MAPK) and starvation for nitrogen. Mei3 inhibits Pat1 kinase, and hence liberates Mei2 from its inhibitory phosphorylation. Additional factors, including the Ste11 transcription factor, are also important.

Figure 2. Synchronous meiosis induced by inactivation of Pat1

(a) Flowchart of the pat1 mutant-induced synchronous meiosis. Cells carrying the pat1-as2 or pat1-114 allele are grown in liquid YES medium to mid-log phase. The cells are collected by centrifugation, resuspended in liquid EMM2 medium lacking a nitrogen source (EMM2-NH₄Cl) and incubated at 25 °C to arrest cells in G₁ phase. Subsequently, the cells are resuspended in fresh EMM2 medium containing a nitrogen source and synchronous meiosis is induced in pat1-as2 cells by adding the inhibitor 1-NM-PP1. Alternatively, Pat1-as2 (or Pat1-114) can be inactivated by shifting the cells to non-permissive temperature 34 °C. (b) Progression of meiosis in diploid strains pat1-114/pat1-114 (JG12209), pat1-as2/pat1-as2 (JG15620), pat1- 114/pat1-114 mat-Pc (JG16328) and pat1-as2/pat1-as2 mat-Pc (JG16113). Cells were cultured to mid-log phase in YES-Ade medium, transferred to EMM2-NH₄Cl medium for 16 h at 25 °C (pat1-114 and pat1-as2) or for 7 h at 25 °C (pat1-114 mat-Pc and pat1-as2 mat- Pc) to synchronize cells in G₁, transferred to EMM2 medium, and incubated at 34 °C or kept at 25 °C with addition of 25 µM 1-NM-PP 1 to inactivate the Pat1-as2 kinase. Progression of meiosis was monitored by flow cytometry for DNA content and by fluorescence microscopy for number of nuclei per cell in samples collected at the indicated time-points after temperature-shift or addition of 1-NM-PP 1.

Figure 2. Synchronous meiosis induced by inactivation of Pat1

(a) Flowchart of the pat1 mutant-induced synchronous meiosis. Cells carrying the pat1-as2 or pat1-114 allele are grown in liquid YES medium to mid-log phase. The cells are collected by centrifugation, resuspended in liquid EMM2 medium lacking a nitrogen source (EMM2-NH₄Cl) and incubated at 25 °C to arrest cells in G₁ phase. Subsequently, the cells are resuspended in fresh EMM2 medium containing a nitrogen source and synchronous meiosis is induced in pat1-as2 cells by adding the inhibitor 1-NM-PP1. Alternatively, Pat1-as2 (or Pat1-114) can be inactivated by shifting the cells to non-permissive temperature 34 °C. (b) Progression of meiosis in diploid strains pat1-114/pat1-114 (JG12209), pat1-as2/pat1-as2 (JG15620), pat1- 114/pat1-114 mat-Pc (JG16328) and pat1-as2/pat1-as2 mat-Pc (JG16113). Cells were cultured to mid-log phase in YES-Ade medium, transferred to EMM2-NH₄Cl medium for 16 h at 25 °C (pat1-114 and pat1-as2) or for 7 h at 25 °C (pat1-114 mat-Pc and pat1-as2 mat- Pc) to synchronize cells in G₁, transferred to EMM2 medium, and incubated at 34 °C or kept at 25 °C with addition of 25 µM 1-NM-PP 1 to inactivate the Pat1-as2 kinase. Progression of meiosis was monitored by flow cytometry for DNA content and by fluorescence microscopy for number of nuclei per cell in samples collected at the indicated time-points after temperature-shift or addition of 1-NM-PP 1.

Figure 3. Sensitivity of cells expressing Pat1-as2 to ATP-analogs

(a) Serial dilutions of wild-type cells (pat1⁺) (JG15458) or pat1-114 (JG11322), pat1-as1 (JG15404) or pat1-as2 (JG15403) cells as well as diploid cells pat1-114/pat1-114 (JG15710), pat1-as2/pat1-as2 (JG16022) and pat1-as2/pat1-as2 matPc (JG16113) were spotted on YES plates containing or lacking the indicated ATP-analogs (25 µM 3-BrB-PP1 or 1-NM-PP1) and grown for 2–3 days at 25 °C or 34 °C as indicated. Colonies of cells with suppressors of pat1 alleles can be seen on YES plate at 34 °C. (b) DIC (differential interference contrast) images and DAPI staining of asci from strains described in (a).

Figure 3. Sensitivity of cells expressing Pat1-as2 to ATP-analogs

(a) Serial dilutions of wild-type cells (pat1⁺) (JG15458) or pat1-114 (JG11322), pat1-as1 (JG15404) or pat1-as2 (JG15403) cells as well as diploid cells pat1-114/pat1-114 (JG15710), pat1-as2/pat1-as2 (JG16022) and pat1-as2/pat1-as2 matPc (JG16113) were spotted on YES plates containing or lacking the indicated ATP-analogs (25 µM 3-BrB-PP1 or 1-NM-PP1) and grown for 2–3 days at 25 °C or 34 °C as indicated. Colonies of cells with suppressors of pat1 alleles can be seen on YES plate at 34 °C. (b) DIC (differential interference contrast) images and DAPI staining of asci from strains described in (a).