Abstract
In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast.
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Introduction
Meiosis is a specialized type of cell division that generates haploid gametes from a diploid cell. In contrast to mitosis, the molecular mechanisms involved in regulating meiosis are much less understood. During meiosis, two rounds of chromosome segregation follow a single round of DNA replication. The switch from mitotic to meiotic cell cycle and the existence of two consecutive rounds of chromosome segregation during the first (meiosis I) and the second meiotic division (meiosis II) without an intervening S phase depend on multiple control mechanisms. These mechanisms include meiosis-specific gene expression and translation, RNA stability control, expression of antisense transcripts, targeted proteolysis, and post-translational modifications of proteins (PTMs)1−7.
Meiosis-specific expression of proteins plays a pivotal role in coordinating key events during meiosis. For example, during the early stages of meiosis, homologous chromosomes need to align in pairs to facilitate the exchange of genetic material through crossing over. In most organisms, these alignments are facilitated by proteins of a synaptonemal complex (SC). The SC helps to stabilize the interactions between homologous chromosomes, facilitating the formation of DNA double-strand breaks (DSBs) and subsequent strand exchange between homologous chromatids8−10. In contrast to other organisms, the fission yeast S. pombe does not form an SC, instead, it develops specific structures known as linear elements (LinEs). Similar to the SC, the LinEs are essential for achieving full meiotic pairing of homologous chromosomes and facilitating meiotic recombination11−13. Furthermore, meiosis-specific recombinase DMC1 and recombinase RAD51 facilitate the recombination events between homologous chromosomes, promoting the exchange of genetic material between them14,15. Another protein whose expression is known to play a crucial role during meiosis is the meiosis-specific subunit of the cohesin complex REC8. It has been shown that timely expression of REC8 during meiosis is crucial for the replacement of its mitotic cohesin counterpart RAD21 in the cohesin complexes along the entire chromosomes16. Furthermore, the cleavage of REC8 in cohesin complexes along chromosome arms during meiosis I allows to segregate the homologous chromosomes in meiosis I and the cleavage of REC8 in cohesin complexes around centromeric regions facilitates segregation of sister chromatids in meiosis II17−20.
Regarding the regulation of meiotic divisions by PTMs, it has been found that multiple kinases, including ATM, ATR, CHK2, Cdc28, DDK, CK1, and PLK, phosphorylate meiosis-specific proteins21−27. On the other hand, protein phosphatases PP1, PP2, and PP4, counteract these kinases to ensure a dynamic balance of phosphorylation events required for proper regulation of chromosome synapsis, recombination, repair of meiotic DSBs, regulation of SC assembly and chromosome segregation28−31. For example, in S. cerevisiae, the coordination between premeiotic DNA replication and the formation of meiotic DSBs involves the phosphorylation of the Spo11-accessory protein Mer222,32. Similarly, the DSB factor Rec104 was shown to be phosphorylated during meiosis, providing a further link between premeiotic DNA replication and the formation of meiotic DSBs33. In C. elegans, meiotic chromosome pairing and movement were shown to be initiated by CHK2-dependent phosphorylation34−36. Similarly, proteins involved in meiotic recombination that are part of the SC have been found to be regulated by phosphorylation26,37,38. Importantly, in S. pombe, Sgo1 and PP2A were found to antagonize CK1-dependent phosphorylation of the meiosis-specific cohesin subunit Rec8 to protect cohesion cleavage at the centromeres39. Furthermore, phosphorylation of Rec8 and Psm3 by Moa1-Plo1 was shown to potentiate the activity of PP2A associated with Sgo1, thus preventing the cleavage of Rec8 by separase and regulating Rec8 cohesin complexes to establish cohesion required for mono-orientation of sister kinetochores17,40−42.
The fission yeast S. pombe shares many molecular, genetic, and biochemical features with multicellular organisms, and many of its proteins are evolutionarily conserved43,44. Generally, entry of S. pombe into meiosis requires both nutrient starvation (especially nitrogen) and proper cell ploidy. Upon nitrogen starvation, S. pombe cells become arrested at the G1phase, and cells with the opposite mating types conjugate to produce a diploid zygote. Expression of the mating type-specific genes of both mating types leads to expression of meiosis inducing protein Mei3, which inactivates Pat1, a protein kinase that acts as a meiotic repressor, leading to activation of the RNA-binding protein Mei2 and the induction of meiosis45. Based on these findings, a strategy for inducing highly synchronous meiosis in S. pombe by combining the nitrogen starvation with the inactivation of a temperature sensitive allele of Pat1 protein kinase (pat1-114) has been developed. Although highly synchronous meiosis in S. pombe can be achieved using the pat1-114 allele, it was found that pat1-114-induced meiosis has many defects, including a reduced frequency of meiotic recombination, aberrant nuclear positioning of centromeres and defects in mono-orientation of sister kinetochores during meiosis I46−48. Some of these defects could be linked to the absence of mating pheromone signaling or to the higher temperature needed to inactivate Pat1-114 kinase46,47,49. To eliminate the negative effects of higher temperature needed to induce synchronous meiosis in pat1-114 cells, we recently introduced a novel conditional ATP analog-sensitive allele of Pat1 protein kinase, referred to as pat1-as2. The pat1-as2 allele allows induction of meiosis at optimal temperature (25 °C) by inhibiting Pat1-as2 kinase using an ATP analog 1-NM-PP1. Importantly, the pat1-as2-induced meiosis has improved fidelity of chromosome segregation during meiosis I and leads to higher spore viability, compared to pat1-114-induced meiosis50−53.
It has been shown that in S. pombe, hundreds of genes are regulated during meiosis in four waves of transcription, with each wave associated with a particular stage of meiosis. These groups of genes correspond to nitrogen-starvation and stress response, early meiosis, middle meiosis and late meiosis-sporulation7. Additionally, analyses of S. pombe meiotic proteomes have revealed that levels of many proteins increase or decrease during the progression of meiosis, while the levels of other proteins remain constant. Moreover, reductions in proteins involved in anabolism and increases in proteins involved in catabolism were observed, suggesting that during meiosis, many of the changes in steady-state protein levels are post-transcriptional54,55.
Despite the findings that progression of S. pombe cells through meiosis is regulated on multiple levels, including PTMs, only few studies analyzed the changes in protein phosphorylation during meiosis56. In this study, we utilized TMTpro 18plex labeling and phosphopeptide enrichment strategies, followed by LC-MS analysis, and compared the expression levels and phosphorylation of proteins in mitotic S. pombe cells with cells progressing through meiosis at optimal (25 °C, pat1-as2), and higher (34 °C, pat1-114) temperatures.
Materials and methods
Chemicals, reagents and kits
The following chemicals and reagents were used: 1-NM-PP1 (4-Amino-1-tert-butyl-3-(1’-naphthylmethyl)pyrazolo[3,4-d]pyrimidine; Toronto Research Chemicals, Toronto, Canada), DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride; Sigma, St. Louis, MO, USA), SYTOX Green (Molecular Probes, Eugene, OR, USA), VECTASHIELD mounting medium for fluorescence (Vector Laboratories, Newark, CA, USA), RNase-free DNase I (New England BioLabs, Ipswich, MA, USA), ProtoScript II first strand cDNA synthesis kit (New England BioLabs, Ipswich, MA, USA), and protease inhibitors (cOmplete, MiniProtease inhibitor cocktail, Roche). Pierce peptide desalting spin columns, TMTpro 18-plex labeling reagents, Pierce quantitative colorimetric peptide assay, High-Select TiO2 phosphopeptide enrichment kit, High-Select Fe-NTA phosphopeptide enrichment kit, and high pH RP fractionation kits were purchased from Thermo Fisher (Thermo Fisher, Waltham, MA, USA). All other chemicals were of analytical or molecular biology grade and obtained from Sigma (Sigma, St. Louis, MO, USA).
Strains and media
The genotypes of the S. pombe strains were as follows: pat1-114 (h−/h−ade6-M210/ade6-M216 pat1-114/pat1-114) and pat1-as2 (h−/h−ade6-M210/ade6-M216 pat1::NatR/pat1::NatRpat1-as(L95A)::HygR/pat1-as(L95A)::HygR). The cells were grown in the following media: YE + 4 S-Ade liquid medium (5.0 g/l yeast extract, 3.0% (w/v) glucose, 0.1 g/l L-leucine, 0.1 g/l L-lysine hydrochloride, 0.1 g/l L-histidine, 0.1 g/l uracil); EMM2-NH4Cl liquid medium (3.0 g/l potassium hydrogen phthalate, 2.2 g/l Na2HPO4, 1.0% (w/v) glucose, supplemented with salts, vitamins and minerals); and EMM2 liquid medium (3.0 g/l potassium hydrogen phthalate, 2.2 g/l Na2HPO4, 5.0 g NH4Cl, 1.0% (w/v) glucose, supplemented with salts, vitamins and minerals). Salts, vitamins and minerals were as described in Anrather et al.57. Solid media were supplemented with 2% agar.
Induction of synchronous meiosis
To induce synchronous meiosis, diploid strains of pat1-114 and pat1-as2 were grown in YE + 4 S-Ade liquid medium at 25 °C until the OD600 reached 0.55. The exponentially growing mitotic cells were then collected by centrifugation (3000 g, 2 min, 25 °C), resuspended in EMM2-NH4Cl medium prewarmed to 25 °C, and incubated at 25 °C for 16 h to arrest cells in G1 phase. Afterward, the cells were collected by centrifugation (3000 g, 2 min, 25 °C) and induced into meiosis. Specifically, the pat1-114 cells were resuspended in fresh EMM2 medium prewarmed to 34 °C, and pat1-as2 cells were resuspended in fresh EMM2 medium prewarmed to 25 °C and supplemented with 25 µM 1-NM-PP1. Subsequently, the cells were incubated at 34 °C (pat1-114) or 25 °C (pat1-as2).
Analysis of progression of cells into meiosis
The progression of cells into meiosis was monitored through fluorescence microscopy and flow cytometry, as well as by analyzing the expression and splicing of reference meiotic genes using RT-PCR. For counting nuclear divisions and flow cytometry analysis, 0.5 ml of culture at the specific time-points was collected by centrifugation (3000 g, 2 min, 4 °C) and fixed in 0.7 ml of 70% ethanol at 4 °C overnight. Nuclear divisions were monitored by counting the number of nuclei after DAPI staining of the fixed cells using fluorescence microscopy (Olympus BX51 fluorescence microscope, OLYMPUS, Tokyo, Japan). The cell cycle profile was analyzed by flow cytometry after staining the fixed cells with SYTOX Green dye. Briefly, the fixed cells were washed with 50 mM sodium citrate buffer and incubated with 0.1 mg/ml of RNase A in 50 mM sodium citrate buffer for 2 h at 37 °C. The samples were then supplemented with SYTOX Green dye (final concentration of 1 µM). Flow cytometry measurements were performed using a FACS Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The SYTOX Green dye was excited with a 488 nm laser, and fluorescence emission was measured using a bandpass filter set of 530 nm. Forward/side light scatter characteristics were used to exclude cell debris from the analysis. For each analysis, 10.000 cells were acquired. The flow cytometry results were analyzed using FlowJo v10.8.1 software (BD Life Sciences, Franklin Lakes, NJ, USA).
To analyze the expression and splicing of reference meiotic genes, 50 ml of culture at the specific time-points was centrifuged (3000 g, 2 min, 4 °C), and total RNA was isolated using the TES (10 mM Tris, pH 7.5, 10 mM EDTA, pH 8.0, 0.5% SDS) and phenol/chloroform (5:1) and chloroform/isoamyl alcohol (24:1) extractions. Subsequently, 1 µg of RNA was incubated with RNase-free DNase I at 37 °C for 30 min and transcribed to cDNA using the ProtoScript II first strand cDNA synthesis kit following the manufacturer´s instructions. The following primers were used for RT-PCR: 5´-caaagtcctaattgcagtgtttcgg-3´ and 5´-ggaggatggatctcttcatacttgc-3´ for amplification of rem1, and 5´-atcagtgttagatttgacacaagtc-3´ and 5´-gtccataaacgtctccaagagatcg-3´ for amplification of mde3. The PCR protocol consisted of initial denaturation (5 min, 95 °C), followed by 30 repeats of 30 s denaturation at 95 °C, 30 s annealing at 60 °C, 30 s extension at 72 °C, and 10 min final extension at 72 °C. The PCR amplicons were analyzed by electrophoretic separation using a 2% agarose gel.
Collection of cells and yeast cell powder preparation
Exponentially growing mitotic cells (OD600 = 0.6−0.8), and cells around meiosis I and meiosis II, were collected by centrifugation (3000 g, 2 min, 4 °C). After removing the supernatant, the yeast cell pellets were snap-frozen in liquid nitrogen and ground into a cell powder using the Freezer/Mill cryogenic grinder cooled with liquid nitrogen (7 cycles, pre-cool 10 min, run 3 min, cool 2 min, rate 15 CPS) (6775 Freezer/Mill, SPEX Sample Prep, Metuchen, NJ, USA).
Sample preparation for proteome and phosphoproteome analyses
For LC-MS/MS analysis, 100 mg of yeast cell powder was resuspended in ice-cold lysis buffer (8 M urea, 25 mM Tris-HCl, pH 8.5, 100 mM TEAB, pH 8.5, 150 mM NaCl) containing phosphatase inhibitors (2.5 mM β-glycerol phosphate, 1 mM KF, 1 mM Na3VO4, and 1 mM Na2H2P2O7), and protease inhibitors (cOmplete, MiniProtease Inhibitor Cocktail). The concentration of proteins in each of the 18 samples was measured by BCA assay, and aliquots of 300 µg were taken for further analysis. Digestion was performed using the single-pot, solid-phase enhanced sample preparation (SP3) method58. Samples were reduced with 10 mM DTT (1 h, 56 °C), and proteins were alkylated with 55 mM iodoacetamide (30 min, RT). Proteins were bound to Sera-Mag beads (GE Healthcare) at a beads-to-protein ratio of 10:1. After washing with 80% ethanol and acetonitrile, proteins were resuspended in 50 mM AmBiC and digested with trypsin (1:25, w/w) for 16 h at 37 °C. The eluted peptides were acidified and desalted using Pierce peptide desalting spin columns. Before TMTpro labeling, the concentration of peptides in samples was measured using the Pierce quantitative colorimetric peptide assay. TMTpro labeling was performed according to the manufacturer’s instructions with minor adjustments. A total of 149 µg of peptides of each sample were labelled with TMTpro (TMT to peptide ratio of 3:1). After testing of the mixing ratio, labelling was stopped by adding 5% hydroxylamine in 100 mM TEAB, and samples were desalted using Pierce peptide desalting spin columns. For phosphopeptide enrichment, the High-Select TiO2 phosphopeptide enrichment and High-Select Fe-NTA phosphopeptide enrichment kits were used according to the manual. Eluates from both kits were pooled and desalted using Pierce peptide desalting spin columns. The prepared phosphoproteomic samples were fractionated using high pH RP fractionation. Fractions containing phosphopeptides were dried in a vacuum concentrator, and peptides were reconstituted in 0.1% TFA before LC-MS/MS analysis.
Offline fractionation of total proteome
The pooled samples were concentrated and desalted using a Pierce Peptide Desalting spin column. Eluates were dried in a vacuum concentrator and reconstituted in a 20 mM ammonia formate buffer (pH 10.0) before fractionation at basic pH. Two-dimensional liquid chromatography was performed by reverse-phase chromatography at high and low pH. In the first-dimension, peptides were separated on a Gemini-NX C18 (150 × 2 mm, 3 μm, 110 A, Phenomenex, Torrance, USA) in a 20 mM ammonia formate buffer (pH 10.0) and eluted over a 44 min gradient from 0 to 60% solvent B (90% acetonitrile, 20 mM ammonium formate, pH 10.0), followed by 5 min at 100% solvent B at 50 µl/min using an Ultimate 3000 RSLC micro system (Thermo Fisher Scientific) equipped with a fraction collector. Thirty-six fractions were collected in a time-based manner (every 30 s) between minute 11.5 and 57. Organic solvent was removed in a vacuum concentrator, and samples were reconstituted with 0.1% TFA.
LC-MS analysis of phosphoproteome and total proteome
Samples were analyzed on an Ultimate 3000 RSLC nano coupled directly to an Exploris 480 with FAIMSpro (both Thermo Fisher Scientific). The samples were injected onto a reversed-phase C18 column (50 cm x 75 μm i.d., packed in-house) and eluted with a gradient of 4–38% mobile phase B (90% acetonitrile, 0.4% formic acid) over 94 min, applying a flow rate of 230 nl/min. MS scans were performed in the range from m/z 375−1650 at a resolution of 120,000 (at m/z = 200). MS/MS scans were conducted for peptide identification and relative quantification of TMT reporter ions with the following parameters: resolution 30,000 with turboTMT for TMTpro reagent; normalized collision energy 33%; isolation width 0.7 m/z; dynamic exclusion 90 s. Two different FAIMS voltages were applied (-40 V and -60 V) with a cycle time of 1.5 s per voltage. FAIMS was operated in standard resolution mode with a static carrier gas flow of 4.6 l/min.
Data and statistical analysis
The MZXML files were extracted from the Thermo raw data files for each FAIMS voltage using FAIMS_MzXML_Generator v 1.0 and were subsequently processed using MaxQuant v 2.3.0.059 employing the built-in Andromeda search engine with the following parameters: 20 ppm peptide tolerance in the first search, 4.5 ppm in the main search upon recalibration, and 20 ppm fragment tolerance. Additionally, 1% peptide and protein false discovery rates (FDR) were set based on reverse decoy database search, along with the match between runs (MBR) feature. The specific parameters for searching included carbamidomethylation (C) and TMTpro on K and peptide N-term as permanent and oxidation (M), acetylation (protein N-term), and phosphorylation (S, T, Y) as variable modifications. Trypsin/P was specified as the proteolytic enzyme, allowing a maximum of two missed cleavage sites and requiring a minimum peptide length of seven amino acids. For the quantification of proteins, reporter ion intensities were utilized, and the search was performed against the target-decoy version of the Schizosaccharomyces pombe UP2485 proteome FASTA (UniProt).
Statistical analysis was performed using Perseus v1.6.15.060. The output tables containing protein groups and phosphorylation sites were filtered to exclude reverse proteins and contaminants. Moreover, low confidence proteins identified only by the site were filtered out from protein groups and the sites with the localization probability lower than 75% from the phosphosites. After log2 transformation of the reporter ion intensities, only proteins and phosphosites with three valid values in at least one experimental group were retained. Consequently, the missing values were imputed from the normal distribution. Principal component analysis (PCA) was employed to evaluate the variability among the biological replicates. An ANOVA test was performed with Permutation based FDR correction, setting a threshold FDR ≤ 0.05. Pairwise differences were evaluated using the Post hoc Tukey’s test at an FDR ≤ 0.05. The Z-score-normalized averages of reporter ion intensities were utilized for hierarchical clustering. For enrichment analysis, the STRING software tool was utilized61, and the results were visualized using the free online platform SRplot available at https://www.bioinformatics.com.cn/62.
Results
Induction of synchronous meiosis in S. pombe
To compare the expression and phosphorylation of proteins in exponentially growing mitotic cells, further referred to as mitotic cells (MIT), and cells progressing through meiotic divisions, diploid pat1-114 and pat1-as2 mutants of S. pombe were first grown exponentially and then arrested in the G1 phase using nitrogen starvation. Note that exponentially growing S. pombe cells are mostly in G2, the longest cell-cycle phase43. The meiosis was induced by either shifting the cell culture to 34 °C (pat1-114) or by adding the 25 µM 1-NM-PP1 and maintaining the cell culture at 25 °C (pat1-as2).
Flow cytometry and nuclei counting analyses confirmed a high level of synchrony in both pat1-114- and pat1-as2-induced meiosis. The pre-meiotic S phase and the entry of cells into meiosis I (MEI1) or meiosis II (MEI2) were delayed by about 2–4 h in pat1-as2 cells, compared to pat1-114 cells (Fig. 1A and B). This difference is in line with the previously observed delay in progression of pat1-as2 cells through meiosis at 25 °C50,51. Additionally, the delayed progression of pat1-as2 cells through meiotic divisions correlates with the expression of meiosis-specific genes, as illustrated by differences in timing of expression and splicing of genes encoding the meiosis-specific cyclin Rem1 and meiotic protein kinase Mde3 in pat1-114 cells (34 °C) and pat1-as2 cells (25 °C) (Fig. 1C)52,63,64.
Progression of diploid pat1-114 and pat1-as2 cells into meiosis. The cells were cultured in YE + 4 S-Ade medium, synchronized in the G1 phase using EMM2-NH4Cl medium, and then shifted into EMM2 medium prewarmed to 34 °C (pat1-114) or resuspended in EMM2 medium prewarmed to 25 °C with the addition of 25 µM 1-NM-PP1 (pat1-as2). The progression of cells into meiosis was monitored by flow cytometry (A), by counting the nuclei (B), and by RT-PCR of reference meiotic genes (C) from samples collected at the indicated time points. Samples for quantitative mass spectrometry analysis were collected at the time points indicated by the pink columns in the nuclei counting data (B). Mitotic cells – exponentially growing mitotic cells, MEI1 – meiosis I, MEI2 – meiosis II, n – number of nuclei, gDNA – genomic DNA, U – PCR amplicon of unspliced pre-mRNA, S – PCR amplicon of spliced mRNA.
For quantitative mass spectrometry analyses, exponentially growing mitotic cells (MIT, OD600 = 0.6–0.8), cells in the meiosis I (MEI1, 4.5 h for pat1-114 and 8.5 h for pat1-as2, Fig. 1B) and cells in the meiosis II (MEI2, 6 h for pat1-114 and 9.5 h for pat1-as2, Fig. 1B) were harvested and processed.
Quantitative proteomic analysis
Quantitative proteomic analysis was performed on samples collected from mitotic (T-MIT), and meiosis I (T-MEI1) and meiosis II (T-MEI2) stages of pat1-114 cells, and from mitotic (I-MIT), and meiosis I (I-MEI1) and meiosis II (I-MEI2) stages of pat1-as2 cells. Out of the total 5134 proteins of S. pombe, 4701 proteins were identified, with 4673 quantified (~ 91.6 of the S. pombe proteome) (Fig. 2A). Principal component analysis (PCA) explained 76.6% of the total variance between analyzed samples, clearly distinguishing between mitotic and meiotic samples (Fig. 2B). Specifically, PC1 explained 59.5% variance, discriminating between mitotic and meiotic samples of both pat1-114 and pat1-as2 samples, while PC2 explained 17.1% variance, discriminating between pat1-114 and pat1-as2 samples.
Consistent with the PC1 results, the heatmap of quantified proteins highlighted two main clusters: one with the mitotic samples of both pat1-114 and pat1-as2 (T-MIT and I-MIT), and the other grouping the meiosis I and meiosis II samples of pat1-114 and pat1-as2 (T-MEI1, T-MEI2, I-MEI1 and I-MEI2) (Fig. 2C). This second cluster was further divided into two subclusters: one with the meiosis I sample of pat1-114 (T-MEI1) and the other with the meiosis II sample of pat1-114 (T-MEI2) and the meiosis I and meiosis II samples of pat1-as2 (I-MEI1 and I-MEI2), which aligns with PC2 results.
To identify proteins with significant quantitative differences in protein levels between analyzed samples, a statistical analysis was performed using ANOVA test (FDR < 0.05). The significantly dysregulated proteins are highlighted in Table S1. When examining differentially abundant proteins between meiosis I and mitotic samples (MEI1 versus MIT), 396 proteins were more abundant, and 901 proteins were less abundant in the meiosis I sample of pat1-114. In contrast, 629 proteins were more abundant, and 728 proteins were less abundant in the meiosis I sample of pat1-as2. Notably, the protein levels of 91 proteins were upregulated, and 273 proteins were downregulated specifically in meiosis I of pat1-114, while the protein levels of 324 proteins were upregulated, and 100 proteins were downregulated specifically in meiosis I of pat1-as2, compared to mitotic samples. Finally, when meiosis II was compared with meiosis I (MEI2 versus MEI1), the protein levels of 244 proteins were found to be upregulated, and 3 proteins were less abundant in the meiosis II sample of pat1-114. In comparison, 117 proteins were more abundant, and 30 proteins were less abundant in the meiosis II sample of pat1-as2, compared to meiosis I samples. Interestingly, 155 proteins were upregulated specifically in meiosis II of pat1-114, while 28 proteins were upregulated, and 27 proteins were downregulated specifically in meiosis II of pat1-as2, compared to meiosis I samples (Fig. 2D, Table S1). To further analyze our proteomics data, we compared the detected changes in the protein levels with the changes in mRNA levels published by Mata et al.7. We found a high correlation between the changes in mRNA levels (UP- or DW-regulated) detected by Mata et al. for pat1-114-induced meiosis and the changes in protein levels detected in our study. Specifically, we observed 76% correlation for pat1-114 cells and 65% correlation for pat1-as2 cells for increased protein and mRNA levels, and 92% correlation for pat1-114 cells and 91% correlation for pat1-as2 cells for decreased protein and mRNA levels when comparing meiosis I and mitotic samples. Further, we observed 92% correlation for pat1-114 cells and 99% correlation for pat1-as2 cells for increased protein and mRNA levels, and 100% correlation for pat1-114 cells and 91% correlation for pat1-as2 cells for decreased protein and mRNA levels when comparing meiosis II and meiosis I samples (Table S1).
These findings indicate that despite considerable similarities in proteomes of pat1-114 and pat1-as2 cells, there are also differences in the expression of specific proteins when meiosis is induced by temperature inactivation of Pat1 kinase (pat1-114, 34 °C), and when meiosis is induced by inhibiting Pat1 kinase using an ATP analog (pat1-as2, 25 °C).
Proteomic profiling of the six subsets of S. pombe samples, including mitotic (T-MIT), meiosis I (T-MEI1), and meiosis II (T-MEI2) samples of pat1-114, and mitotic (I-MIT), meiosis I (I-MEI1), and meiosis II (I-MEI2) samples of pat1-as2 cells, respectively (n = 3; three biological replicates for each sample). (A) Summary of the number of proteins quantified in the analyzed samples. (B) Two-dimensional principal component analysis (PCA) evaluating differences in the quantified proteins among the analyzed samples. (C) Heatmap depicting the abundance patterns of 4673 proteins quantified in the analyzed samples. Each column and row represent an individual sample and protein, respectively. Relative protein levels are depicted in color scale: red indicates more abundance, and blue indicates less abundance. (D) Venn diagrams depicting the number of proteins that were identified to be specifically or similarly upregulated and downregulated when comparing the particular stages of meiosis in pat1-114 and pat1-as2 cells.
To assess functional relationships between the proteins that were significantly dysregulated in pat1-114- and pat1-as2-induced meiosis, the web-based STRING database tool was employed61.
The STRING analysis revealed that proteins whose levels were upregulated during meiosis I in both pat1-114 and pat1-as2 cells were enriched for GO terms (GO Biological Process, GO-BP) mainly related to the meiotic cell cycle, reproductive process, ascospore formation, cellular component assembly involved in morphogenesis, macroautophagy and autophagy, meiotic recombination, cell cycle, meiotic cell cycle process, meiotic nuclear division, and meiosis I, among others. We did not detect any GO terms enrichment for proteins upregulated specifically in pat1-114 cells during meiosis I. Regarding the proteins specifically upregulated during meiosis I in pat1-as2 cells, these proteins were enriched for GO terms mainly linked to establishment of localization, transport, localization, vesicle-mediated transport, transmembrane transport, macromolecule localization, and cellular localization, and others. On the other hand, proteins whose levels were downregulated in both pat1-114 and pat1-as2 cells were enriched for GO terms related to anabolism, represented by organonitrogen compound biosynthetic process, carboxylic acid metabolic process, ribonucleoprotein complex biogenesis, ribosome biogenesis, and cellular nitrogen compound metabolic process, among others. In the case of proteins whose expression was specifically downregulated during meiosis I in pat1-114 cells, these proteins were enriched for GO terms related to the regulation of gene expression, ncRNA processing and ncRNA metabolic process (Fig. 3, Table S2).
More specifically, among the proteins whose levels were found to be significantly upregulated during meiosis I in both pat1-114- and pat1-as2 cells, we identified the MBF transcription factor activator Rep1, meiotic DNA-binding fork head transcription factor Mei4 or meiosis specific cohesin subunit Rec11, among others. Furthermore, we found that the levels of Cut2, Mde2, Mde3, Mde10, Meu14, Spo4, Spo6, Spn5 and Spn7 proteins were also significantly elevated during meiosis I. On the other hand we detected decreased protein levels of MBF transcription factor activator Rep2, ribonucleoside reductase large subunit Cdc22, and Mde4, counterpart of the S. cerevisiae monopolin complex subunit Lrs4, among others. Regarding the proteins whose levels were found to be significantly upregulated during meiosis I either in pat1-114- or in pat1-as2 cells, the proteins involved in the regulation of faithful meiotic divisions were identified. For instance, the protein levels of anaphase-promoting complex subunits Hcn1, Cut20, and Nuc2 and meiotic cohesin complex kleisin subunit Rec8 were significantly upregulated during meiosis I specifically in pat1-114 cells. On the other hand, the protein levels of cohesin loading factors Mis4 and Ssl3, cohesin loader subunit Pds5, shelterin complex telomere-binding subunit Rap1, RecA family ATPase Rlp1, mitotic spindle checkpoint protein Mad2, and protein kinases Hhp1 and Hhp2 were found to be specifically upregulated during meiosis I in pat1-as2 cells (Table S1).
Concerning the proteins whose levels were specifically downregulated during meiosis I in pat1-114 cells, the shelterin complex subunit Tpz1, MCM complex subunit Mcm4, MIS12/MIND complex Mis14 and CENP-U ortholog Mis17 were detected, among others. Regarding the proteins whose levels were specifically downregulated during meiosis I in pat1-as2 cells, we detected Mis6-Sim4 complex subunit Fta4, MCM complex subunit Mcm3, DNA polymerase delta small subunit Cdc1, serine/threonine protein phosphatase Clp1, and FANCM-MHF complex subunit Mhf2, among others (Table S1).
GO enrichment analysis of proteins dysregulated in meiosis I when comparing meiosis I and mitotic samples (MEI1 versus MIT) in S. pombe pat1-114 and pat1-as2 cells. The scatter plot of pat1-114 & pat1-as2, MEI1 versus MIT (downregulated) includes the top 20 most enriched biological process categories (for the complete list of GO enrichment, refer to Table S2). The X-axis label represents the rich factor, and the Y-axis label shows the GO terms (biological process categories). The rich factor reflects the proportion of proteins in a given GO term, with a higher rich factor indicating greater enrichment. The color of the dots represents the enrichment score [–log10(p-value)], where red indicates high enrichment, and green indicates low enrichment. Dot size corresponds to the number of proteins in the respective GO term, with larger dots indicating a greater number of proteins.
Similarly, significant differences in the expression level of several proteins were observed when comparing meiosis II and meiosis I samples (MEI2 versus MEI1) in pat1-114 and pat1-as2 cells. In this case, the STRING analysis revealed that proteins upregulated during meiosis II in both pat1-114 and pat1-as2 cells were enriched for GO terms (GO-BP) mainly related to the meiotic cell cycle, cell cycle, sporulation resulting in formation of a cellular spore and ascospore formation, among others. No GO terms enrichment was observed for proteins upregulated during meiosis II specifically in pat1-114 or pat1-as2 cells. Concerning the proteins downregulated during meiosis II, the STRING analysis revealed that in pat1-as2-induced meiosis, the GO terms were enriched for proteins participating in cell wall organization or biogenesis, fungal type cell organization and biogenesis, cellular carbohydrate metabolic process and protein galactosylation and glycosylation, among others (Fig. 4, Table S2).
More specifically, among the proteins whose levels increased significantly when pat1-114 and pat1-as2 cells progressed into meiosis II, the RNA-binding protein Mde7, meiotic APC activator Mfr1, HECT-type ubiquitin-protein ligase E3 Mug30, spore wall 1,3-beta-glucan synthase catalytic subunit Bgs2, spore wall 1,3-beta-glucanosyltransferase Gas4, ortholog of S. cerevisiae Spo71 protein Mug56, RhoGEF for Rho1 Rgf2 protein, and septins Spn2, Spn5, Spn6 and Spn7 were detected, among others. Interestingly, among the proteins whose levels increased significantly when pat1-114 cells enter meiosis II, we identified protein kinases Ppk31 and Cka1, APC substrate recognition subunit Apc10, APC platform subcomplex scaffold subunit Cut4, and Mde4, counterpart of the S. cerevisiae monopolin complex subunit Lrs4, among others. In terms of the proteins whose levels increased significantly when pat1-as2 cells entered meiosis II, we detected DNA repair protein Fan1, meiotic fizzy-related APC coactivator Fzr2, DASH complex subunit Spc34, and Spo4-Spo6 kinase complex subunit Spo6, among others. Regarding the proteins downregulated during meiosis II specifically in pat1-as2 cells, we identified the cohesin complex ATPase subunit Psm3, the Hsk1-Dfp1 kinase complex regulatory subunit Dfp1, the meiotic recombination protein Rec10, the Cds1/Rad53/Chk2 family protein kinase Mek1, and the sister chromatid separation inhibitor securin Cut2, among others (Table S1).
GO enrichment analysis of proteins dysregulated in meiosis II when comparing meiosis II and meiosis I samples (MEI2 versus MEI1) in S. pombe pat1-114 and pat1-as2 cells. The X-axis label represents the rich factor, and the Y-axis label shows the GO terms (biological process categories). The rich factor reflects the proportion of proteins in a given GO term, with a higher rich factor indicating greater enrichment. The color of the dots represents the enrichment score [–log10(p-value)], where red indicates high enrichment, and green indicates low enrichment. Dot size corresponds to the number of proteins in the respective GO term, with larger dots indicating a greater number of proteins.
Quantitative phosphoproteomic analysis
Applying quantitative phosphoproteomics analysis to mitotic (T-MIT), meiosis I (T-MEI1), and meiosis II (T-MEI2) samples of pat1-114, and mitotic (I-MIT), meiosis I (I-MEI1), and meiosis II (I-MEI2) samples of pat1-as2 cells, we identified and quantified a total of 7164 phosphosites (Fig. 5A). PCA explained 79.6% of the total variance and clearly discriminated between mitotic and meiotic samples (Fig. 5B). Specifically, PC1 explained 62.9% variance, discriminating between mitotic and meiotic samples of both pat1-114 and pat1-as2 samples, while PC2 explained 16.7% variance, discriminating between pat1-114 and pat1-as2 samples.
Consistent with the results of the PC1, the heatmap of quantified phosphosites highlighted two main clusters: one with the mitotic samples of both pat1-114 and pat1-as2 (T-MIT and I-MIT), and the other grouping the meiosis I and meiosis II samples of pat1-114 and pat1-as2 (T-MEI1, T-MEI2, I-MEI1 and I-MEI2) (Fig. 5C). The second cluster further divided into two subclusters: one with the meiosis I sample of pat1-114 (T-MEI1) and the other with the meiosis II sample of pat1-114 (T-MEI2) and meiosis I and meiosis II samples of pat1-as2 (I-MEI1 and I-MEI2), consistent with PC2 results.
To identify proteins whose phosphorylation significantly oscillated during the progression of S. pombe cells through meiosis, a statistical analysis of quantified phosphosites was performed using an ANOVA test (FDR < 0.05). The amino acid residues that were found to be differently phosphorylated during progression of cells through meiotic divisions are summarized in Table S3.
The analysis of differences between meiosis I and mitotic samples (MEI1 versus MIT) led to the identification of 993 amino acid residues that were more phosphorylated and 130 amino acid residues that were less phosphorylated in the meiosis I samples of pat1-114 and pat1-as2, compared to mitotic samples. Interestingly, 287 amino acid residues were more phosphorylated and 201 amino acid residues were less phosphorylated specifically in meiosis I of pat1-114, while 831 amino acid residues were more phosphorylated and 25 amino acid residues were less phosphorylated specifically in meiosis I of pat1-as2, compared to mitotic samples. When meiosis II was compared with meiosis I (MEI2 versus MEI1), 225 amino acid residues were found to be more phosphorylated, and 8 amino acid residues were less phosphorylated in the meiosis II samples of pat1-114 and pat1-as2. Furthermore, the phosphorylation of 346 amino acid residues was found to be upregulated, and 9 amino acid residues were detected to be less phosphorylated specifically in meiosis II of pat1-114, while the phosphorylation of 94 amino acid residues was found to be upregulated, and 83 amino acid residues were found to be downregulated specifically in meiosis II of pat1-as2, compared to meiosis I samples (Fig. 5D, Table S3).
Phosphoproteomic profiling of the six subsets of S. pombe samples, including mitotic (T-MIT), meiosis I (T-MEI1), and meiosis II (T-MEI2) samples of pat1-114, and mitotic (I-MIT), meiosis I (I-MEI1), and meiosis II (I-MEI2) samples of pat1-as2 cells, respectively (n = 3; three biological replicates for each sample). (A) Summary of the number of quantified phosphosites in the analyzed samples. (B) Two-dimensional principal component (PC) analysis evaluating differences in the quantified phosphosites among the analyzed samples. (C) Heatmap depicting the abundance patterns of 7164 quantified phosphosites in the analyzed samples. Each column and row represent an individual sample and phosphosite, respectively. The relative degree of phosphorylation of particular phosphosites is depicted in a color scale: red indicates a higher degree of phosphorylation, and blue indicates a lower degree of phosphorylation. (D) Venn diagrams depicting the number of sites whose phosphorylation was found to be specifically or similarly upregulated and downregulated when comparing particular stages of meiosis in pat1-114 and pat1-as2 samples.
To evaluate functional relationship among proteins with significantly dysregulated phosphorylation in pat1-114- and pat1-as2-induced meiosis, the STRING analysis was utilized. We found that proteins with upregulated phosphorylation during meiosis I in both pat1-114 and pat1-as2 cells were enriched for GO terms (GO-BP) primarily related to the cell cycle, meiotic cell cycle, reproductive process, reproduction, localization, chromosome attachment to the nucleus envelope, centromere clustering and chromosome localization, among others. For proteins with phosphorylation specifically upregulated during meiosis I only in pat1-114 cells, the GO term enrichment was related to the meiotic cell cycle, cell cycle, reproductive process, microtubule cytoskeleton organization involved in mitosis, mitotic spindle elongation and microtubule-based movement, among others. Regarding proteins with phosphorylation specifically upregulated during meiosis I in pat1-as2 cells, the GO term enrichment was related to the regulation of biological process, biological regulation, regulation of cellular process, localization and signaling, among others (Fig. 6, Table S4).
On the other hand, proteins with downregulated phosphorylation in both pat1-114 and pat1-as2 cells were enriched for proteins involved in rRNA processing, regulation of biological process and regulation of cellular process and organonitrogen compound biosynthetic process, among others. No enrichment in GO terms was found for proteins with downregulated phosphorylation specifically during meiosis I in pat1-114 or pat1-as2 cells (Fig. 6, Table S4).
Interestingly, among the proteins whose phosphorylation was found to be significantly increased during meiosis I in both pat1-114 and pat1-as2 cells, compared to mitotic cells, those required for proper meiotic chromosome segregation, meiotic recombination, and regulation of microtubule kinetochore attachment were identified. For instance, increased phosphorylation of the Pcs1, counterpart of the S. cerevisiae monopolin complex subunit Csm1, at S70 was detected. Additionally, phosphorylation of Pcs1 at S73 was specifically upregulated during meiosis I in pat1-as2 cells. In the case of cohesin subunits, increased phosphorylation of the meiotic cohesin complex kleisin subunit Rec8 at S366, S377 and S378 was detected in both pat1-114 and pat1-as2 cells. Regarding the cohesin regulators, increased phosphorylation of the cohesin release factor Wpl1 at S61, S64, S65, and S155 in pat1-as2 cells, the cohesin loading factor Mis4 at T193 in pat1-114 cells, and Mis4 at S183 and S204 in pat1-as2 cells was detected. Furthermore, we detected the meiosis I-specific increase in the phosphorylation of proteins involved in meiotic recombination. For example, we observed significantly increased phosphorylation of the DNA recombination mediator Sfr1 at S26, T63 and S165, and the meiotic recombination protein Rec10 at S424 and S529 in both pat1-114 and pat1-as2 cells. Additionally, an increase in the phosphorylation of Rec10 at T436 and S468 during meiosis I was specifically detected in pat1-as2 cells. Additionally, increased phosphorylation of the meiosis specific kinetochore regulator meikin (Moa1) at T150 and S154, integral nuclear inner membrane protein Bqt4 at T302 and S358, kinetochore scaffold protein Cnp20 at T164, spindle pole body SUN domain protein Sad1 at T5 and T63, sister chromatid separation inhibitor Cut2 at T18 and S109, and cohesin protector protein Sgo2 at S7, S145 and S177 in both pat1-114 and pat1-as2 cells was detected during meiosis I. Interestingly, we found that while in pat1-114 cells, the phosphorylation of the kinesin-5 family plus-end directed microtubule motor protein Cut7 at T1011 and kinesin-14 family minus-end directed microtubule motor protein Pkl1 at S279 and S358 is specifically increased, in pat1-as2 cells, increased phosphorylation of Cnp20 at S219, Ndc80 complex subunit Spc25 at S109, Ndc80 complex subunit Ndc80 at S367, MIS12/MIND complex protein Mis13 at S302, Sgo2 at S240 and T292, outer kinetochore KNL network protein Spc7 at S638, and Bqt4 at S256 and S263 was detected (Table S3).
GO enrichment analysis of proteins whose phosphorylation was dysregulated in meiosis I when comparing meiosis I and mitotic samples (MEI1 versus MIT) in S. pombe pat1-114 and pat1-as2 cells. The scatter plots of pat1-114 & pat1-as2, pat1-114, and pat1-as2, all MEI1 versus MIT (upregulated) include the top 20 most enriched biological process categories (for the complete list of GO enrichment, refer to Table S4). The X-axis label represents the rich factor, and the Y-axis label shows the GO terms (biological process categories). The rich factor reflects the proportion of proteins in a given GO term, with a higher rich factor indicating greater enrichment. The color of the dots represents the enrichment score [–log10(p-value)], where red indicates high enrichment, and green indicates low enrichment. Dot size corresponds to the number of proteins in the respective GO term, with larger dots indicating a greater number of proteins.
Concerning the proteins whose phosphorylation was downregulated during meiosis I compared to mitotic cells, we found only a few proteins necessary for proper meiotic chromosome segregation and meiotic recombination. We found that the phosphorylation of the cohesin protector protein Sgo2 at S322 was downregulated in both pat1-114 and pat1-as2 cells, while phosphorylation of the cohesin release factor Wpl1 at S56, and the DNA recombination mediator protein Swi2 at S261, was significantly decreased in meiosis I specifically in pat1-114 cells (Table S3).
Similarly, we observed significant differences in the phosphorylation of proteins when comparing meiosis II and meiosis I samples. In this case, we found that proteins with upregulated phosphorylation during meiosis II in both pat1-114 and pat1-as2 cells were enriched for GO terms (GO-BP) primarily related to reproduction, meiotic cell cycle, sporulation resulting in formation of a cellular spore, and ascospore formation, among others. Regarding proteins with phosphorylation specifically upregulated during meiosis II only in pat1-114 cells, the GO terms were enriched for processes related to biological regulation and cellular process. Furthermore, the GO terms enrichment for proteins whose phosphorylation was specifically upregulated during meiosis II only in pat1-as2 cells was linked to processes involved in ascospore formation, vesicle-mediated transport, sexual reproduction, Golgi vesicle transport, and ascospore-type prospore assembly, among others. Concerning the proteins with downregulated phosphorylation specifically during meiosis II, the enrichment for a single GO term related to cell cycle was identified in pat1-as2 cells (Fig. 7, Table S4).
More specifically, among the proteins that were more phosphorylated during meiosis II compared to meiosis I in both pat1-114 and pat1-as2 cells, we identified the phosphorylation of cohesin release factor Wpl1 at S56. Additionally, increased phosphorylation during meiosis II was detected for the spore wall 1,3-beta-glucan synthase catalytic subunit Bgs2 at S175, S937, S943 and S947, as well as the sporulation protein Spo3 at S28 and S112 in both pat1-114 and pat1-as2 cells. The cell wall and ascospore endo-1,3-beta-glucanase Eng2 exhibited increased phosphorylation at S635, and the sporulation protein Spo3 at S449 in pat1-114 cells. Additionally, the cell wall alpha-1,3-glucan synthase Mok13 showed increased phosphorylation at S759 and S772 in pat1-as2 cells. Interestingly, phosphorylation of septins involved in the spore membrane bending pathway was found to be increased during meiosis II. This includes the phosphorylation of Spn7 at S325, S330 and S385, and S1307 in both pat1-114 and pat1-as2 cells, Spn2 at S314 and S321, and Spn6 at S354 in pat1-114, and Spn5 at S56 and S67, and Spn7 at S292, S297, S299, S326, S337, S342 and S350 in pat1-as2 cells (Table S3).
GO enrichment analysis of proteins whose phosphorylation was dysregulated in meiosis II when comparing meiosis II and meiosis I samples (MEI2 versus MEI1) in S. pombe pat1-114 and pat1-as2 cells. The X-axis label represents the rich factor, and the Y-axis label shows the GO terms (biological process categories). The rich factor reflects the proportion of proteins in a given GO term, with a higher rich factor indicating greater enrichment. The color of the dots represents the enrichment score [–log10(p-value)], where red indicates high enrichment, and green indicates low enrichment. Dot size corresponds to the number of proteins in the respective GO term, with larger dots indicating a greater number of proteins.
Regarding the proteins whose phosphorylation decreased during meiosis II compared to meiosis I, we observed a downregulation in the phosphorylation of the meiotic RNA-binding protein Spo5 at S484 in pat1-114 cells. Additionally, decreased phosphorylation of Spo5 at T6, the RNA-binding protein involved in meiosis Mei2 at S736, the cohesin loading factor Mis4 at S204, the meiotic cohesin complex kleisin subunit Rec8 at S378, the meiotic recombination protein Rec10 at S375, the meiosis specific kinetochore regulator meikin (Moa1) at T150 and S154, and the Smc5-6 complex subunit Smc6 at S449, among others, was detected during meiosis II in pat1-as2 cells (Table S3).
Discussion
To investigate how the expression and phosphorylation of proteins change in S. pombe cells during meiosis, we induced highly synchronous meiosis using pat1-114 and pat1-as2 mutants. Then, we performed quantitative proteomics and phosphoproteomics analyses of mitotic cells and cells collected from meiosis I or meiosis II stages (Fig. 1B). Using TMTpro labeling and phosphopeptide enrichments strategies, followed my mass spectrometry analysis, we quantified 4673 proteins (∼91% of the predicted proteome of S. pombe) and 7172 phosphosites (Figs. 2A and 5A). Among the quantified proteins and phosphosites, the level of 57.4% of proteins (2680 proteins) and the phosphorylation of 55.8% peptides (4005 phosphosites) significantly changed when comparing protein levels and phosphorylation events in mitotic cells and cells in meiosis I and meiosis II stages (Table S1, Table S2). To our knowledge, this is one of the largest proteomics and phosphoproteomics datasets describing differences in protein levels and phosphorylation of proteins in S. pombe cells during meiosis, both at elevated (34 °C, pat1-114) and optimal (25 °C, pat1-as2) temperatures.
In the last years, several studies analyzed the changes in the protein levels and phosphorylation of proteins during mitotic and meiotic divisions in S. pombe54,55,65−67. These studies proposed the multiple regulatory mechanisms controlling the progression of S. pombe cells through particular stages of mitosis and meiosis, including those regulating the protein functions through their expression and phosphorylation.
Taken together, our analysis revealed that after induction of meiosis in S. pombe, either by higher temperature (34 °C, pat1-114), or using 1-NM-PP1 (25 °C, pat1-as2), the levels of 934 proteins significantly changed during meiosis I. Additionally, the levels of 91 proteins significantly changed when cells enter meiosis II. We also found that the levels of 364 proteins significantly changed during meiosis I and the levels of 155 proteins significantly changed during meiosis II specifically in pat1-114 cells. Finally, we detected significant differences in the levels of 424 proteins during meiosis I and 55 proteins during meiosis II specifically in pat1-as2 cells (Fig. 2D). The proteins whose levels significantly increased during meiosis I in both pat1-114 and pat1-as2 cells were enriched for the proteins involved in meiotic cell cycle, reproductive process, ascospore formation, cellular component assembly involved in morphogenesis, macroautophagy and autophagy, reciprocal meiotic recombination, cell cycle, meiotic cell cycle process, meiotic nuclear division, and meiosis I, among others. On the other hand, proteins whose levels were downregulated in both pat1-114 and pat1-as2 cells were enriched for the proteins involved in anabolism processes, represented by organonitrogen compound biosynthetic process, carboxylic acid metabolic process, ribonucleoprotein complex biogenesis, ribosome biogenesis, and cellular nitrogen compound metabolic process, among others. A similar switch from anabolic to catabolic processes during meiosis has been previously observed for both S. cerevisiae and S. pombe54,68,69. Additionally, we found that the proteins specifically upregulated or downregulated during meiosis I in pat1-as2 or pat1-114 cells were enriched for those involved in the establishment of localization, vesicle-mediated and transmembrane transports, macromolecule and cellular localizations and in the regulation of gene expression, ncRNA processing and ncRNA metabolic process, respectively (Fig. 3, Table S2).
In line with this, we found that the levels of MBF transcription factor activator Rep1 and meiotic DNA-binding fork head transcription factor Mei4 increased and the level of MBF transcription factor activator Rep2 significantly decreased during meiosis I in both pat1-114 and pat1-as2 mutants. Previously it has been shown that Rep1 factor is important for timely expression of genes involved in pre-meiotic S phase and meiotic recombination, such as the ribonucleoside reductase large subunit Cdc22 and meiosis specific cohesin subunits Rec8 and Rec11. On the other hand, Rep2 factor was found to repress the expression of Rec8 and Rec1170. In line with these findings, we found that in meiosis I both pat1-114 and pat1-as2 cells had significantly increased level of Rec11, the meiosis specific cohesin subunit required for meiotic recombination and segregation of homologous chromosomes71,72. Furthermore, we detected significantly decreased protein level of Cdc22 in meiosis I. This suggests that Cdc22 is quickly degraded when cells complete pre-meiotic S phase and enter meiosis I. It is known that Cdc22 is required for DNA synthesis and mutations in cdc22 were shown to block pre-meiotic DNA replication73. Furthermore, we found that the levels of proteins involved in meiotic DNA DSBs formation, such as Hop1, Mde2, Mug20 and Rec27 and sister chromatid separation inhibitor Cut2, were significantly elevated during meiosis I in both pat1-114 and pat1-as2 cells. The increased levels of these proteins during meiosis I suggest that the expression of these proteins has to be tightly controlled to secure their proper function in meiotic recombination and in protection of meiotic cohesin from premature cleavage by separase Cut1 19,74,75,76,77,78. It has been shown previously, that after meiotic recombination, the transcription factor Mei4 activates the expression of genes important for meiotic nuclear divisions and early steps of spore formation79−84. In line with this, we detected significantly increased levels of Mde3, Mde10, Meu14, Spo4, Spo6, Spn5 and Spn7 proteins in meiosis I, and Mde5, Mde7, Spn5 and Spn7 proteins in meiosis II, respectively. Among the proteins whose levels significantly decreased in meiosis I in both pat1-114 and pat1-as2 samples, compared to mitotic samples, we detected Mde4, counterpart of the S. cerevisiae monopolin complex subunit Lrs4. These data are consistent with previous observations that Mde4 is needed for faithful sister-chromatid segregation during mitosis and meiosis II. However, unlike its S. cerevisiae counterpart Lrs4, Mde4 is dispensable for segregation of homologous chromosomes during meiosis I85,86 (Table S1).
Despite considerable similarity between meiotic proteomes of pat1-114 and pat1-as2 cells, we detected also some differences. For example, we found that in pat1-114 cells the protein levels of APC subunits Hcn1, Cut20, and Nuc2, and meiotic cohesin complex kleisin subunit Rec8 are significantly upregulated during meiosis I. It has been shown previously that S. cerevisiae cells lacking CDC26, ortholog of Hcn1, are sensitive to higher temperature87. It is possible that increased levels of APC subunits during meiosis I might help pat1-114 cells to deal with the elevated temperature used to induce meiosis. Furthermore, APC is known to degrade the Cut2, which inhibits the separase Cut1 responsible for Rec8 cleavage76,77,78. Therefore, it is plausible that the elevated level of Rec8 in pat1-114 cells during meiosis I may represent a compensatory response of these cells to increased Cut1 activity that results from the degradation of its inhibitor Cut2 by APC. This is in contrast to pat1-as2-induced meiosis where no specific increase in the protein levels of any APC subunits or Rec8 was detected. On the other hand, the protein levels of cohesin loading factors Mis4 and Ssl3, cohesin loader subunit Pds5, shelterin complex telomere-binding subunit Rap1, RecA family ATPase Rlp1, mitotic spindle checkpoint protein Mad2, and protein kinases Hhp1 and Hhp2, among others, were specifically upregulated in pat1-as2 mutant. These proteins are known to have important roles during meiosis, such as Pds5, Mis4 and Ssl3 regulate the loading of cohesin complexes88, Rap1 is part of meiotic spindle pole body89, Rlp1 and Mad2 are involved in processes of meiotic recombination and meiosis I spindle assembly checkpoint signaling90,91, and protein kinases Hhp1 and Hhp2 are known to phosphorylate Rec8 and Rec1139,72,92. Regarding the proteins whose levels were specifically downregulated during meiosis I in pat1-114 cells, the shelterin complex subunit Tpz1, MCM complex subunit Mcm4, MIS12/MIND complex Mis14 and CENP-U ortholog Mis17 were identified. Tpz1 is known to regulate telomere length homeostasis93, Mcm4 was shown to be important for pre-meiotic DNA replication94,95, and Mis14 is part of meiotic kinetochores96. The proteins whose levels were found to be downregulated during meiosis I specifically in pat1-as2 cells included the Mis6-Sim4 complex subunit Fta4, serine/threonine protein phosphatase Clp1, DNA polymerase delta small subunit Cdc1, MCM complex subunit Mcm3, and FANCM-MHF complex subunit Mhf2, among others (Table S1). Fta4 is known to participate in the formation of centromere-specific chromatin97, Clp1 regulates spindle elongation by dephosphorylating Mde4, Ase1 and Klp985,98,99, Cdc1 is involved in pre-meiotic DNA replication73, and Mhf2 is known for its role in resolution of meiotic recombination intermediates100. Given the findings regarding the changes in protein levels mentioned above, further studies are needed to explore how the detected alterations in protein levels affect the mechanisms regulating meiosis I in pat1-114 and pat1-as2 cells.
Similarly, significant differences in the expression level of several proteins were observed when comparing meiosis II and meiosis I samples. In this case, we found that proteins upregulated during meiosis II in both pat1-114 and pat1-as2 cells were enriched for GO terms related to the meiotic cell cycle, cell cycle, sporulation, and ascospore formation, among others. No GO term enrichment was observed for proteins upregulated during meiosis II specifically in pat1-114 or pat1-as2 cells. Regarding the proteins downregulated during meiosis II, we found that in pat1-as2 cells, the GO terms were enriched for proteins participating in cell wall organization or biogenesis, fungal type cell organization and biogenesis, cellular carbohydrate metabolic process, and protein galactosylation and glycosylation, among others (Fig. 4, Table S2).
Among the proteins whose levels increased significantly when pat1-114 and pat1-as2 cells progressed into meiosis II and sporulation we identified proteins known to participate in catabolic processes, such as RNA-binding protein Mde7, meiotic APC activator Mfr1 and HECT-type ubiquitin-protein ligase E3 Mug30, spore wall 1,3-beta-glucan synthase catalytic subunit Bgs2, spore wall 1,3-beta-glucanosyltransferase Gas4, ortholog of S. cerevisiae Spo71 protein Mug56, spore formation protein Rgf2 and septins Spn2, Spn5, Spn6 and Spn7 regulating spore membrane bending pathway56,81,101−104. Furthermore, when pat1-114 cells enter meiosis II, we found the protein levels of kinases Ppk31 and Cka1, APC substrate recognition subunit Apc10, APC platform subcomplex scaffold subunit Cut4 and Mde4, counterpart of the S. cerevisiae monopolin complex subunit Lrs4, among others, to be significantly elevated. Our finding that protein levels of Ppk31 and Cka1 kinases which are known to be involved in regulation of autophagy and pre-mRNA splicing56,105, APC complex subunits Apc10 and Cut4 participating in catabolic processes54,106,107, and Mde4 involved in prevention of merotelic kinetochore orientation85,86, are upregulated during meiosis II is consistent with the known functions of these proteins in regulation of later stages of meiosis. On the other hand, the group of proteins whose levels increased significantly when pat1-as2 cells entered meiosis II, was represented by DNA repair protein Fan1, which plays a role in recombinational interstrand cross-link repair108, meiotic fizzy-related APC coactivator Fzr2 participating in APC complex regulated catabolic processes54,56,101, DASH complex subunit Spc34 involved in formation of meiotic kinetochores96, and Spo4-Spo6 kinase complex subunit Spo6, which regulates meiosis II and sporulation84,109, among others. Regarding the proteins downregulated during meiosis II specifically in pat1-as2 cells, we identified the cohesin complex subunit Psm3, Hsk1-Dfp1 kinase complex regulatory subunit Dfp1, protein kinase Mek1, meiotic recombination protein Rec10, and the sister chromatid separation inhibitor Cut2, among others (Table S1). The decreased levels of these proteins, are in line with their role in regulating the early stages of meiosis27,33,42,110. Based on the aforementioned findings regarding the changes in protein levels, future studies are necessary to investigate how the detected alterations in protein levels during meiosis II impact the mechanisms of meiotic cell divisions. Furthermore, these studies should explore whether differences between proteomes of pat1-114 and pat1-as2 cells during meiosis I and meiosis II can account for the observed phenotypes related to meiotic chromosome segregation and spore viability in pat1-114 and pat1-as2 cells50.
Phosphorylation, a reversible post-translational modification, plays a pivotal role in regulating protein functions. Through the reversible addition of phosphate groups, proteins may undergo structural and functional alterations, thereby regulating their activities and interaction networks. It has been demonstrated that phosphorylation plays a significant role in regulating the functions of proteins involved in specific stages of meiosis, such as pre-meiotic DNA replication, homologous recombination, chromosome segregation, and spore formation21−42,111−114.
Regarding the analysis of changes in protein phosphorylation in S. pombe during meiosis, we conducted quantitative phosphoproteomics analysis and compared differences in phosphoproteomes between mitotic cells and cells in meiosis I or meiosis II in pat1-114 and pat1-as2 cells. In total, we identified and quantified 7164 phosphosites (Fig. 5A). By analyzing differences between meiosis I and mitotic samples we detected changes in phosphorylation of 1123 amino acid residues during meiosis I in both pat1-114 and pat1-as2 cells, compared to mitotic samples. Furthermore, we found that phosphorylation of 488 amino acid residues was dysregulated during meiosis I specifically in pat1-114 cells, while phosphorylation of 856 amino acid residues changed during meiosis I specifically in pat1-as2 cells, compared to mitotic cells. Regarding the phosphoproteomes of meiosis II samples (MEI1 versus MEI2), we detected significant difference in phosphorylation of 233 amino acid residues in both pat1-114 and pat1-as2 cells. Additionally, we found that the phosphorylation of 355 amino acid residues changed during meiosis II specifically in pat1-114 cells. Finally, 177 amino acid residues were found to be differently phosphorylated during meiosis II specifically in pat1-as2 cells, compared to cells in meiosis I (Fig. 5D, Table S3). These findings may provide insights into functional links between phosphorylation and protein functions during meiotic divisions in S. pombe, as previously illustrated in this yeast for protein functions during mitosis65,66.
Among the proteins whose phosphorylation significantly changed during meiosis we detected several proteins that are known to be involved in regulation of meiotic recombination, meiotic chromosome segregation and regulation of microtubule kinetochore attachment. For example, we found that during meiosis I, the phosphorylation of DNA recombination mediator Sfr1 at S26, T73 and S165 was increased in both pat1-114 and pat1-as2 cells (Table S3). Previously, the phosphorylation of Sfr1 was found to be important for promoting Rad51-dependent DNA repair115. Additionally, it has been shown that phosphomimetic sfr1-13D mutant only partially rescued the homologous non-disjunction phenotype of sfr1Δ cells116. These findings suggest that the function of Sfr1 during meiosis may be regulated by phosphorylation. Similarly, we detected increased level of phosphorylation of the meiotic recombination protein Rec10 at S424 and S529 in both pat1-114 and pat1-as2 cells, and Rec10 at T436 and S468 specifically in pat1-as2 cells during meiosis I. Additionally, we found that phosphorylation of the DNA recombination mediator protein Swi2 at S261 was significantly decreased during meiosis I specifically in pat1-114 cells (Table S3). This is in line with the previous studies implying that functions of meiotic recombination factors may be regulated by phosphorylation117,118.
Regarding the changes in phosphorylation of proteins regulating kinetochore assembly and attachment we detected increased phosphorylation of Pcs1, counterpart of the S. cerevisiae monopolin complex subunit Csm1, at S70 during meiosis I in both pat1-114 and pat1-as2 cells and Pcs1 at S73 specifically in pat1-as2 cells (Table S3). It is plausible that phosphorylation of Pcs1 at S70 and S73 may regulate the function of this protein during meiosis I as was shown for its partner Mde4, whose timely phosphorylation by Cdc2 is required to prevent its precocious localization to the metaphase spindle86,119. Additionally, we detected increased phosphorylation of the meiotic kinetochore protein meikin Moa1 at T150 and S154 during meiosis I (Table S3), suggesting possible regulation of its function by phosphorylation. Moa1 was previously shown to control both mono-orientation of sister kinetochores and cohesion protection 40–42,41,,120. In the case of cohesin subunits, increased phosphorylation of the Rec8 at S366, S377 and S378 was detected in both pat1-114 and pat1-as2 cells (Table S3). Phosphorylation of Rec8 is known to play an important role for separase-mediated cleavage of Rec839,78,92,121−124. It has been found previously that phosphorylation of Rec8 by Hhp1 and Hhp2 kinases is required for efficient removal of Rec8 at the onset of anaphase I. These CK1 kinases were shown to be required for full levels of Rec8 phosphorylation, including the phosphorylation of S366, the residue which phosphorylation we found to be upregulated during meiosis I92. Furthermore, it has been shown that phosphorylation of Rec8 at S412, one of the CK-1 dependent phosphorylation sites, is required for timely cleavage of Rec8 and promotes homologue separation at anaphase I. Additional six putative CK1 target sites (S400, S402, S404, S410, T414 and S416) contribute to timely cleavage of Rec8 by separase. N-terminal phosphorylation of Rec8 at S40, S63, T113, S176, S186, S187, T193, T198, T219, T222, S230 and S233 also contributes to efficient segregation of homologues during meiosis I39. Regarding cohesin regulators, we detected increased phosphorylation of Wpl1 at S61, S64, S65, and S155 in pat1-as2 cells, and decreased phosphorylation of Wpl1 at S56 in pat1-114 cells during meiosis I. Wpl1 is a cohesin release factor which plays important role during dissociation of cohesin from chromatin and its meiosis specific phosphorylation was found to promote Rec8-cohesin release125. In the case of cohesin loading factor Mis4, we detected its increased phosphorylation at T193 in pat1-114 cells, and at S183 and S204 in pat1-as2 cells (Table S3). Our findings raise the possibility that phosphorylation of the cohesin and cohesin regulators Mis4 and Wpl1 during meiosis I may regulate their meiotic functions.
In addition to the increased phosphorylation during meiosis I of proteins involved in meiotic recombination and chromosome segregation, we also detected increased phosphorylation of several other proteins important for meiotic divisions, such as integral nuclear inner membrane protein Bqt4 at T302 and S358, spindle pole body SUN domain protein Sad1 at T5 and T63, kinetochore scaffold protein Cnp20 at T164, and sister chromatid separation inhibitor Cut2 at T18 and S109. Interestingly, we found that the phosphorylation of Sgo2 at S7, S145 and S177 was increased, while phosphorylation of Sgo2 at S322 was decreased during meiosis I in both pat1-114 and pat1-as2 cells (Table S3). These proteins are known for their roles in meiotic telomere tethering, kinetochore assembly, regulation of Rec8 cleavage and protection of centromeric cohesion78,103,126,127. We also found that phosphorylation of the kinesin-5 family plus-end directed microtubule motor protein Cut7 at T1011 and kinesin-14 family minus-end directed microtubule motor protein Pkl1 at S279 and S358 was specifically increased during meiosis I in pat1-114 cells. These proteins are known for their role in meiotic spindle assembly128. Interestingly, we detected increased phosphorylation of kinetochore scaffold protein Cnp20 at S219, Ndc80 complex subunit Spc25 at S109, Ndc80 complex subunit Ndc80 at S367, MIS12/MIND complex protein Mis13 at S302, Sgo2 at S240 and T292, outer kinetochore KNL network protein Spc7 at S638, and Bqt4 at S256 and S263 during meiosis I specifically in pat1-as2 cells. These proteins are known for their role in regulation of kinetochore’s assembly and function49,120,126,129 (Table S3). These findings suggest significant differences in the phosphorylation of proteins regulating spindle assembly and kinetochore functions during meiosis I in pat1-114 and pat1-as2 cells. Therefore, further studies are necessary to understand how these phosphorylation events affect meiotic functions of these proteins.
Similarly, we observed significant changes in the phosphorylation of proteins when cells enter meiosis II (MEI2 versus MEI1). In this case, the proteins with increased phosphorylation during meiosis II in both pat1-114 and pat1-as2 cells were enriched for GO terms linked to reproduction, meiotic cell cycle, sporulation resulting in formation of a cellular spore and ascospore formation, among others. Regarding the proteins with elevated phosphorylation during meiosis II specifically in pat1-114 cells, the GO terms were enriched for processes related to biological regulation and cellular process. Furthermore, the GO terms enrichment for proteins whose phosphorylation was upregulated during meiosis II specifically in pat1-as2 cells was linked to ascospore formation, vesicle-mediated transport, sexual reproduction, Golgi vesicle transport, and ascospore-type prospore assembly, among others. Regarding the proteins with downregulated phosphorylation specifically during meiosis II, the enrichment for a single GO term related to cell cycle was identified only in pat1-as2 cells (Fig. 7, Table S4). Among the proteins whose phosphorylation increased during meiosis II in both pat1-114 and pat1-as2 cells, we detected the phosphorylation of Wpl1 at S56. A previous study has shown that in S. cerevisiae, meiosis-specific phosphorylation of Wpl1 promotes the release of Rec8-cohesin125. Our findings showing significant phosphorylation increases of Wpl1 at S61, S64, S65, and S155 during meiosis I, and Wpl1 at S56 during meiosis II, are consistent with meiosis-specific regulation of Wpl1 in S. pombe. Additionally, we found that phosphorylation of spore wall 1,3-beta-glucan synthase catalytic subunit Bgs2 at S175, S937, S943 and S947, and sporulation protein Spo3 at S28 and S112 occurred in both pat1-114 and pat1-as2 cells during meiosis II. Moreover, we detected increased phosphorylation of cell wall and ascospore endo-1,3-beta-glucanase Eng2 at S635, and Spo3 at S449 in pat1-114 cells during meiosis II. Furthermore, the cell wall alpha-1,3-glucan synthase Mok13 showed increased phosphorylation at S759 and S772 specifically in pat1-as2 cells. These findings suggest that phosphorylation may also regulate the functions of proteins involved in spore membrane formation. This is further supported by our finding that phosphorylation of proteins involved in the spore membrane bending pathway also increased during meiosis. This includes phosphorylation of Spo7 at T12, S213, S680, and S1307 during meiosis I, and Spn7 at S325, S330 and S385 during meiosis II in both pat1-114 and pat1-as2 cells, Spo7 at T159, S368, T571 and T953, Spn2 at S314 and S321, and Spn6 at S354 during meiosis II specifically in pat1-114 cells, and Spn5 at S56 and S67, and Spn7 at S292, S297, S299, S326, S337, S342 and S350 during meiosis II specifically in pat1-as2 cells (Table S3). Previous studies have shown that Spo7 regulates the formation of the leading edge and initiation of forespore membrane assembly, while septins Spn2, Spn5, Spn6 and Spn7 form a scaffold structure that assists in guiding the oriented extension of the forespore membrane83,130. Phosphorylation of proteins involved in the spore membrane formation and spore membrane bending pathway identified in our study suggests that their functions may be regulated by phosphorylation.
Finally, among the proteins whose phosphorylation decreased when cells entered meiosis II, we identified the meiotic RNA-binding protein Spo5. While the phosphorylation of Spo5 at T6 and S484 was upregulated during meiosis I in both pat1-114 and pat1-as2 cells, the phosphorylation of Spo5 at S484 and T6 significantly dropped in meiosis II in pat1-114 and pat1-as2 cells, respectively. Similarly, a significant decrease in phosphorylation of Mei2 at S736, Mis4 at S204, Rec8 at S378, Rec10 at S375, Moa1 at T150 and S154, and Smc6 at S449 was detected during meiosis II specifically in pat1-as2 cells (Table S3). The finding that phosphorylation of these proteins decreased in meiosis II may indicate and support the important role of their phosphorylation during earlier stages of meiosis.
Although our results do not reveal new mechanistic insights into meiotic processes, our data provide a solid basis for further analyses. Changes during the progression of meiosis in fission yeast S. pombe have been studied by various approaches including transcriptional and ribosome profiling7,131. While in this manuscript, we provide a brief comparison of our proteomic data with results obtained from transcriptional profiling, comparison with ribosome profiling remains an important aim for future studies. Moreover, similar studies of meiosis have been performed in other organisms132,133,134,135. Extending such analyses to other organisms will be important to decipher which phenomena are S. pombe specific and which are more general. Ribosome profiling and other studies of meiotic S. pombe cells identified new ORFs encoding proteins that have not been previously described55,131. Searching our mass-spectrometry data for peptides corresponding to these novel ORFs may provide further support for the presence of these proteins in fission yeast cells. In addition, detection of protein isoforms may reveal new meiotic regulatory pathways. Simultaneous multi-omics technologies performed in single cells are undergoing rapid development and it is likely that such approaches will help overcome some of the challenges we are currently facing136,137,138.
Interestingly, by comparing pat1-114 with pat1-as2 induced meiosis, we were able to assess the impact of elevated temperature (25 °C, pat1-as2 versus 34 °C, pat1-114) on meiosis. It will be important to validate and further analyze these results. Recently, the negative effects of elevated temperature on meiosis have been highlighted139,140. Studies on the impact of increased temperature on meiosis have become even more important in the context of current climate change, when sexually reproducing organisms face rising temperatures.
In summary, our study provides an overview of changes in the levels and phosphorylation of proteins during the progression of S. pombe cells through meiotic divisions. Whether the specific changes in the levels and phosphorylation of proteins identified in this study are important for the proper progression of S. pombe cells through meiosis is an intriguing question for future investigation.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD053920.
References
Bolte, M. et al. Inhibition of APC-mediated proteolysis by the meiosis-specific protein kinase Ime2. Proc. Natl. Acad. Sci. U S A. 99 (7), 4385–4390 (2002).
Benjamin, K. R. et al. Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2. Genes Dev. 17 (12), 1524–1539 (2003).
Yamamoto, M. The selective elimination of messenger RNA underlies the mitosis-meiosis switch in fission yeast. Proc. Jpn Acad. Ser. B Phys. Biol. Sci. 86 (8), 788–797 (2010).
Kim Guisbert, K. S. et al. Meiosis-induced alterations in transcript architecture and noncoding RNA expression in S. Cerevisiae. RNA. 18 (6), 1142–1153 (2012).
Okaz, E. et al. Meiotic prophase requires proteolysis of M phase regulators mediated by the meiosis-specific APC/CAma1. Cell. 151 (3), 603–618 (2012).
Normant, V., Beaudoin, J. & Labbé, S. An antisense RNA-mediated mechanism eliminates a meiosis-specific copper-regulated transcript in mitotic cells. J. Biol. Chem. 290 (37), 22622–22637 (2015).
Mata, J. et al. The transcriptional program of meiosis and sporulation in fission yeast. Nat. Genet. 32 (1), 143–147 (2002).
Dunce, J. M. et al. Structural basis of meiotic chromosome synapsis through SYCP1 self-assembly. Nat. Struct. Mol. Biol. 25 (7), 557–569 (2018).
Syrjanen, J. L., Pellegrini, L. & Davies, O. R. A molecular model for the role of SYCP3 in meiotic chromosome organisation. eLife. 3, e02963 (2014).
Crichton, J. H. et al. Structural maturation of SYCP1-mediated meiotic chromosome synapsis by SYCE3. Nat. Struct. Mol. Biol. 30 (2), 188–199 (2023).
Scherthan, H., Bähler, J. & Kohli, J. Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast. J. Cell. Biol. 127 (2), 273–285 (1994).
Lorenz, A. et al. S. Pombe meiotic linear elements contain proteins related to synaptonemal complex components. J. Cell. Sci. 117 (Pt 15), 3343–3351 (2004).
Loidl, J. S. Pombe linear elements: the modest cousins of synaptonemal complexes. Chromosoma. 115 (3), 260–271 (2006).
Cloud, V. et al. Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science. 337 (6099), 1222–1225 (2012).
Hinch, A. G. et al. The configuration of RPA, RAD51, and DMC1 binding in meiosis reveals the nature of critical recombination intermediates. Mol. Cell. 79 (4), 689–701 (2020).
Watanabe, Y. et al. Pre-meiotic S phase is linked to reductional chromosome segregation and recombination. Nature. 409 (6818), 359–363 (2001).
Sakuno, T. & Watanabe, Y. Studies of meiosis disclose distinct roles of cohesion in the core centromere and pericentromeric regions. Chromosome Res. 17 (2), 239–249 (2009).
Ogushi, S. et al. Loss of sister kinetochore co-orientation and peri-centromeric cohesin protection after meiosis I depends on cleavage of centromeric REC8. Dev. Cell. 56 (22), 3100–3114e4 (2021).
Kitajima, T. S. et al. Distinct cohesin complexes organize meiotic chromosome domains. Science. 300 (5622), 1152–1155 (2003).
Ishiguro, K. et al. A new meiosis-specific cohesin complex implicated in the cohesin code for homologous pairing. EMBO Rep. 12 (3), 267–275 (2011).
Clyne, R. K. et al. Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I. Nat. Cell. Biol. 5 (5), 480–485 (2003).
Henderson, K. A. et al. Cyclin-dependent kinase directly regulates initiation of meiotic recombination. Cell. 125 (7), 1321–1332 (2006).
Carballo, J. A. et al. Phosphorylation of the axial element protein Hop1 by Mec1/Tel1 ensures meiotic interhomolog recombination. Cell. 132 (5), 758–770 (2008).
Cartagena-Lirola, H. et al. Role of the Saccharomyces cerevisiae Rad53 checkpoint kinase in signaling double-strand breaks during the meiotic cell cycle. Mol. Cell. Biol. 28 (14), 4480–4493 (2008).
Penedos, A. et al. Essential and checkpoint functions of budding yeast ATM and ATR during meiotic prophase are facilitated by Differential Phosphorylation of a meiotic adaptor protein, Hop1. PLoS ONE. 10 (7), e0134297 (2015).
Nadarajan, S. et al. Polo-like kinase-dependent phosphorylation of the synaptonemal complex protein SYP-4 regulates double-strand break formation through a negative feedback loop. eLife. 6, e23437 (2017).
Tougan, T. et al. The Mek1 phosphorylation cascade plays a role in meiotic recombination of Schizosaccharomyces Pombe. Cell. Cycle. 9 (23), 4688–4702 (2010).
Falk, J. E. et al. A Mec1- and PP4-dependent checkpoint couples centromere pairing to meiotic recombination. Dev. Cell. 19 (4), 599–611 (2010).
Nolt, J. K. et al. PP2A (cdc)⁵⁵ is required for multiple events during meiosis I. Cell. Cycle. 10 (9), 1420–1434 (2011).
Sato-Carlton, A. et al. Protein phosphatase 4 promotes chromosome pairing and synapsis, and contributes to maintaining crossover competence with increasing age. PLoS Genet. 10 (10), e1004638 (2014).
Barbosa, P. et al. SCF-Fbxo42 promotes synaptonemal complex assembly by downregulating PP2A-B56. J. Cell. Biol. 220 (2), e202009167 (2021).
Murakami, H. & Keeney, S. Temporospatial coordination of meiotic DNA replication and recombination via DDK recruitment to replisomes. Cell. 158 (4), 861–873 (2014).
Kee, K. et al. Spatial organization and dynamics of the association of Rec102 and Rec104 with meiotic chromosomes. EMBO J. 23 (8), 1815–1824 (2004).
Kim, Y., Kostow, N. & Dernburg, A. F. The chromosome Axis mediates Feedback Control of CHK-2 to ensure crossover formation in C. Elegans. Dev. Cell. 35 (2), 247–261 (2015).
Penkner, A. M. et al. Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1. Cell. 139 (5), 920–933 (2009).
Woglar, A. et al. Matefin/SUN-1 phosphorylation is part of a surveillance mechanism to coordinate chromosome synapsis and recombination with meiotic progression and chromosome movement. PLoS Genet. 9 (3), e1003335 (2013).
Jordan, P. W., Karppinen, J. & Handel, M. A. Polo-like kinase is required for synaptonemal complex disassembly and phosphorylation in mouse spermatocytes. J. Cell. Sci. 125 (Pt 21), 5061–5072 (2012).
Nadarajan, S. et al. The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis. eLife. 5, e12039 (2016).
Ishiguro, T. et al. Shugoshin-PP2A counteracts casein-kinase-1-dependent cleavage of Rec8 by separase. Nat. Cell. Biol. 12 (5), 500–506 (2010).
Ma, W. et al. Meikin synergizes with shugoshin to protect cohesin Rec8 during meiosis I. Genes Dev. 35 (9–10), 692–697 (2021).
Yokobayashi, S. & Watanabe, Y. The kinetochore protein Moa1 enables cohesion-mediated monopolar attachment at meiosis I. Cell. 123 (5), 803–817 (2005).
Liu, Y. et al. Phosphorylation of Rec8 cohesin complexes regulates mono-orientation of kinetochores in meiosis I. Life Sci. Alliance. 7 (5), e202302556 (2024).
Hayles, J. & Nurse, P. Introduction to Fission Yeast as a Model System. Cold Spring Harb Protoc. ; 2018(5). (2018).
Vo, T. V. et al. A proteome-wide fission yeast interactome reveals Network Evolution principles from yeasts to Human. Cell. 164 (1–2), 310–323 (2016).
Watanabe, Y. et al. Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast. Nature. 386 (6621), 187–190 (1997).
Bähler, J. et al. Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells. Curr. Genet. 19 (6), 445–451 (1991).
Yamamoto, A. & Hiraoka, Y. Monopolar spindle attachment of sister chromatids is ensured by two distinct mechanisms at the first meiotic division in fission yeast. EMBO J. 22 (9), 2284–2296 (2003).
Chikashige, Y. et al. Meiosis induced by inactivation of Pat1 kinase proceeds with aberrant nuclear positioning of centromeres in the fission yeast Schizosaccharomyces Pombe. Genes Cells. 9 (8), 671–684 (2004).
Asakawa, H. et al. Dissociation of the Nuf2-Ndc80 complex releases centromeres from the spindle-pole body during meiotic prophase in fission yeast. Mol. Biol. Cell. 16 (5), 2325–2338 (2005).
Cipak, L. et al. ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature. Cell. Cycle. 11 (8), 1626–1633 (2012).
Cipak, L. et al. Synchronized fission yeast meiosis using an ATP analog-sensitive Pat1 protein kinase. Nat. Protoc. 9 (1), 223–231 (2014).
Guerra-Moreno, A. et al. Chemical genetic induction of meiosis in Schizosaccharomyces Pombe. Cell. Cycle. 11 (8), 1621–1625 (2012).
Nambu, M. et al. Direct evaluation of cohesin-mediated sister kinetochore associations at meiosis I in fission yeast. J. Cell. Sci. 135 (1), jcs259102 (2022).
Krapp, A. et al. Analysis of the S. Pombe Meiotic Proteome reveals a switch from anabolic to catabolic processes and extensive post-transcriptional regulation. Cell. Rep. 26 (4), 1044–1058e5 (2019).
Huraiova, B. et al. Proteomic analysis of meiosis and characterization of novel short open reading frames in the fission yeast Schizosaccharomyces Pombe. Cell. Cycle. 19 (14), 1777–1785 (2020).
Rutherford, K. M., Lera-Ramírez, M. & Wood, V. PomBase: a global core Biodata Resource-growth, collaboration, and sustainability. Genetics. 227 (1), iyae007 (2024).
Anrather, D. et al. SILAC-Based proteomic analysis of meiosis in the fission yeast Schizosaccharomyces Pombe. Methods Mol. Biol. 2603, 19–29 (2023).
Hughes, C. S. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 14 (1), 68–85 (2019).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26 (12), 1367–1372 (2008).
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 13 (9), 731–740 (2016).
Szklarczyk, D. et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 51 (D1), D638–D646 (2023).
Tang, D. et al. SRplot: a free online platform for data visualization and graphing. PLoS One. 18 (11), e0294236 (2023).
Malapeira, J. et al. A meiosis-specific cyclin regulated by splicing is required for proper progression through meiosis. Mol. Cell. Biol. 25 (15), 6330–6337 (2005).
Averbeck, N. et al. Negative control contributes to an extensive program of meiotic splicing in fission yeast. Mol. Cell. 18 (4), 491–498 (2005).
Carpy, A. et al. Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces Pombe (Fission yeast). Mol. Cell. Proteom. 13 (8), 1925–1936 (2014).
Swaffer, M. P. et al. Quantitative Phosphoproteomics reveals the Signaling Dynamics of Cell-Cycle Kinases in the fission yeast Schizosaccharomyces Pombe. Cell. Rep. 24 (2), 503–514 (2018).
Sivakova, B. et al. Label-free quantitative phosphoproteomics of the fission yeast Schizosaccharomyces Pombe using strong Anion Exchange- and porous Graphitic Carbon-based fractionation strategies. Int. J. Mol. Sci. 22 (4), 1747 (2021).
Wen, F. P. et al. Distinct temporal requirements for autophagy and the proteasome in yeast meiosis. Autophagy. 12 (4), 671–688 (2016).
Becker, E. et al. The protein expression landscape of mitosis and meiosis in diploid budding yeast. J. Proteom. 156, 5–19 (2017).
Cunliffe, L., White, S. & McInerny, C. J. DSC1-MCB regulation of meiotic transcription in Schizosaccharomyces Pombe. Mol. Genet. Genomics. 271 (1), 60–71 (2004).
Sakuno, T. & Watanabe, Y. Phosphorylation of cohesin Rec11/SA3 by casein kinase 1 promotes homologous recombination by assembling the meiotic chromosome axis. Dev. Cell. 32 (2), 220–230 (2015).
Phadnis, N. et al. Casein kinase 1 and phosphorylation of Cohesin Subunit Rec11 (SA3) promote meiotic recombination through Linear element formation. PLoS Genet. 11 (5), e1005225 (2015).
Forsburg, S. L. & Hodson, J. A. Mitotic replication initiation proteins are not required for pre-meiotic S phase. Nat. Genet. 25 (3), 263–268 (2000).
Kariyazono, R. et al. Conserved HORMA domain-containing protein Hop1 stabilizes interaction between proteins of meiotic DNA break hotspots and chromosome axis. Nucleic Acids Res. 47 (19), 10166–10180 (2019).
Gregan, J. et al. Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast. Curr. Biol. 15 (18), 1663–1669 (2005).
Funabiki, H. et al. Cut2 proteolysis required for sister-chromatid separation in fission yeast. Nature. 381 (6581), 438–441 (1996).
Funabiki, H., Kumada, K. & Yanagida, M. Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes. EMBO J. 15 (23), 6617–6628 (1996).
Kitajima, T. S. et al. Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast. EMBO J. 22 (20), 5643–5653 (2003).
Abe, H. & Shimoda, C. Autoregulated expression of Schizosaccharomyces Pombe meiosis-specific transcription factor Mei4 and a genome-wide search for its target genes. Genetics. 154 (4), 1497–1508 (2000).
Okuzaki, D. et al. Fission yeast meu14 + is required for proper nuclear division and accurate forespore membrane formation during meiosis II. J. Cell. Sci. 116 (Pt 13), 2721–2735 (2003).
Nakamura, T. et al. ADAM family protein Mde10 is essential for development of spore envelopes in the fission yeast Schizosaccharomyces Pombe. Eukaryot. Cell. 3 (1), 27–39 (2004).
Nakamura, T. et al. Novel fission yeast Cdc7-Dbf4-like kinase complex required for the initiation and progression of meiotic second division. Mol. Cell. Biol. 22 (1), 309–320 (2002).
Onishi, M. et al. Role of septins in the orientation of forespore membrane extension during sporulation in fission yeast. Mol. Cell. Biol. 30 (8), 2057–2074 (2010).
Nakamura, T., Kishida, M. & Shimoda, C. The Schizosaccharomyces Pombe spo6 + gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation. Genes Cells. 5 (6), 463–479 (2000).
Gregan, J. et al. The kinetochore proteins Pcs1 and Mde4 and heterochromatin are required to prevent merotelic orientation. Curr. Biol. 17 (14), 1190–1200 (2007).
Choi, S. H. et al. Phosphorylation state defines discrete roles for monopolin in chromosome attachment and spindle elongation. Curr. Biol. 19 (12), 985–995 (2009).
Hartwell, L. H. et al. Genetic control of the Cell Division cycle in yeast: V. Genetic analysis of cdc mutants. Genetics. 74 (2), 267–286 (1973).
Murayama, Y. & Uhlmann, F. DNA entry into and exit out of the Cohesin Ring by an Interlocking Gate mechanism. Cell. 163 (7), 1628–1640 (2015).
Chikashige, Y. & Hiraoka, Y. Telomere binding of the Rap1 protein is required for meiosis in fission yeast. Curr. Biol. 11 (20), 1618–1623 (2001).
Grishchuk, A. L. & Kohli, J. Five RecA-like proteins of Schizosaccharomyces Pombe are involved in meiotic recombination. Genetics. 165 (3), 1031–1043 (2003).
Bernard, P., Maure, J. F. & Javerzat, J. P. Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation. Nat. Cell. Biol. 3 (5), 522–526 (2001).
Rumpf, C. et al. Casein kinase 1 is required for efficient removal of Rec8 during meiosis I. Cell. Cycle. 9 (13), 2657–2662 (2010).
Jun, H. I. et al. Tpz1 controls a telomerase-nonextendible telomeric state and coordinates switching to an extendible state via Ccq1. Genes Dev. 27 (17), 1917–1931 (2013).
Murakami, H. & Nurse, P. Regulation of premeiotic S phase and recombination-related double-strand DNA breaks during meiosis in fission yeast. Nat. Genet. 28 (3), 290–293 (2001).
Lindner, K. et al. Essential role of MCM proteins in premeiotic DNA replication. Mol. Biol. Cell. 13 (2), 435–444 (2002).
Hayashi, A. et al. Reconstruction of the kinetochore during meiosis in fission yeast Schizosaccharomyces Pombe. Mol. Biol. Cell. 17 (12), 5173–5184 (2006).
Shiroiwa, Y. et al. Mis17 is a regulatory module of the Mis6-Mal2-Sim4 centromere complex that is required for the recruitment of CenH3/CENP-A in fission yeast. PLoS One. 6 (3), e17761 (2011).
Fu, C. et al. Phospho-regulated interaction between kinesin-6 Klp9p and microtubule bundler Ase1p promotes spindle elongation. Dev. Cell. 17 (2), 257–267 (2009).
Meadows, J. C. et al. Identification of a Sgo2-Dependent but Mad2-Independent Pathway Controlling Anaphase Onset in Fission yeast. Cell. Rep. 18 (6), 1422–1433 (2017).
Yan, Z. et al. A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability. Mol. Cell. 37 (6), 865–878 (2010).
Kimata, Y. et al. Mes1 controls the meiosis I to meiosis II transition by distinctly regulating the anaphase-promoting complex/cyclosome coactivators Fzr1/Mfr1 and Slp1 in fission yeast. Mol. Biol. Cell. 22 (9), 1486–1494 (2011).
Liu, J., Tang, X., Wang, H. & Balasubramanian, M. Bgs2p, a 1,3-beta-glucan synthase subunit, is essential for maturation of ascospore wall in Schizosaccharomyces Pombe. FEBS Lett. 478 (1–2), 105–108 (2000).
de Medina-Redondo, M. et al. The beta-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces Pombe. Mol. Microbiol. 68 (5), 1283–1299 (2008).
García, P. et al. Fission yeast rgf2p is a rho1p guanine nucleotide exchange factor required for spore wall maturation and for the maintenance of cell integrity in the absence of rgf1p. Genetics. 181 (4), 1321–1334 (2009).
Mikolaskova, B. et al. Identification of Nrl1 domains responsible for interactions with RNA-Processing factors and regulation of Nrl1 function by phosphorylation. Int. J. Mol. Sci. 22 (13), 7011 (2021).
Yamashita, Y. M. et al. 20S cyclosome complex formation and proteolytic activity inhibited by the cAMP/PKA pathway. Nature. 384 (6606), 276–279 (1996).
Kominami, K., Seth-Smith, H. & Toda, T. Apc10 and Ste9/Srw1, two regulators of the APC-cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell-cycle arrest in fission yeast. EMBO J. 17 (18), 5388–5399 (1998).
Fontebasso, Y. et al. The conserved fanconi anemia nuclease Fan1 and the SUMO E3 ligase Pli1 act in two novel Pso2-independent pathways of DNA interstrand crosslink repair in yeast. DNA Repair. (Amst). 12 (12), 1011–1023 (2013).
Kovacikova, I. et al. A knockout screen for protein kinases required for the proper meiotic segregation of chromosomes in the fission yeast Schizosaccharomyces Pombe. Cell. Cycle. 12 (4), 618–624 (2013).
Ogino, K. et al. Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase. J. Biol. Chem. 276 (33), 31376–31387 (2001).
Wan, L. et al. Cdc28-Clb5 (CDK-S) and Cdc7-Dbf4 (DDK) collaborate to initiate meiotic recombination in yeast. Genes Dev. 22 (3), 386–397 (2008).
Matos, J. et al. Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis. Cell. 147 (1), 158–172 (2011).
Galander, S. et al. Reductional Meiosis I chromosome segregation is established by coordination of Key Meiotic Kinases. Dev. Cell. 49 (4), 526–541e5 (2019).
Krapp, A. et al. The Schizosaccharomyces Pombe septation initiation network (SIN) is required for spore formation in meiosis. J. Cell. Sci. 119 (Pt 14), 2882–2891 (2006).
Liang, P. et al. Phosphoregulation of DNA repair via the Rad51 auxiliary factor Swi5-Sfr1. J. Biol. Chem. 299 (8), 104929 (2023).
Sevcovicova, A. et al. Mapping and analysis of Swi5 and Sfr1 Phosphorylation sites. Genes (Basel). 12 (7), 1014 (2021).
Ogino, K. et al. Hsk1 kinase is required for induction of meiotic dsDNA breaks without involving checkpoint kinases in fission yeast. Proc. Natl. Acad. Sci. U S A. 103 (21), 8131–8136 (2006).
Cromie, G. & Smith, G. R. Meiotic recombination in Schizosaccharomyces Pombe: a paradigm for genetic and molecular analysis. Genome Dyn. Stab. 3, 195 (2008).
Rumpf, C. et al. Laser microsurgery provides evidence for merotelic kinetochore attachments in fission yeast cells lacking Pcs1 or Clr4. Cell. Cycle. 9 (19), 3997–4004 (2010).
Miyazaki, S. et al. Meikin-associated polo-like kinase specifies Bub1 distribution in meiosis I. Genes Cells. 22 (6), 552–567 (2017).
Kitajima, T. S., Kawashima, S. A. & Watanabe, Y. The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis. Nature. 427 (6974), 510–517 (2004).
Watanabe, Y. Shugoshin: guardian spirit at the centromere. Curr. Opin. Cell. Biol. 17 (6), 590–595 (2005).
Sakuno, T. et al. Rec8 cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination. Nucleic Acids Res. 50 (7), 3799–3816 (2022).
Sakuno, T. & Hiraoka, Y. Rec8 cohesin: a structural platform for shaping the meiotic chromosomes. Genes (Basel). 13 (2), 200 (2022).
Challa, K. et al. Meiosis-specific prophase-like pathway controls cleavage-independent release of cohesin by Wapl phosphorylation. PLoS Genet. 15 (1), e1007851 (2019).
Hu, C. et al. Structural insights into chromosome attachment to the nuclear envelope by an inner nuclear membrane protein Bqt4 in fission yeast. Nucleic Acids Res. 47 (3), 1573–1584 (2019).
Chikashige, Y. et al. Meiotic proteins bqt1 and bqt2 tether telomeres to form the bouquet arrangement of chromosomes. Cell. 125 (1), 59–69 (2006).
Loncar, A. et al. Kinesin-14 family proteins and microtubule dynamics define S. Pombe mitotic and meiotic spindle assembly, and elongation. J. Cell. Sci. 133 (11), jcs240234 (2020).
Kawashima, S. A. et al. Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin. Science. 327 (5962), 172–177 (2010).
Nakamura-Kubo, M. et al. The fission yeast pleckstrin homology domain protein Spo7 is essential for initiation of forespore membrane assembly and spore morphogenesis. Mol. Biol. Cell. 22 (18), 3442–3455 (2011).
Duncan, C. D. & Mata, J. The translational landscape of fission-yeast meiosis and sporulation. Nat. Struct. Mol. Biol. 21 (7), 641–647 (2014).
Wettstein, R. et al. Waves of regulated protein expression and phosphorylation rewire the proteome to drive gametogenesis in budding yeast. Dev. Cell. 59 (13), 1764–1782e8 (2024).
Koch, L. B. et al. Rewiring of the phosphoproteome executes two meiotic divisions in budding yeast. EMBO J. 43 (7), 1351–1383 (2024).
Bhagwat, N. R. et al. SUMO is a pervasive regulator of meiosis. Elife. 10, e57720 (2021).
Cheng, Z. et al. Pervasive, coordinated protein-level changes driven by transcript isoform switching during meiosis. Cell. 172 (5), 910–923e16 (2018).
Guo, Y. et al. Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in in vitro-matured oocytes. Mol. Cell. Proteom. 21 (8), 100267 (2022).
Jiang, Y. R. et al. Simultaneous deep transcriptome and proteome profiling in a single mouse oocyte. Cell. Rep. 42 (11), 113455 (2023).
Matzinger, M. & Mechtler, K. Improving single cell proteomics experiments: how can we best utilize latest-generation data acquisition and MS instrument architecture? Expert Rev. Proteom. 20 (10), 193–195 (2023).
Crhak Khaitova, L. et al. Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis. Elife. 12, RP90253 (2024).
De Jaeger-Braet, J. & Schnittger, A. Heating up meiosis - chromosome recombination and segregation under high temperatures. Curr. Opin. Plant. Biol. 80, 102548 (2024).
Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50 (D1), D543–D552 (2022).
Funding
This study was supported by the Slovak Research and Development Agency Contract No. APVV-20-0141 and Slovak Grant Agency VEGA, grant no. 2/0021/22. Furthermore, this research was supported by the Operational Program Integrated Infrastructure for the project: Center for Biomedical Research - BIOMEDIRES - II. stage, ITMS: 313011W428, co-financed by the European Regional Development Fund. The work was co-financed by the National Scholarship Programme of the Slovak Republic (https://www.stipendia.sk/). J.G. was supported by the Austrian Science Fund (FWF) (grant P30516).
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B.S.: investigation, methodology and data analysis; A.W.: investigation and methodology; M.K.: investigation and data analysis; J.J.: Investigation and methodology; J.G.: funding acquisition, writing—review and editing; K.K.: methodology, data analysis, supervision and funding acquisition, writing—review and editing; P.B.: methodology, data analysis, supervision, funding acquisition, writing—review and editing; L.C.: investigation, methodology, formal analysis, data analysis, conceptualization and writing—original draft, review and editing, supervision and funding acquisition.
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Sivakova, B., Wagner, A., Kretova, M. et al. Quantitative proteomics and phosphoproteomics profiling of meiotic divisions in the fission yeast Schizosaccharomyces pombe. Sci Rep 14, 23105 (2024). https://doi.org/10.1038/s41598-024-74523-0
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DOI: https://doi.org/10.1038/s41598-024-74523-0