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First published online 14 April 2008
doi: 10.1242/jcs.022830

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Mug27 is a meiosis-specific protein kinase that functions in fission yeast meiosis II and sporulation

Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan

View larger version (33K): [in this window] [in a new window]   Fig. 1. Mug27 is a meiosis-specific serine/threonine protein kinase that is conserved in various species. (A) Schematic representation of Mug27 and of other members of the NDR family of proteins. The predicted protein-kinase domains (black box) are indicated. The nuclear-localization signal of Mug27 is also indicated (gray box). These motifs were identified by PSORT II (http://psort.nibb.ac.jp/) and Pfam (http://www.sanger.ac.uk/Software/Pfam). Hs, Homo sapien; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans. (B) The phylogenetic tree of Mug27. The relationships between the orthologous proteins were inferred by the neighbor-joining method. The numbers represent the phylogenetic distance. Both the sequence and phylogenetic analyses were performed using a GENETYX program (Software Development Co., Ltd). Tb, Trypanosoma brucei. (C) Western blot analysis of the production of Mug27-9Myc and Meu13 (meiotic timing control) proteins during the synchronous meiosis of strain SOP015. Tubulin levels were also examined as a loading control. (D) Northern blot analysis of mug27+, sid2+ and leu1+ (loading control) expression. Total RNA was extracted from CD16-1 (h+/h–) and CD16-5 (h–/h–) cells at the indicated times after nitrogen starvation. The former but not the latter strain enters meiosis upon nitrogen starvation. The RNA was blotted and probed with the ORFs of mug27+ and leu1+. The graphs indicates the meiotic profiles of the cells used for RNA extraction. The progression of meiosis was monitored every 2 hours after nitrogen starvation. The cells with one, two, three or four nuclei were enumerated by counting the Hoechst-33342-stained nuclei. At least 200 cells were counted under the microscope.

View larger version (76K): [in this window] [in a new window]   Fig. 2. Mug27 is a meiosis-specific SPB-associated protein. (A,B) Mug27 subcellular localization during meiosis relative to Sid4-mRFP (A) and Sad1-mCherry (B) expression as observed by fluorescence microscopy. The mug27+-gfp sid4+-mrfp strain (SOP028; A) and the mug27+-gfp sad1+-mCherry strain (SOP062; B) were induced to enter meiosis by nitrogen starvation. 10 hours later, the cells were collected and stained with Hoechst 33342 to visualize the DNA (blue). The GFP signal is green, and the mRFP and mCherry signals are red. (B; lowermost panels) Enlarged views of the merged images at early anaphase II (left) and late anaphase II (right) at the areas indicated by the rectangles are shown. (C) Mug27-GFP does not localize to the SPB in mitotic cells. Mitotic cells were transformed with empty pREP1-GFP (GFP expression vector; SOP065) or pREP1-mug27+-GFP (Mug27-GFP expression vector; SOP066), which overproduce (OP) the encoded protein. The sid2+-gfp sad1+-mCherry strain (SOP067) served as an SPB colocalization control.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Accurate positioning of Mug27 at anaphase II depends on proper FSM formation. (A) The homothallic haploid strain SOP069 (h90 mug27+-gfp sad1+-mCherry spo15) was cultured in EMM2 with appropriate supplements and then transferred to EMM-N to induce meiosis. 10 hours later, cells at different stages of meiosis were stained with Hoechst 33342. The bar graphs were drawn by determining the Mug27-GFP localization in 69 spo15+ and 87 spo15 cells, and then plotting the frequencies of the indicated patterns. (B) The haploid strains SOP044 (h– mug27+-gfp pat1-114; i) and SOP079 (h– mug27+-gfp cdc11-123 pat1-114; ii) were monitored after shifting the temperature to induce meiosis. 5 hours later, cells at different stages of meiosis were stained with Hoechst 33342 (blue). Scale bars: 5 µm. (C) The bar graphs were drawn by determining the Mug27-GFP localization in 114 pat1-114 and 112 cdc11-123 pat1-114 cells, and then plotting the frequencies of the indicated patterns.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Mug27 is required for proper spore formation. (A,B) The mug27 and mug27(KD) mutants generate small spores in a temperature-sensitive manner, with a significant loss of viability. The mug27+ (SOP091w), mug27 (SOP095), mug27-3HA (SOP101) and mug27(KD)-3HA (SOP100) strains were induced to enter meiosis at 33°C (high temperature), 28°C (suitable temperature) or 25°C (low temperature). (A) DIC images of the asci are shown. The mean lengths of the spores and the standard deviations analyzed by MetaMorph software (Universal Imaging Corp.) are indicated. Scale bars: 10 µm. The bar graph is a histogram of the spore diameter. (B) Spore viability was measured by random spore analysis. (C) Abnormal nuclei and FSMs are observed frequently in mug27 spores in a temperature-sensitive manner. GFP-Psy1-expressing mug27+ (SOP091w) and mug27 (SOP095) strains were induced to enter meiosis at 33°C, 28°C or 25°C by nitrogen starvation. After 10 hours, the cells were stained with Hoechst 33342 (blue) and observed by fluorescence microscopy. (i) Typical images of sporulating cells are shown. Scale bar: 10 µm. (ii) Enlarged images of the rightmost panels in i. Each nucleus is successfully engulfed by the FSM in mug27+ cells (arrow), whereas some nuclei protrude (asterisk) or escape (triangle) from the FSM in mug27 cells. (iii) Quantitative analysis of the mug27+ and mug27 cells at sporulation is summarized by the bar graph. Each colored area represents the cell populations harboring the indicated number of haploid nuclei that showed normal or abnormal localizations. The bar graphs were generated by counting the nuclei of 124 mug27+ (28°C), 139 mug27 (33°C), 126 mug27 (28°C) and 125 mug27 (25°C) cells. (D) SPBs are abnormally separated from the FSM in mug27 cells. Cells expressing GFP-Psy1, Meu14-GFP and Sad1-mCherry (mug27+, SOP117w; mug27, SOP118) were induced to enter meiosis by nitrogen starvation at 25°C. After 9.5 (i), 10 (ii) or 12 (iii) hours, the cells were stained with Hoechst 33342 and observed by fluorescence microscopy. Typical images are shown. Dark green is GFP-Psy1, bright green is Meu14-GFP, red is Sad1-mCherry and blue is DNA stained by Hoechst 33342. (ii) Frequency of SPB association with the FSM at late anaphase II. The pink and red areas in the graph indicate the population of cells that harbor SPBs associating or not associating with the FSM, respectively. (iii) Frequency of cells that harbor SPBs separated from the FSM at sporulation. Asci were classified into three types (type I, II and III) and their relative frequencies are shown by pink (type I), orange (type II), red (type III) or green (type II plus type III), respectively.

View larger version (42K): [in this window] [in a new window]   Fig. 5. Mug27 is required for FSM development. (A) The formation of the FSM in mug27+ (SOP091w) and mug27 (SOP095) cells during spore formation was visualized by examining the behavior of GFP-Psy1. Graphs show that the frequency of abnormal FSM growth in mug27+, mug27-3HA (SOP101), mug27 and mug27(KD)-3HA (SOP100) cells 20 hours after meiosis was increased. Typical images are shown in the right-hand panels. Abnormal localization of GFP-Psy1 to the cell cortex in a mug27 cell is indicated by the arrowheads. (B) Time-lapse images of the GFP-Psy1 and Meu14-GFP proteins in mug27+ (SOP091w; i) and mug27 (SOP095; ii) cells undergoing FSM formation at 28°C. These images show a subset of the GFP images that were captured every 2 minutes. The time in minutes, with 0 minutes being the time at which FSM formation begins, is indicated at the bottom of each photograph. Scale bars: 10 µm. (C,D) Comparison of the time taken for the FSM to form in mug27+ and mug27 cells. (C) Graph shows the time taken for the cells to proceed from metaphase II to closure of the Meu14 ring (n=50 for each cell type); (D) graph shows the duration of the two FSM-formation phases depicted in the upper panel of D. The FSM-formation process was divided into two phases as follows: phase I, from the initiation of FSM formation to the time at which the diameter of the Meu14-GFP ring is maximal; phase II, the time at which the large Meu14-GFP ring begins to reduce in size until FSM formation is complete. The depictions indicate how GFP-Psy1 and Meu14-GFP move during FSM formation in S. pombe. (E) Comparison between mug27+ and mug27 cells of the maximum diameter of the Meu14 ring. The maximum diameter of Meu14 was measured by MetaMorph software (n=84 for each cell type).

View larger version (59K): [in this window] [in a new window]   Fig. 6. Vacuole morphology during sporulation. GFP-Ypt7-expressing mug27+ (SOP122w) and mug27 (SOP123) cells were cultured in EMM2, then induced to enter meiosis by nitrogen starvation at 25°C. 10 hours later, cells at different stages of meiosis were stained with FM4-64 and Hoechst 33342. The formation of the FSM in mug27+ (i) and mug27 (ii) cells during spore formation was visualized using fluorescence microscopy to monitor the behavior of FM4-64 and the vacuole membranes. Scale bar: 10 µm. (iii) Cells in which vacuole-membrane fusion was observed (yellow) or not (green) at late sporulation in mug27+ (n=161) or mug27 (n=162) cells, respectively. Typical images are shown in the upper panel.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Overexpression of sid2+ suppressed the abnormal phenotypes of mug27 cells. (A,B) The mug27+ (NP40-1C), mug27 (SOP023), mug27 pREP1-sid2-GFP (SOP115) and mug27 pREP1-GFP (SOP114) strains were cultured in EMM2 containing 1 µg/ml thiamine with supplements and then transferred to EMM2 without thiamine to induce the expression of Sid2-GFP or GFP proteins. Subsequently, 20 hours after the first medium replacement, the cells were transferred to fresh EMM2-N without thiamin to induce meiosis at 28°C. (A) DIC images of the asci are shown. Scale bar: 10 µm. Histogram shows spore diameter. The mean lengths of the spores and the standard deviations were analyzed by MetaMorph software (Universal Imaging Corp.). (B) Spore viability, measured by random spore analysis.

View larger version (31K): [in this window] [in a new window]   Fig. 8. A model for the function of Mug27 during FSM formation. During sporulation in S. pombe, the Mug27 complex localizes to the SPB as a complex that is partially distinct from the SIN. Unlike the SIN, which always localizes to the SPB, Mug27 also localizes to the extending FSM and regulates the normal formation of the FSM by phosphorylation of unknown targets.

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