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

Journal of Cell Science 121, 1547-1558 (2008)
Published by The Company of Biologists 2008
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Research Article

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

Ayami Ohtaka, Daisuke Okuzaki and Hiroshi Nojima*

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

* Author for correspondence (e-mail: snj-0212{at}biken.osaka-u.ac.jp)

Accepted 18 February 2008


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Several meiosis-specific proteins of Schizosaccharomyces pombe play essential roles in meiotic progression. We report here that a novel meiosis-specific protein kinase, Mug27 (also known as Ppk35), is required for proper spore formation. This kinase is expressed by the mug27+ gene, which is abruptly transcribed after horsetail movement. This transcription is maintained until the second meiotic division. Green fluorescent protein (GFP)-tagged Mug27 appears at the start of prometaphase I, localizes to the spindle pole body (SPB) and then translocates to the forespore membrane (FSM) at late anaphase II. In the mug27{Delta} strain, smaller spores are produced compared with those of the mug27+ strain. Moreover, spore viability was reduced by half or more compared with that of the mug27+ strain. The protein-kinase activity of Mug27 appears to be important for its function: the putative kinase-dead Mug27 mutant had similar phenotypes to mug27{Delta}. Our results here indicate that the Mug27 kinase localizes at the SPB and regulates FSM formation and sporulation.

Key words: Meiosis, Kinase, Forespore membrane, Spindle pole body (SPB), Schizosaccharomyces pombe


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The fission yeast Schizosaccharomyces pombe is an ideal model system in which to study the meiotic process at the molecular level because it is easy to induce meiosis in this species and meiotic progress can be analyzed at the single-cell level. Fission yeast cells, unlike cells of the budding yeast Saccharomyces cerevisiae, are most stable in the haploid state and are essentially asexual under rich nutritional conditions. However, when fission yeast cells are starved of nutrients, especially nitrogen, haploid cells with opposite mating types form zygotes (Egel, 1989Go), undergo meiosis and then form a double-layered forespore membrane (FSM). FSMs begin to form on the cytoplasmic side of the modified spindle pole body (SPB) and extend by fusing with membranous vesicles derived from the endoplasmic reticulum via the Golgi network, going on to wrap each divided nucleus (Nakamura et al., 2001Go). Vacuole fusion in the late stage of sporulation might be important for spore maturation (Kashiwazaki et al., 2005Go). The inner membrane of the FSM becomes the plasma membrane of the newborn spore (reviewed in Shimoda, 2004Go). After the spore wall is successfully organized, the mature spores are released from the ascus by autolysis of the ascus cell wall. When these spores are placed in rich nutritional conditions, they keep growing and do not undergo conjugation unless they are starved of nutrients. To permit error-free segregation of the chromosomes during meiotic cell division, internal compartmentalization and meiotic nuclear division must be properly coordinated. A key structure that links these two events is the SPB. The SPB is required not only for meiotic spindle assembly, in which it acts as a microtubule-organizing center, but also for FSM formation, because sporulation is totally abolished when SPB modification is blocked by a mutation of the SPB component Spo15 (Ikemoto et al., 2000Go). The SPB might therefore serve as a platform that coordinates nuclear division and pre-spore formation.

A number of the genes that are required for the meiotic events described above have been cloned, and their functions during meiosis and/or sporulation have been analyzed (Shimoda, 2004Go; Ohtaka et al., 2007aGo). However, compared with the mitotic cell cycle, our knowledge of the molecular mechanisms that regulate meiosis and sporulation is limited. To alleviate this problem, we sought to comprehensively identify the meiosis-specific protein kinase using the S. pombe genome database (Wood et al., 2002Go) (http://www.genedb.org/genedb/pombe/index.jsp) and found 182 open reading frames (ORFs) that encode proteins containing a Ser/Thr kinase domain. According to the S. pombe genome-wide transcriptome analysis (Mata et al., 2002Go), expression of 34 of these 182 ORFs is upregulated during meiosis. Among these, we focused on the functional analysis of mug27+ (ppk35+) (Martín-Castellanos et al., 2005Go; Bimbó et al., 2005Go) because it is a homolog of Sid2, which is required for mitotic exit.

Mug27 belongs to the evolutionarily conserved nuclear Dbf2-related (NDR) kinase family, members of which are essential components of the pathways that control morphological changes, mitotic exit, cytokinesis, cell proliferation and apoptosis (reviewed by Hergovich et al., 2006Go). The NDR family members of Saccharomyces cerevisiae (Dbf2 and Dbf20) participate in the mitotic exit network (MEN), whereas S. pombe NDR family members (Sid2) act in a similar signaling pathway, known as the septation initiation network (SIN) (reviewed by Bardin and Amon, 2001Go; McCollum and Gould, 2001Go; Bosl and Li, 2005Go). MEN and SIN regulate crucial events in the cell cycle. For example, the SIN components in fission yeast localize to the SPB and function both at the end of mitosis, when they trigger the contraction of the acto-myosin ring, and in meiosis, when they regulate spore formation (reviewed by Wolfe and Gould, 2005Go; Krapp et al., 2004Go; Krapp et al., 2006Go). The core components of the SIN are three protein kinases (Cdc7, Sid1 and Sid2) and their associated regulatory and/or targeting subunits (Spg1, Cdc14 and Mob1), which assemble at the SPB on a scaffold composed of Sid4 and Cdc11. In particular, Sid2 plays a pivotal role in the SIN (McCollum and Gould, 2001Go). However, little is known about the function of Mug27, a meiotic counterpart of Sid2, during meiosis. Here we report that Mug27 kinase localizes to the SPB and plays a pivotal role in FSM formation and sporulation.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Mug27 is a meiosis-specific protein kinase that is conserved in a variety of species
Mug27 consists of 624 amino acids and harbors a putative protein-kinase domain (amino acids 162-465) and a nuclear-localization signal (89-95; PETRRKR) (Fig. 1A). Using the BLAST algorithm (http://www.genome.ad.jp/), we found that Mug27 is a putative ortholog of the NDR kinase family. The NDR family is evolutionarily conserved, and members can be found in Drosophila melanogaster [Trc (tricornered) and Lats (also known as Warts)], Caenorhabditis elegans [sensory axon guidance-1 (SAX-1) and LATS], S. cerevisiae (Dbf2, Dbf20 and Cbk1), S. pombe (Sid2 and Orb6), and other fungi, protozoa and plants (Fig. 1A). The phylogenic tree constructed by the neighbor-joining method (Saitou and Nei, 1987Go) indicates that the amino acid sequences of these Mug27 orthologs are closely related to one another (Fig. 1B). These observations suggest that Mug27 might play a conserved role in different species.


Figure 1
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  		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.

 

To accurately examine the expression of the Mug27 protein during meiosis, we constructed the mug27+-9myc strain, which expresses Mug27 protein tagged with nine copies of the Myc epitope at its C-terminal end. To attain synchronous meiosis, we used the pat1-114 temperature-sensitive strain, which enters meiosis in a highly synchronous manner when it is shifted to the restrictive temperature (Iino and Yamamoto, 1985Go). Thus, we replaced the mug27+ gene of the pat1-114 strain with the mug27+-9myc fusion gene. pat1-114 mug27+-9myc diploid cells were then induced to enter synchronized meiosis by a temperature shift, and their lysates were subjected to western blot analysis using the anti-Myc antibody (PL14). We first confirmed that the pat1-114 mug27+-9myc and pat1-114 diploid cells underwent meiotic progression in a similar fashion and produced similar-looking spores (data not shown). The western blot analysis showed that the Mug27-9Myc protein was the expected size and was only expressed during meiosis, with a peak being observed at 4-5 hours after the temperature shift, i.e. during the meiotic nuclear divisions (Fig. 1C). As a control that showed the timing of meiosis, we used the meiosis-specific protein Meu13, which plays a pivotal role in homologous pairing and meiotic recombination at meiosis I (Nabeshima et al., 2001Go), and regulates the meiotic recombination checkpoint (Shimada et al., 2002Go).

To examine the meiosis-specific transcription of mug27+, we also performed northern blot analysis of RNA obtained from CD16-1 (h+/h–) and CD16-5 (h–/h–) cells harvested at various times after the induction of meiosis by nitrogen starvation. In this experiment, we took advantage of the fact that the heterozygous CD16-1 strain initiates meiosis upon nitrogen starvation, whereas the homozygous CD16-5 strain does not. This analysis revealed that mug27+ displays meiosis-specific transcription that peaks at about 10 hours after the medium change (Fig. 1D). However, it is likely that the Mug27-9Myc protein-expression pattern shown in Fig. 1C is more accurate than this mug27+ gene-expression pattern because highly synchronized meiosis can be achieved when the pat1-114 strain is used.

Mug27-GFP localizes at the SPB and at the FSM-like structure during nuclear division
We first examined the subcellular localization of Mug27 by constructing a Mug27-GFP-expressing strain in the h90 genetic background and by inducing it to undergo meiosis by nitrogen starvation. As shown in the fluorescence microscope images of Fig. 2A (top), no GFP signal was detected during mitosis. Upon mating, however, the Mug27-GFP fusion protein appeared as a dot near the edge of the nucleus during metaphase I to metaphase II (Fig. 2A, rows 3-6). The dot colocalized with the fluorescence signal of Sid4-mRFP, which is known to localize at the SPB (Fig. 2A). Thus, Mug27 is expressed during the nuclear divisions of meiosis alone and also localizes at the SPB. Sid4 is the most upstream component of the SIN, which regulates spore formation (Krapp et al., 2006Go). Like other SIN proteins, Sid4 does not exhibit the characteristic crescent shape that many SPB antigens adopt during the late stages of meiosis when the outer spindle plaque is remodeled to promote spore formation (Krapp et al., 2006Go) (Fig. 2A). Mug27 also does not appear to exhibit this crescent shape (Fig. 2A). An example of this crescent shape is shown in Fig. 2B and supplementary material Fig. S1A by mCherry-tagged Sad1, which is a component of the SPB (Hagan and Yanagida, 1995Go).


Figure 2
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  		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.

 
Next, to examine the localization of Mug27-GFP in more detail, we compared the subcellular localization of Mug27 with mCherry-tagged Sad1. During meiosis II, Sad1 undergoes a morphological transformation from a compact dot into a multilayered expanded structure (Hagan and Yanagida, 1995Go) (Fig. 2B). We confirmed that Mug27, similar to Sid2 (supplementary material Fig. S1A) and its scaffold protein Sid4 (Fig. 2A), localizes at the SPB with a non-crescent morphology when the SPB itself is transformed to a crescent morphology in meiosis II (Fig. 2B). However, unlike the SIN components, Mug27-GFP localized at the FSM-like structure when the FSM started to extend from the SPB (Fig. 2B). The Mug27-GFP signal then grew and eventually formed a sac that completely engulfed the haploid nucleus at anaphase II. Enlarged views of the cell at anaphase clearly indicate that Mug27 localizes on the outer side of Sad1 at that point (Fig. 2B, lower panels).

Mug27 functions at the SPB in a meiosis-specific manner
To examine whether Mug27-GFP localizes at the SPB when it is artificially expressed during the mitotic growth phase, we co-expressed Mug27-GFP (using the nmt1 promoter in the pREP1-GFP expression vector) and Sad1-mCherry (using the native promoter of sad1+) during mitosis. Unlike Mug27 and Sad1 expression in meiosis (Fig. 2B), and Sid2 and Sad1 expression in mitosis (Sparks et al., 1999Go), Mug27-GFP and Sad1-mCherry did not colocalize (Fig. 2C). Thus, Mug27-GFP does not localize at the SPB during the mitotic growth phase. This suggests that Mug27 localization to the SPB requires an unknown recruiting factor that is expressed or modified in a meiosis-specific manner.

To confirm whether Mug27 has the same function as its homolog Sid2 in mitosis, we examined whether Mug27 could rescue the lethal phenotype of a sid2 mutant at a restrictive temperature of 37°C. Briefly, we transformed the temperature-sensitive sid2-250 strain (Sparks et al., 1999Go) with pREP1-GFP, pREP1-sid2-GFP or pREP1-mug27-GFP and grew the resulting strains on selecting plates at 25°C before shifting them to 37°C (supplementary material Fig. S1B). However, only the sid2 gene could rescue the sid2-250 strain at 37°C. This indicates that, under these conditions, enough Sid2-GFP was expressed to rescue the sid2-250 mutant. However, the Mug27-GFP protein could not rescue the mutant under the same circumstances. Thus, the function of Mug27 is different from Sid2 or is inactivated in mitotic conditions.

Accurate positioning of Mug27 at anaphase II depends on proper FSM formation
The subcellular localization of Mug27-GFP during meiosis suggested that Mug27 might play a role in FSM formation. Thus, we examined the subcellular localization of Mug27-GFP in spo15{Delta} cells, in which structural conversion of the SPB does not occur and no spore formation is observed (Ikemoto et al., 2000Go). We found that Mug27-GFP localized normally at the SPB from metaphase I to metaphase II (data not shown). After anaphase II, however, Mug27-GFP did not accumulate at the SPB or at the FSM-like structure (Fig. 3A). Instead, many of the Mug27-GFP signals were randomly scattered away from the peri-nucleus. To further investigate whether Cdc11, a scaffold protein of SIN (Krapp et al., 2001Go), is also required for the SPB localization of Mug27-GFP, haploid meiosis was induced by inactivation of Pat1 using the pat1-114 mutation (Iino and Yamamoto, 1985Go; Nurse, 1985Go) in haploid cdc11-123 temperature-sensitive strains. We found that, similar to in spo15{Delta} cells, the SPB localization of Mug27-GFP was initially normal but became abnormal around the time that the FSM formed (Fig. 3B,C). These results suggest that the SPB localization of Mug27 requires proper FSM formation but not Cdc11 itself.


Figure 3
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  		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{Delta}) 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{Delta} 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.

 
The spores of the mug27{Delta} strain are abnormal
Morphological examination revealed that spores of mug27{Delta} cells were smaller than those of mug27+, to a degree dependent on the temperature at which meiosis was induced. We examined 50 mug27{Delta} cells in which meiosis was induced at 28°C, and analyzed stained nuclei of more than 100 mug27{Delta} cells in which meiosis was induced at various temperatures. Almost all of the cells examined possessed four properly closed FSMs, although not all of them grew to the cell wall. However, the average diameters of mug27+ and mug27{Delta} spores were 3.4 µm and 2.0 µm by meiotic induction at 33°C; 3.4 µm and 1.4 µm at 28°C; and 3.2 µm and 1.1 µm at 25°C, respectively (Fig. 4A). As shown in Fig. 4B, the spore viability of the mug27{Delta} mutant was halved when a temperature of 33°C or 28°C was used for meiotic induction, whereas the spores of mug27{Delta} cells induced at 25°C were even less viable. Thus, mug27{Delta} cells generate abnormally small spores, and these are significantly less viable than mug27+ spores.


Figure 4
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  		Fig. 4. Mug27 is required for proper spore formation. (A,B) The mug27{Delta} and mug27(KD) mutants generate small spores in a temperature-sensitive manner, with a significant loss of viability. The mug27+ (SOP091w), mug27{Delta} (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{Delta} spores in a temperature-sensitive manner. GFP-Psy1-expressing mug27+ (SOP091w) and mug27{Delta} (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{Delta} cells. (iii) Quantitative analysis of the mug27+ and mug27{Delta} 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{Delta} (33°C), 126 mug27{Delta} (28°C) and 125 mug27{Delta} (25°C) cells. (D) SPBs are abnormally separated from the FSM in mug27{Delta} cells. Cells expressing GFP-Psy1, Meu14-GFP and Sad1-mCherry (mug27+, SOP117w; mug27{Delta}, 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.

 

Spore size is basically determined by the development of the FSM (Nakamura et al., 2001Go). To understand why the smaller spores show lower viability in mug27{Delta} cells, we compared the morphologies of the FSM and the nucleus in mug27+ and mug27{Delta} cells during sporulation by examining GFP-Psy1 and Meu14-GFP localization. Psy1 is a fission yeast homolog of mammalian syntaxin 1A, which is a t-SNARE (soluble NSF attachment protein receptor) protein on the plasma membrane (Nakamura et al., 2001Go). During meiosis II, Psy1 translocates from the plasma membrane to the nascent FSM. Meu14 localizes at the leading edge of the FSM during meiosis II and is essential for accurate FSM formation (Okuzaki et al., 2003Go). The localization of GFP-Psy1 (Nakamura et al., 2001Go) and Meu14-GFP (Okuzaki et al., 2003Go) during meiosis has been well documented previously. Thus, monitoring the movement of GFP-Psyl and Meu14-GFP allows us to visualize the process of FSM formation. We found that the small FSMs of mug27{Delta} cells could engulf only a part of the nucleus (asterisk in Fig. 4Ci,ii) or completely failed to capture the nucleus (triangle in Fig. 4Ci,ii). By contrast, the FSM of mug27+ cells always enclosed the entire nucleus (arrow in Figs 4Ci,ii). The abnormality of mug27{Delta} cells was more conspicuous when the temperature at which meiosis was induced was decreased from 33°C to 28°C or 25°C (Fig. 4Ciii). These results suggest that failure of proper nuclear enclosure causes the decreased spore viability observed in mug27{Delta} cells.

Mug27 is required for proper engulfment of the haploid nucleus into the FSM envelope
We next undertook a more detailed examination of the defects in spore morphogenesis in mug27{Delta} cells. The modification of the SPB from a compact plaque to a multilayered structure during meiosis II is a prerequisite to sporulation (Hirata and Shimoda, 1992Go; Ikemoto et al., 2000Go). Because Mug27 is already present from the early stage of FSM assembly, we suspected that modification of the SPB is impaired in mug27{Delta} cells. We constructed mug27+ and mug27{Delta} strains that can express GFP-Psy1, Meu14-GFP and Sad1-mCherry proteins, and examined their subcellular localization using fluorescence microscopy. We found that the modified crescent-shaped SPBs (Sad1-mCherry, red) were normally observed in mug27{Delta} cells at a frequency comparable to that seen in mug27+ cells during the second meiotic division, from metaphase II to early anaphase II (Fig. 4Di). This indicates that the sporulation defect observed in mug27{Delta} cells is not due to a failure in modification of the SPB structure during meiosis II.

At late anaphase II, the FSM of mug27+ cells continued to envelop the nucleus (stained with Hoechst 33342, blue in Fig. 4) by associating with the SPB (GFP-Psy1, dark green in Fig. 4) until its leading edge (Meu14-GFP, bright green in Fig. 4) properly closed. By contrast, in mug27{Delta} cells, the SPB was separated from the FSM in nearly 30% of asci, allowing the nucleus to escape engulfment (Fig. 4Dii). At sporulation phase, mug27+ asci formed normal spores with the haploid nucleus successfully encapsulated into the FSM, and the SPB was properly attached to the FSM (Fig. 4Diii, type I). However, abnormal spores were observed in more than 90% of the mug27{Delta} cell asci. Either the SPB barely attached, allowing the nucleus to protrude from the FSM envelope (Fig. 4Diii, type II), or the SPB was completely separated from the FSM, allowing the nucleus to escape entirely (Fig. 4Diii, type III). These results indicate that Mug27 plays a role in the proper association of the SPB with the FSM and that it is required for successful engulfment of the haploid nucleus into the FSM envelope.

Mug27 is required for proper development of the FSM
Next, to investigate whether Mug27 regulates spore size, we monitored the growth profiles of the FSM in mug27{Delta} cells by examining the movements of GFP-Psy1 and Meu14-GFP. Time-lapse observation revealed that, although FSM formation was correctly initiated during meiosis II in mug27{Delta} cells, the membrane did not grow properly (Fig. 5B). In mug27+ cells, GFP-Psy1 was completely translocated from the cell cortex to the FSM, where Meu14 rings were closed. By contrast, in mug27{Delta} cells, GFP-Psy1 localized not only at the FSM but also at the cell cortex (Fig. 5A). These results indicate that Mug27 is required for normal development of the FSM.


Figure 5
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  		Fig. 5. Mug27 is required for FSM development. (A) The formation of the FSM in mug27+ (SOP091w) and mug27{Delta} (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{Delta} 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{Delta} cell is indicated by the arrowheads. (B) Time-lapse images of the GFP-Psy1 and Meu14-GFP proteins in mug27+ (SOP091w; i) and mug27{Delta} (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{Delta} 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{Delta} 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).

 
mug27{Delta} cells show delayed FSM formation
To examine the effect of the mug27{Delta} mutation on FSM development, we performed time-lapse observations of live cells by visualizing GFP-Psy1 (dark green in Fig. 5, FSM) and Meu14-GFP (bright green in Fig. 5, the leading edge of the FSM). We calculated the duration of FSM formation, measuring the timing of its beginning and completion by observing the movement of Meu14-GFP. We found that the duration of Meu14 expression was longer in mug27{Delta} cells (Fig. 5B,C), despite the smaller size of the mug27{Delta} spores (see Fig. 4A). When we divided the period of FSM formation into two phases according to the behavior of the Meu14-GFP ring, we found that phase I was almost normal but phase II was longer in mug27{Delta} cells compared with mug27+ cells (Fig. 5D). By contrast, the maximum diameter of the Meu14-GFP ring at the leading edge of the FSM was normal in mug27{Delta} cells (Fig. 5E). These results indicate that Mug27 is required for accurate FSM development but not for the formation of the FSM leading edge.

Mug27 is required for proper vacuole fusion during sporulation
We next examined the effect of the mug27{Delta} mutation on the changes in vacuolar morphology that take place during sporulation. Defective vacuole fusion might affect spore formation (Kashiwazaki et al., 2005Go). The morphology of mug27{Delta} cell vacuoles during spore formation was observed using the vacuolar membrane marker GFP-Ypt7 (Kashiwazaki et al., 2005Go). The FSM was simultaneously visualized by staining with the fluorescent styryl dye FM4-64 (Vida and Emr, 1995Go), which is taken up via the endocytic pathway in S. pombe (Gachet and Hyams, 2005Go) and stains the FSM when added during meiosis I (Jun Kashiwazaki and Taro Nakamura, unpublished observation). In mug27+ cells at the late stage of sporulation, when spore wall materials accumulate between the two layers of the FSM, there was extensive fusion of the vacuoles, forming a few large membranous compartments that occupied the entire cytoplasm (Kashiwazaki et al., 2005Go) (Fig. 6i, lower panel). The vacuolar membrane was found to make close contact with the nascent spores and with the plasma membrane of the mother cell. By contrast, the mug27{Delta} cells did not exhibit this remarkable vacuolar enlargement (Fig. 6ii,iii). Thus, the extensive vacuolar fusion that occurs in the late stage of sporulation, which reflects the generation of new vacuole membranes to build an appropriate FSM, is highly dependent on Mug27 signaling.


Figure 6
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  		Fig. 6. Vacuole morphology during sporulation. GFP-Ypt7-expressing mug27+ (SOP122w) and mug27{Delta} (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{Delta} (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{Delta} (n=162) cells, respectively. Typical images are shown in the upper panel.

 
The kinase activity of Mug27 is needed for proper spore formation
Sequence analysis of the mug27+ gene using the GeneDB databank (http://www.genedb.org/genedb/pombe/index.jsp) predicts that the Mug27 protein functions as a Ser/Thr protein kinase. To determine whether the protein-kinase function of Mug27 is required in meiosis, we created a mutant strain that expresses a 3HA-tagged kinase-dead version of Mug27 [Mug27(KD)-3HA]. In this strain, a point mutation was introduced into the proposed ATP-binding site of the kinase, such that lysine 191 was changed to arginine (K191R). A 3HA epitope tag was then inserted in frame at the 3' end of the chromosomal mug27+ gene. We used western blot analysis to confirm that the Mug27(KD)-3HA protein was expressed (data not shown). As shown in Fig. 4A,B and Fig. 5A, the cells carrying mug27(KD)-3ha had a similar phenotype to mug27{Delta} cells with regards to their FSM formation and sporulation. Thus, the kinase activity of Mug27 is required for proper FSM development and spore formation.

Sid2 can rescue the abnormal phenotypes of mug27{Delta} mutants
To examine whether overexpression of Sid2 can rescue the abnormal meiotic phenotypes of mug27{Delta} cells, we transformed pREP1-Sid2-GFP or pREP1-GFP (vector alone) into mug27{Delta} cells and induced them to enter meiosis. In mug27{Delta} cells expressing Sid2-GFP proteins, spore size (Fig. 7A) and spore viability (Fig. 7B) were restored to levels almost equal to those of mug27+ cells. Overexpression of the pREP1-GFP vector alone did not rescue the abnormal phenotypes of mug27{Delta} cells. These results suggest that Mug27 and Sid2 function within the same signaling pathway during meiosis.


Figure 7
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  		Fig. 7. Overexpression of sid2+ suppressed the abnormal phenotypes of mug27{Delta} cells. (A,B) The mug27+ (NP40-1C), mug27{Delta} (SOP023), mug27{Delta} pREP1-sid2-GFP (SOP115) and mug27{Delta} 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.

 


   Discussion
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Mug27 is required for accurate FSM formation during meiosis II
The SPB in yeast functions not only to nucleate and organize microtubules, but it also acts as a signaling center that coordinates mitotic and meiotic cell cycle events. Although several S. pombe SPB components have been identified, it remains unclear how the SPB regulates spore formation. In the present study, we showed that Mug27 is a novel protein kinase that is only expressed during the nuclear divisions of meiosis (Fig. 1) and that localizes to the SPBs during prometaphase I to metaphase II. Moreover, we found that Mug27-GFP translocated to an FSM-like structure at anaphase II, which is when FSM formation starts (Fig. 2). We also found that the deletion of mug27 (mug27{Delta}) was associated with the formation of abnormally small spores (Fig. 4A) and reduced spore viability (Fig. 4B), which occurred in a manner dependent on low temperature. In both budding and fission yeasts, vesicles are transported to the vicinity of the SPB and fused to each other to generate bilayered prospore membrane (PSM) or FSM, which then encapsulate the haploid nuclei and serve as platforms for spore wall deposition (Shimoda, 2004Go). Endocytosis appears to be important for this process, because endocytosis-defective mutants show severe defects in sporulation (Morishita and Engebrecht et al., 2005; Iwaki et al., 2004Go). Of note is that abnormal spores of mug27{Delta} cells (Fig. 4) resemble those of fission yeast endocytosis-defective-mutant ypt7{Delta} cells (Iwaki et al., 2004Go) (Fig. 4). Indeed, inhibition of endocytosis by the addition of NaN3 during FSM formation resulted in the generation of small spores in mug27+ cells at a level similar to that observed in mug27{Delta} cells (supplementary material Fig. S3). As such, the results reported here suggest that Mug27 might play a pivotal role in this vesicle-trafficking event including endocytosis.

Notably, even though mug27 deletion reduced the spore size, these cells needed more time to close the FSM (Fig. 5B,C). Indeed, when the period of FSM formation was divided into two phases according to the behavior of Meu14-GFP (Fig. 5D), mug27{Delta} cells required more time to complete the posterior phase (Fig. 5D). Nonetheless, the maximal diameter of the Meu14-GFP ring at the leading edge of the FSM was normal in mug27{Delta} cells (Fig. 5E). These results suggest that, although the mechanisms that generate the FSM leading edge are normal in mug27{Delta} cells, the extension of the FSM occurs abnormally. Moreover, 83.9% of the mug27{Delta} cells displayed GFP-Psy1 signals at the membranes of both the ascus and the spores when the FSM engulfed the four haploid nuclei that had been generated (Fig. 5A). By contrast, during mug27+ cell sporulation, all GFP-Psy1 signals were translocated to the spore membrane only. This disparity probably arose because of abnormal translocation in mug27{Delta} cells, causing some GFP-Psy1 molecules to fail to translocate to the spore membrane, remaining instead as dots in the ascus membrane (arrowheads in Fig. 5A). This possibility is supported by the fact that, in both mug27{Delta} and mug27+ cells, the GFP-Psy1 signal accumulated normally at the SPB during metaphase II, which is when FSM formation starts. By contrast, only mug27{Delta} cells showed the later accumulation of GFP-Psy1 in the ascus membrane (Fig. 5A).

Several reports have indicated that the FSM is elongated by fusion with vesicles that are derived from the endoplasmic reticulum via the Golgi network (Nakase et al., 2001Go; Nakamura et al., 2001Go; Nakamura-Kubo et al., 2003Go; Shimoda, 2004Go; Nakamura et al., 2005Go). We show here that, during sporulation, Mug27 plays an essential role in the vacuolar fusion that is pivotal for spore formation. Vacuolar protein sorting (vps) mutations also result in defective sporulation. For example, the Sec1 family protein Vps33 (Iwaki et al., 2003Go) and the phosphatidylinositol 3-kinase Vps34 (Pik3) participate in FSM assembly (Onishi et al., 2003Go). In addition, the retromer components Vps5, Vps17 and Vps29, which are involved in retrograde transport from the endosomes to the Golgi network, are also required for the normal development of the FSM (Koga et al., 2004Go). Because sporulation is a process of dynamic cell remodeling, it requires the degradation of large amounts of pre-existing proteins in vacuoles. In fact, null mutations of the isp6 gene, which encodes vacuolar proteinase B in fission yeast, have a drastic blocking effect on spore formation (Sato et al., 1994Go). Because the mug27{Delta} mutation does not completely block spore formation, but rather specifically impairs FSM assembly, it is unlikely that reduced protease activity is a major cause of the sporulation defects observed in mug27{Delta} cells. Moreover, deletion of mug27+ did not affect meiotic progression (supplementary material Fig. S2A) or chromosome segregation (supplementary material Fig. S2B). This indicates that Mug27 is not important for nuclear division. Rather, it is only required for the proper formation of the FSM.


Figure 8
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  		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.

 
Mug27 is a novel type of FSM-formation regulator
Fig. 8 illustrates our working hypothesis about the role of Mug27 during FSM formation. Similar to Mug27, several SIN components display a non-crescent morphology at the SPB and their mutants show defects in spore formation. However, upon closer inspection, these SIN mutants and mug27{Delta} differ phenotypically (supplementary material Fig. S1C). For example, the cdc11-123 mutant is defective in spore formation because its FSM cannot properly engulf the spore nuclei (Krapp et al., 2006Go) (Fig. 3Bii). By contrast, we show here that mug27{Delta} cells can form spores, albeit small ones (Fig. 4A). Moreover, Cdc11 is not a scaffold for Mug27, because Mug27-GFP can localize at the SPB in cdc11-123 mutant cells before anaphase II, which is when abnormal sporulation begins (Fig. 3B). By contrast, most SIN components use Cdc11 as a scaffold (Morrell et al., 2004Go). Nonetheless, overexpression of Sid2, which is a Mug27 homolog and one of the SIN components, partially suppressed the abnormal phenotypes of mug27{Delta} cells (Fig. 7). Thus, although Mug27 is not a meiotic SIN component, it appears to be a novel regulator for FSM formation that cross-talks with SIN components. Supporting this is the observation that mutations of Rab GTPases encoding a vacuolar protein (e.g. ypt7) have effects similar to those observed in mug27{Delta} cells, in that they result in the formation of small spores, and produce defects in vacuole fusion at late sporulation (Kashiwazaki et al., 2005Go). Similar to the mug27{Delta} strain, cells lacking Spo3, which localizes to the FSM and is required for FSM formation, cannot properly engulf the haploid nucleus, although they do exhibit normal structural change of the SPB and timely initiation of FSM development (Nakamura et al., 2001Go). Future experiments will be needed to characterize the mechanism by which Mug27 regulates FSM formation. In particular, it will be of interest to determine the phosphorylation targets of Mug27 kinase.

In metazoan cells, the formation of the nuclear envelope (NE) around chromatin occurs at the end of cell division. A recent report using fractionated Xenopus egg and sperm chromatin shows that NE formation after mitosis was achieved via the sliding and flattening of the tubular endoplasmic reticulum (ER) network (Anderson and Hetzer, 2007Go). Notably, GTP{gamma}S, which specifically inhibits ER-tubule formation without blocking vesicle fusion, caused the generation of small nuclei because of a block in nuclear transport, and the phenotype of the small nuclei resembled the of mug27{Delta} spores (Fig. 4). Considering that Sid2 suppressed the abnormal sporulation of mug27{Delta} cells (Fig. 7), and that the Sid2 mutant generated abnormal spore membranes during sporulation at a restrictive temperature (data not shown), Sid2 might also be involved in membrane formation. Because NE formation and spore membrane formation are partly similar, the results presented here might help elucidate the mechanisms regulating de novo formation of membrane structure more generally.

In conclusion, we have identified and characterized the protein kinase Mug27, which is a novel component of the S. pombe SPB, and found it to be an essential regulator of FSM formation and sporulation.


   Materials and Methods
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Yeast strains, media and molecular biology
The S. pombe strains and plasmids used in this study are listed in Table 1. The media used were complete-media yeast extract-peptone-dextrose (YPD) or yeast extract plus histidine (YEH) (75 µg/ml), the synthetic Edinburgh minimal medium 2 (EMM2), and the sporulation medium molt extract (ME) or EMM2-nitrogen (EMM2-N). The induction of synchronous meiosis was assessed as described previously (Shimada et al., 2002Go). We used the high-copy plasmid pREP1 driven by its nmt1 promoter for overproduction experiments (Okuzaki et al., 2003Go).


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  		Table 1. Strains used in this study

 

Gene disruption of mug27+
To disrupt the mug27+ gene by replacing it with the ura4+ gene, we used the polymerase chain reaction (PCR) to obtain a DNA fragment carrying the 5' upstream and 3' downstream regions of the mug27+ gene. For this purpose, we synthesized the following four oligonucleotides and used them as primers: mug27 5F-KpnI (–500), 5'-GGTACCAGAACATAAATAATAATCTGGATAGTTTGC-3'; mug27 5R-XhoI (–1), 5'-GCCTCGAGTCTATGTTTGTGGAATCGTTCTATAAAATTG-3'; mug27 3F-PstI-XhoI-TAA (+1), 5'-CTGCAGCTCGAGTAAATCCAGATAATAACGCATCAATTACC-3'; mug27 3R-SacI (+501), 5'-GCGAGCTCATGGTTATAATGCCTTCTATCTTCTATTTC-3'. The underlined sequences denote the artificially introduced restriction enzyme sites for KpnI, XhoI, PstII and SacI, respectively. These PCR products and the 1.8 kb HindIII fragment containing the ura4+ gene were inserted into the pBluescriptII KS (+) vector via the KpnI-XhoI, SmaI-SacI or HindIII sites. The mug27 5F-KpnI (–500) and mug27 3R-SacI (+501) primers generated a 2.8-kb PCR product containing the ura4+ cassette. This plasmid construct was digested with KpnI and SacI, and the resulting construct was introduced into the h90 wild-type (WT) strain (AO193). The Ura+ transformants were then screened by PCR analysis to identify the disrupted strain.

Construction of strains harboring integrated mug27+-tag genes
To prepare each construct, we followed the previously described method (Saito et al., 2004Go). Thus, we performed PCR using the WT (TP4-5A) genome as the template and obtained a DNA fragment carrying the ORF and 3' downstream regions of the mug27+ gene. The following primers were used to obtain the C-terminal region of the mug27 ORF: mug27 Integ-SalI (260), 5'-GTCGACCCGAGACAAGAAGAAAGCGG-3' and mug27-C, 5'-GCGGCCGCGGGAGCAAAAATTCATACAGGTCTTTGC-3'. The underlined sequences denote the artificially introduced restriction enzyme sites for NdeI and NotI, respectively. The following primers were used to obtain the 3' downstream region: mug27 3F-PstI-XhoI-TAA (+1), 5'-CTGCAGCTCGAGTAAATCCAGATAATAACGCATCAATTACC-3' and mug27 3R-SacI (+501), 5'-GAGCTCATGGTTATAATGCCTTCTATCTTCTATTTC-3'. The underlined sequences denote the artificially introduced restriction enzyme sites for PstI and XhoI, and SacI, respectively. The 3' downstream region was inserted into the tag-containing pREP vector via XhoI-SacI and then the C-terminal region of the ORF was inserted into this vector via SalI-NotI. The construct was then cut out by using SalI-SacI and inserted into the LEU2+-containing pT7Blue T vector, which was subsequently digested with NotI and introduced into the h90 WT strain (AO193). The Leu+ transformants were then screened by PCR.

Fluorescent microscopic observation
Fluorescent microscopic observations were performed as described previously (Saito et al., 2005Go). Cells were cultured in 10 ml EMM2 with supplements [adenine (75 µg/ml), histidine (75 µg/ml), leucine (250 µg/ml), lysine (75 µg/ml) and uracil (75 µg/ml)] until they reached mid-log phase at 28°C. The cells were collected by centrifugation, washed three times with 1 ml EMM2-N and then induced to enter meiosis by incubation in EMM2-N at 28°C for 10 hours. For live observations, we added 0.5 µg/ml Hoechst 33342 to 200 µl of the cells and an aliquot was observed under a fluorescence microscope (Olympus BX51). Fluorescence images were acquired by using Photoshop 7.0 (Adobe).

For time-lapse observations, SOP091w and SOP095 cells, which expressed GFP-Psy1 and Meu14-GFP, were cultured in 10 ml EMM2 plus supplements until they reached mid-log phase at 28°C. They were then induced to enter meiosis by incubation in EMM2-N at 28°C. After 10 hours of nitrogen starvation, live cells were placed on a glass-bottomed dish coated with 0.2% concanavalin A. Images under a fluorescence microscope (Olympus IX71) were recorded every 2 minutes (2.5 second of exposure time) with three optical sections made at 200-µm intervals for each time point. The projected images obtained with Meta Morph software were analyzed.

FM4-64 staining
To visualize the fission yeast vacuole and FSM, we labeled the cells with the lipophilic dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide] (Molecular Probes, Eugene, OR) (Vida and Emr, 1995Go), with some modifications of the previous report (Kashiwazaki et al., 2005Go). Briefly, cells were induced to enter meiosis by nitrogen starvation. After 9 hours, FM4-64 was added at a final concentration of 2 µl/ml (diluted from 10 µg/µl stock solution in DMSO) and incubated with shaking at room temperature for 30 minutes. Then, cells were collected by centrifugation, inoculated into fresh EMM2-N medium and cultured for an additional 2 hours before being observed by fluorescence microscopy.

Spore viability
Spore viability was determined as described previously (Ohtaka et al., 2007bGo). Briefly, h90 haploid strains were grown on YPD plates at 33°C. Cells were mated and sporulated on EMM2-N plates at 25°C, 28°C and 33°C for 3-4 days. At the end of the culture, the ascal walls dissolved spontaneously and single spores were liberated. The spores were separated on YEH agar plates by using a micromanipulator (Singer Instruments, Somerset, UK). The plates were incubated at 30°C for 5 days, after which spore viability was calculated.


   Acknowledgments
 
We thank D. McCollum, C. Shimoda, T. Nakamura, M. Yamamoto, Y. Hiraoka, W. Z. Cande and J. Kashiwazaki, and the National BioResource Project – Yeast (http://yeast.lab.nig.ac.jp/nig/), for S. pombe strains; and R. Y. Tsien for plasmids. We are also indebted to N. Nakamura for teaching us an unpublished technique for FM4-64 staining and to P. Hughes for critically reading the manuscript. This work was supported in part by Innovation Plaza Osaka of the Japan Science and Technology Agency (JST), and by grants-in-aid for Scientific Research on Priority Areas `Applied Genomics', Scientific Research (S), Exploratory Research, and the Science and Technology Incubation Program in Advanced Regions from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H.N. A.O. is a Research Fellow of the Japan Society for the Promotion of Science.


   Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/9/1547/DC1


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 Discussion
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H. Yan, W. Ge, T. G. Chew, J. Y. Chow, D. McCollum, A. M. Neiman, and M. K. Balasubramanian
The Meiosis-Specific Sid2p-related Protein Slk1p Regulates Forespore Membrane Assembly in Fission Yeast
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