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First published online 20 May 2003
doi: 10.1242/jcs.00496

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Fission yeast meu14⁺ is required for proper nuclear division and accurate forespore membrane formation during meiosis II

Daisuke Okuzaki^*,1, Wataru Satake^*,1, Aiko Hirata² and Hiroshi Nojima^1,

¹ Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
² Department of Integrated Biosciences, Graduate School of Frontier Sciences Tokyo University, Bldg. FSB-101/601, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan

View larger version (73K): [in a new window]   Fig. 1. Expression of meu14+ and its paralogs during meiosis and structural comparison of their gene products. (A) The meu14+ gene is specifically transcribed during meiosis. Diploid h+/h- (CD16-1) and h-/h- (CD16-5) cells were exposed to nitrogen starvation, which triggers meiosis in the former but not the latter strain, and cells were collected at 2 hour intervals for RNA extraction. Upper panels: Total RNA was blotted and probed by radiolabeled meu14+, mfp1+, mfp2+ and aro3+ DNA fragments. The intensities of the bands obtained by using the aro3+ probe and ethidium bromide staining were used as loading controls. Numbers at the bottom represent the cell populations harvested at each 2 hour interval and assessed for the percentages of cells that had four nuclei (counted after staining the cells with DAPI). (B) meu14+ gene expression is under the control of the sporulation-specific Mei4 transcription factor. Diploid h-/h- pat1-114/pat1-114 (JZ670) and h-/h- pat1-114 mei4 pat1-114 mei4 (AB4) cells were synchronized to enter the G1 phase by nitrogen starvation, and then induced to meiosis by temperature shift. Nitrogen was then added, and the cells were incubated at 34°C and collected at 2 hour intervals for RNA analysis as in panel A. (C) Structure of Meu14. (i) Conserved structural motifs. The coiled-coil region (C-C) is predicted using the COILS program with a 21-residue window setting (Lupas et al., 1991). (ii) Alignment of the predicted amino acid sequence of Meu14 with S. pombe and S. cerevisiae homologues. Homologous proteins were identified in a BLAST search using the protein sequence of Meu14. Hyphens represent gaps inserted to attain maximal homology. Amino acids identical to those in Meu14 are highlighted by a gray background. Amino acid numbers are denoted at the right-hand side of each sequence. The extended coiled-coil motif found in Meu14 is indicated by a solid line. (D) The phylogenetic tree of the Meu14 homologues. The tree was constructed based on the whole amino acid sequence of each protein by Genetyx-Mac software (Software Development, Tokyo, Japan) using the neighbor joining method (Saitou and Nei, 1987). The genomic DNA sequences used are recorded in the DDBJ/EMBL/GenBank database (accession nos. CAA97088, AAB68101, CAB16733 and CAA19279, respectively).

View larger version (45K): [in a new window]   Fig. 2. Phenotypes of meu14 cells. (A) The meu14 disruptant is defective in spore formation. Shown are DIC (differential interference contrast) and fluorescence photographs of Hoechst 33342-stained cells of the wild-type (TP4D-5A/TP4D-1D) and the meu14 mutant (YDO100) strains after 16 hours of nitrogen starvation. Bar, 10 µm. (B) Absence of spore walls in meu14 cells induced to sporulate. Wt and meu14 cells were streaked on a sporulation plate (EMM-N) and incubated at 28°C for 4 days. They were then exposed for 5 minutes to iodine vapor, which stains cells that have sporulated dark brown. (C) Progression of meiosis in wild-type and meu14 cells. Up to 1x107 cells/ml cultured overnight in liquid growth medium (EMM2) were incubated with shaking at 30°C in liquid sporulation medium (EMM-N). A portion of the culture was taken every 2 hours and stained with DAPI. Cells were classified based on the numbers of nuclei. , interphase (mononucleate); , Horse-tail; , binucleate; , tetranucleate. For each sample, approximately 200 cells were counted. Values depict one representative result of four independent experiments. (D) The meu14 cells frequently produce abnormal tetranucleate cells. Tetranucleate cells were classified according to the number and position of the four nuclei per cell. 1, normal pattern; 2, unequally segregated nuclei; 3, missegregated nuclei; 4, unsegregated nuclei; 5, abnormally distributed nuclei; 6, distorted nuclei. The percentages of Wt and meu14 cells in each category after 12 hours of nitrogen starvation were compared. At least 200 cells were counted.

View larger version (61K): [in a new window]   Fig. 3. Meu14 is required for the proper segregation of SPBs. (A) Chromosomal DNA, SPBs, microtubules in meu14 (YDO100) cells were stained by Hoechst 33342 (blue), anti-Sad1 antibody (green) and anti--tubulin antibody TAT-1 (red), respectively. Merged images are also shown. The following typical images are displayed. (i) Microtubule extension between the two pairs of dividing nuclei at meiosis II is not synchronized. (ii) Multiple foci of Sad1 staining are observed. (iii) Microtubules between the two nuclei are not detected. (iv) Localization of microtubules is abnormal and there are more than four Sad1 foci. (v,vi) meu14 (YDO100) cells at metaphase I (v) or anaphase I (vi). (B) Sad1 colocalizes with the SPB protein Spo15 during meiosis II. The following typical images are displayed for meu14 (YDO111) (i-iii) or wild-type (iv,v) cells. (i) One of the four SPBs is abnormal. (ii) More than four SPBs are observed. (iii) Nuclei are not properly divided at meiosis II. (iv,v) Wild-type cells at metaphase II (iv) and anaphase II (v). Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 4. Subcellular localization of Meu14-GFP in fixed cells. (A) Detection of Meu14-GFP protein by western blotting, which is expressed in a meiosis-specific manner. A band corresponding to Meu14-GFP with the expected size (shown by an arrowhead) is detected predominantly 4-8 hours after nitrogen starvation, after which it suddenly disappears and cannot be detected at all at 10 hours and 12 hours (uppermost panels). In contrast, a band for GFP alone is detected primarily at 6-12 hours (middle panels), probably because the Meu14 portion of Meu14-GFP is easily degraded, releasing the GFP portion. Western blot probed by an anti-Cdc2 antibody was used as a loading control. Asterisks indicate nonspecific bands. The extracts of wild-type cells (TP4D-5A/TP4D-1D) transformed with pRGT81-meu14+ (meu14+-GFP) or pRGT81 (GFP vector) are used to identify the expressed Meu14-GFP protein. These extracts are denoted by 14 or G, respectively (rightmost lanes). (B) Fluorescence from Meu14-GFP observed at various stages of meiosis. Cells were immunostained with the TAT1 antibody to mark the microtubules (red). Cells bearing meu14+-gfp (YDO120) were induced to enter meiosis and then chemically fixed and analyzed by fluorescence microscopy for DNA (blue), Meu14-GFP (green), and microtubules (red). The three images are merged and shown in the rightmost panels. (C) Correlation of the distance between the two Meu14-GFP rings (c) with either (a) the distance between two nuclei or (b) the length of the microtubules. (D) meu14+-gfp (YDO120) cells immunostained by the anti-Sad1 antibody to visualize the SPB at various stages of meiosis. It appears that Meu14-GFP is located on the cytoplasmic side of the SPB (white arrow). (E) meu14+-gfp (YDO120) cells immunostained by the anti-NPC antibody mAB414. Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 5. Dynamic movement of Meu14-GFP in live meiotic cells as revealed by time lapse observations. (A) Cells (YDO120) passing from prometaphase II to metaphase II. (i) Merged images of Meu14-GFP (green) and nuclei stained by Hoechst 33342 (blue) recorded at 2 minute intervals and shown at 6 minute intervals. (ii) An enlarged Meu14-GFP image at 0 minutes is shown to highlight the appearance of the Meu14-GFP dot/ring structure (arrowhead) at the end of the nucleus. (B) Cells passing from metaphase II to anaphase II. (C) Three-dimensional images of the Meu14-GFP structure during anaphase II. (D) Enlarged views of Meu14-GFP localization at the late stage of meiosis II. A thin pouch-like image containing trace amounts of Meu14-GFP is observed, at the end of which a strong band of Meu14-GFP is detected. Bar, 10 µm. Please refer to Movies 1-4 for the dynamic Meu14-GFP profile and Movie 5 for three-dimensional images of Meu14-GFP rings.

View larger version (51K): [in a new window]   Fig. 6. Meu14 is localized at the leading edge of the forespore membrane and is required for its extension. (A) Localization of Meu14-GFP, Spo3-HA, and Sad1 during meiosis II. meu14+-gfp spo3::HA diploid cells (YDO121) at metaphase II (panel i and ii) and anaphase II (panels iii-v) were analyzed by immunofluorescence microscopy. Hoechst 33342 staining is blue, Meu14-GFP is green, anti-HA staining is red, and anti-Sad1 staining is yellow. (B) Psy1, a forespore membrane component, localizes abnormally in h90meu14psy1+-gfp cells (YDO150). h90psy1+-gfp cells (YN68) (panel (i) and h90meu14psy1+-gfp cells (panels ii-iv) harboring the integrated psy1+-gfp gene were induced to enter meiosis II and were analyzed by immunofluorescence microscopy. Hoechst 33342 staining is blue, anti-Sad1 staining is red, and Psy1-GFP is green. (C) Frequency of the abnormal forespore formation in h90meu14 cells. Stained cells were classified into the following 7 classes: class I, all four forespore membranes were formed next to nuclei; class II, all four membranes were formed next to nuclei but were crushed; class III, one or two forespore membranes alone were formed normally; class IV, membranes were formed but they do not properly enclose the nucleus; class V, one or two membranes were formed but others formed next to the nuclei; class VI, all four membranes were crushed and formed aggregates near the nuclei; class VII, all four membranes were formed normally. At least 250 cells were counted. (D) Localization of Meu14-GFP in the absence of Spo3 or Spo15. h90meu14+-gfp cells (YDO50; panels i and ii), h90 meu14+-gfp spo3::ura4+ cells (YDO130; panels iii and iv), and h90 meu14+-gfp spo15::ura4+ cells (YDO10; panels v-vii) undergoing meiosis II were analyzed by immunofluorescence microscopy. Hoechst 33342 staining is blue, Meu14-GFP is green, and anti-Sad1 staining is red. Bar, 10 µm.

View larger version (155K): [in a new window]   Fig. 7. Thin section electron microscopy of meu14 cells. (A) EM images of the meu14 (YDO100) (i) and the wild-type (TP4D-5A/TP4D-1D) (ii) strains, and enlarged views (iii-vi) after spore wall formation had commenced. (B) EM images of the meu14 (i) and the wild-type (iv) strains, and enlarged views (ii and iii) to show the abnormal encapsulation of the nuclei in the meu14 cells.

View larger version (123K): [in a new window]   Fig. 7. Thin section electron microscopy of meu14 cells. (C) EM images of the meu14 cells (i and iv), and enlarged views (ii, iii, v and vi) to show the abnormal localization of the SPBs in the meu14 cells. (D) An EM image of the meu14 cells shows that the forespore membrane abnormally encloses the spindle (i). An example (ii) demonstrates that the absence (iii) or presence (iv) of SPB affects the proper formation of the forespore membrane in the meu14 cells. Pictures of wild-type cells are encircled by black lines. White arrowheads (spindle), white arrows (SPB) and black arrows (forespore membrane). White scale bar, 1 µm. Black scale bar, 0.5 µm.