Localization of Mcp4 during meiosis. The merged images are depicted schematically in the rightmost panels. (A) Comparison of the subcellular localizations of Mcp4, SPBs, and DNA during the vegetative cell cycle, meiosis, and sporulation. The homothallic haploid cell line AO64 (h⁹⁰ mcp4⁺-3ha) was cultured in EMM with appropriate supplements and either left in EMM or transferred to EMM-N to induce meiosis. The cells were fixed chemically at the vegetative growth phase or at different stages of meiosis and then stained with Hoechst 33342, anti-HA antibody, and anti-Sad1 antibody to detect the DNA (blue), Mcp4-3HA (red), and SPBs (green), respectively. The fluorescence microscopic images of these three cellular components are merged in the middle panels. The images indicated by arrowheads were enlarged and depicted schematically in the lower panels. (B) Comparison of the subcellular localizations of Mcp4-3HA, tubulin, and DNA. Chemically fixed AO64 cells at late anaphase II were stained with Hoechst 33342, anti-HA antibody, and anti-TAT1 antibody to visualize the DNA (blue), Mcp4-3HA (red), and tubulin (green), respectively. Mcp4 appears to aggregate close to both ends of the microtubules. (C) Comparison of the subcellular localizations of Mcp4-3HA, Meu14-GFP, and DNA. meu14⁺-gfp mcp4⁺-3ha cells (AO187) in anaphase II were chemically fixed and stained with Hoechst 33342 and anti-HA antibody. In the merged image, Meu14 is shown in green. The Mcp4 rings (red) are flanked by chromatin (blue) on one side and Meu14 rings on the other during late meiosis II. (D) Comparison of the subcellular localizations of F-actin and Meu14-GFP. AO185 cells were chemically fixed and stained with Hoechst 33342 and rhodamine-phalloidin to visualize the chromatin (blue) and F-actin (red), respectively. The F-actin occupies the space between the Meu14 (green) and Mcp4 rings shown in panel C. (E) Comparison of the subcellular localizations of Mcp4-3HA and GFP-Psy1. pREP81-gfp-psy1⁺mcp4⁺-3ha cells (AO194) in anaphase II and during sporulation were chemically fixed and stained with Hoechst 33342 and anti-HA antibody to visualize the chromatin (blue) and Mcp4 (red), respectively. GFP-Psy1 is shown in green. Mcp4 was localized at the leading edge of the FSM during anaphase II. Bars, 10 μm.

Accurate positioning of Mcp4 at anaphase II depends on proper FSM formation. (A and B) Microscopic analysis of Mcp4-3HA localization in meu14⁺ (A) and meu14Δ (B) cells during meiosis. The homothallic haploid strains AO185 (h⁹⁰ meu14Δ mcp4⁺-3ha) and AO64 (h⁹⁰ mcp4⁺-3ha) were cultured in EMM with appropriate supplements and then transferred to EMM-N to induce meiosis. After 10 h of incubation, the cells were chemically fixed and stained at different stages of meiosis with Hoechst 33342, the anti-HA antibody, and the anti-TAT1 antibody to detect the DNA (blue), Mcp4-3HA (red), and tubulin (green), respectively. (i) Typical images at metaphase II, anaphase II, and sporulation. The images are merged in the right panels. (ii) Enlarged views (left) of the cells at anaphase II denoted by white arrowheads in panels i and their schematic renditions (right). The red arrows indicate the Mcp4-3HA dots. (C and D) Typical images of F-actin in meu14⁺ (AO64) (C) and meu14Δ (AO185) (D) cells at metaphase II, anaphase II, and sporulation. F-actin and DNA were stained with rhodamine-phalloidin (red) and Hoechst 33342 (blue), respectively. The yellow arrows indicate the aggregated F-actin patches. Bars, 10 μm.

The C-terminal coiled-coil domain of Mcp4 is required for proper localization of this protein during meiosis. (A) Schematic depiction of Mcp4 and Mcp4ΔC. The coiled-coil motif is indicated by the black box. (B) Comparison of the localization patterns in mitotic cells of ectopically expressed Mcp4ΔC-GFP and CFP-labeled Rer1. Rer1 is an ER/Golgi complex shuttling protein (17, 24). Mitotic AO168 (mcp4ΔC-GFP rer1⁺-cfp) cells were cultured in EMM without thiamine. The merged images are shown in the rightmost panel. (C to E) Comparison of the localization patterns of ectopically expressed Mcp4-GFP (i) and Mcp4ΔC-GFP (ii) in mitotic (C) and meiotic (D and E) pREP41 (mcp4⁺-gfp or mcp4ΔC-gfp) cells. (C) The cells were cultured in EMM without thiamine and stained with FM4-64 (red). This allowed us to visualize the Golgi complex/endosome 5 min after uptake and then the vacuole 60 min later. (D and E) The cells were cultured in EMM containing 1 μg/ml thiamine with supplements and then transferred to EMM without thiamine to induce Mcp4-GFP expression. Subsequently, 10 h after the first medium exchange, the cells were transferred to fresh EMM-N without thiamine to induce meiosis. After incubation for 10 h, live cells were observed under a fluorescence microscope in the presence of FM4-64 (red) to visualize the localization of the Golgi complex/endosome and the vacuole (D), or the cells were chemically fixed and stained with rhodamine-phalloidin (red) to detect F-actin (E). The green and blue signals are from Mcp4ΔC-GFP and Hoechst 33342-stained DNA, respectively. The merged images are shown in the rightmost panels. The images denoted by the white arrowheads in panel E are enlarged and depicted schematically in the bottom panels. Bars, 10 μm.

Mcp4 is a meiosis-specific coiled-coil protein. (A) Schematic depiction of Mcp4 (355 amino acids). The predicted coiled-coil motif was identified by PSORT II (http://psort.nibb.ac.jp/) and is indicated by the black box. (B) Meiotic expression of mcp4⁺. h⁺/h⁻ (CD16-1) and h⁻/h⁻ (CD16-5) diploid cells were subjected to nitrogen starvation, which induces CD16-1 but not CD16-5 cells to enter meiosis. The cells were collected at 2-h intervals, and the total RNAs were blotted and probed with the mcp4⁺ ORF. The RNAs were also probed with the aro3⁺ ORF as a loading control. (C) Meiotic expression of Mcp4-3HA. The h⁻/h⁻ pat1-114 mcp4⁺-3ha strain (AO89) was induced to enter meiosis synchronously by a temperature shift, and the cells were collected at 30-min intervals for protein extraction, blotting, and probing with anti-HA antibody. Meu13 expression was also analyzed by using an anti-Meu13 antibody to help identify the meiotic stage at each time point. The tubulin levels were also examined as a loading control. At each time point in panels B and C, the frequency of cells with one, two, three, or four nuclei was determined by counting at least 200 Hoechst 33342-stained cells under a microscope. The upper panels show the stages of meiosis.

Mcp4-GFP localizes side by side with F-actin during meiosis. (A) Microscopic analysis of Mcp4 and F-actin localization during meiosis. The homothallic haploid strain AO192 (h⁹⁰ mcp4⁺-3ha crn1⁺-gfp), which expresses Crn1-GFP, was induced to enter meiosis, after which the cells were chemically fixed and stained with Hoechst 33342 and anti-HA antibody to detect DNA (blue) and Mcp4-3HA (red), respectively. Crn1-GFP, which marks the F-actin complex, was visualized directly through its GFP-derived fluorescence. The merged images are shown schematically in the rightmost panels. (B) Enlarged view (left) and its schematic depiction (right) of the merged image at late anaphase II indicated by the white arrowhead in panel A. The localization of Meu14 that was revealed in Fig. 2C and D is also shown. (C) Microscopic analysis of the meiotic localization patterns of F-actin and ectopically expressed Mcp4-GFP. The homothallic haploid strain AO00, which bears the mcp4⁺-gfp plasmid and expresses Mcp4-GFP under the control of the nmt41 promoter, was cultured in EMM containing 1 μg/ml thiamine with supplements and then transferred to EMM without thiamine to induce the expression of Mcp4-GFP. Subsequently, 10 h after the first medium replacement, the cells were transferred to fresh EMM-N without thiamine to induce meiosis. Cells at various stages of meiosis were chemically fixed and stained with Hoechst 33342 and 4 μM rhodamine-phalloidin to visualize the DNA (blue) and F-actin (red), respectively. Mcp4-GFP is shown in green. The merged images are shown in the rightmost panels. (D) Mcp4-GFP localization is independent of F-actin polymerization. mcp4Δ cells were transformed with the Mcp4-GFP-expressing construct pRGT41-mcp4⁺, thus generating the AO189 strain. These cells were cultured in EMM containing 1 μg/ml thiamine with supplements and then transferred to EMM without thiamine to induce Mcp4-GFP expression. Subsequently, 10 h after the first medium change, the cells were transferred to fresh EMM-N without thiamine to induce meiosis and incubated for another 10 h. Two hours after the addition of dimethyl sulfoxide solvent (DMSO) (i) or 50 μM latrunculin A in dimethyl sulfoxide (ii), the cells were chemically fixed and stained with rhodamine-phalloidin. Bars, 10 μm. The images indicated with arrowheads were enlarged and depicted schematically in the lower panels.

Phenotypes of mcp4Δ cells during meiosis. (A and B) Mcp4 is required for proper formation of the F-actin structure at anaphase II. GFP-Psy1-expressing mcp4⁺ (YN68) (A) and mcp4Δ (AO125) (B) strains were induced to enter meiosis by nitrogen starvation. After 10 h, the cells were chemically fixed and stained with rhodamine-phalloidin to detect F-actin. (i) Typical fluorescence microscope images at metaphase II and anaphase II. The images for F-actin (red), GFP-Psy1 (green), and DNA (blue) are merged in the right panels. (ii) Enlarged views of the images indicated by the arrowheads in panels i and their schematic renditions. F-actin forms a dumbbell shape in mcp4⁺ cells (red arrows in panel A, part ii) but a balloon shape in mcp4Δ cells (red arrowhead in panel B, part ii). Bars, 10 μm. (C) Quantitative analysis of the cell populations harboring zero to four dumbbell or balloon shapes in the mcp4⁺ or mcp4Δ strain at anaphase II. The bar graphs were drawn by counting the F-actin shapes in 30 mcp4⁺ and 31 mcp4Δ cells. (D to F) mcp4Δ spores are more salt sensitive than mcp4⁺ spores. mcp4⁺ (YN68) and mcp4Δ (AO125) spores were separated by a manipulator onto YEH agar plates, with or without 0.1 M or 0.17 M NaCl. The plates were incubated at 30°C for 5 days. (D) Sizes of colonies of mcp4Δ spores after 2 or 5 days on a YEH plate in the absence (0 mM) or presence (175 mM) of NaCl. (E) Viability of mcp4Δ spores under high NaCl conditions. Spores of mcp4⁺ and mcp4Δ cells were dissected by a micromanipulator. The data shown are the average values for at least three independent assays (at least 80 spores were dissected per assay). The standard deviations are indicated by the error bars. (F) Quantitative analysis of spore populations that could not make colonies when returned to the YEH plate under high sodium salt conditions. Typical images of the germinated spores for mcp4⁺ and mcp4Δ strains are shown in the insets. The data shown are the average values for at least three independent assays. At least 8 or 22 spores were dissected per assay for the mcp4⁺ or mcp4Δ strain, respectively. The standard deviations are indicated by the error bars.

Schematic presentations summarizing the subcellular localization of Mcp4 at metaphase II and anaphase II of S. pombe meiosis. The relative positions of the cell cortex, F-actin, nuclear envelope (NE), nucleus, ER, SPBs, microtubule, Psy1 at the FSM, and Meu14 at the leading edge of the FSM are depicted. The F-actin ring is sandwiched between the Meu14 and Mcp4 rings at anaphase II. See the text for details.