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First published online January 14, 2005
doi: 10.1242/10.1242/jcs.01629

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Mcp6, a meiosis-specific coiled-coil protein of Schizosaccharomyces pombe, localizes to the spindle pole body and is required for horsetail movement and recombination

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

View larger version (32K): [in a new window]   Fig. 1. Mcp6 is a meiosis-specific coiled-coil protein that harbours three homologous regions conserved in other proteins that are involved in the movement of subcellular components. (A) Schematic representation of Mcp6. The locations of the two coiled-coil motifs and the NLSs are indicated [as identified by PSORT II (http://psort.nibb.ac.jp/)]. (B) Schematic representation of Mcp6-related proteins of other species. The locations of homologous domains in these proteins are denoted by vertical white, grey and black arrowheads. Multiple sequence alignment shows homologous domains between Mcp6 from S. pombe (denoted Sp) and the related proteins from other species. Gaps inserted in the alignment to attain maximal homology are indicated by hyphens. The amino acids that are identical or similar among four or more of the seven species examined are shaded in black or grey. Abbreviations: MHC, myosin heavy chain; SMC, structural maintenance of chromosomes; Sp, Schizosaccharomyces pombe; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Mm, Mus musculus; Xl, Xenopus laevis; Hs, Homo sapiens. The analysis of DNA sequences was performed by using the GENETYX program (Software Development, Tokyo, Japan). (C) Northern blot analysis of mcp6+ and aro3+ (loading control). Total RNA was extracted from CD16-1 (h+/h-) and CD16-5 (h-/h-) cells at the indicated times after the induction of meiosis by nitrogen starvation. RNA was blotted and probed with the ORFs of mcp6+ and aro3+. The graph below indicates the meiotic profiles of the cells used for RNA extraction. The progression of meiosis was monitored every 2 hours after nitrogen starvation. The numbers of cells that bear one, two, three or four nuclei were assessed by counting the nuclei stained with Hoechst 33342. At least 200 cells were counted under the microscope. (D) Western blot analysis of the production of Mcp6-GFP and Meu13 (meiotic timing control) proteins during the synchronous meiosis of strain ST194. Tubulin levels were also examined as a loading control.

View larger version (51K): [in a new window]   Fig. 3. Nuclear movement is abnormal during the horsetail phase in mcp6 cells. (A) Profiles of the meiotic progression in pat1 (JZ670) and pat1 mcp6 (TT405) diploid cells (azygotic meiosis). The progression of meiosis was monitored every 30 minutes (3-7 hours) or 1 hour (0-2 hours and 7-8 hours) after the temperature shift, depending on the phase of meiosis. At least 200 cells were counted under a microscope to assess the frequencies of Hoechst-33342-stained cells that bear a horsetail, one nucleus, two nuclei and more than three nuclei. Each point denotes the average value of at least three independent experiments. Standard deviations are indicated as error bars. (B) Time-lapse images of pat1 and pat1 mcp6 diploid cells during meiosis I. The nuclei were stained with Hoechst 33342. Images of a single cell were obtained at 2.5-minute intervals. The numbers at the bottom of each photograph represent the timing in minutes, with 0 minute being 2 hours after temperature shift to induce azygotic meiosis. Bar, 5 µm. (C) Time-lapse observation of wild-type (WT) (CT026-1) and mcp6 (ST193) cells during meiosis I. The nuclei were visualized by the fluorescence of a Pol-GFP fusion construct. Images of a single cell were obtained at 5 minute intervals. The numbers at the bottom of each photograph represent the timing in minutes, with 0 minutes being when nuclear fusion (karyogamy) occurs. The white arrowheads indicate the putative trailing edge of the moving nucleus. Bar, 5 µm. (D) The duration of meiotic prophase, meiosis I (MI) and meiosis II (MII) in mcp6 and WT cells. The average values were calculated from ten independent cells observed under a microscope. Standard deviations are shown as error bars.

View larger version (69K): [in a new window]   Fig. 2. Mcp6 is a meiosis-specific SPB-associated protein. (A) Microscopic analysis of Mcp6 localization during meiosis. The mcp6+-gfp dsred-sad1+ strain (ST142) was induced to enter meiosis by nitrogen starvation. After 6 hours of incubation, the cells were collected and fixed with methanol for microscopic observation. The GFP signal is green, the DsRed signal is red and Hoechst 33342 staining is blue. (B) Mcp6-GFP localizes to the SPB but not to the telomeres in mitotic cells. Overproduction by transforming mitotic cells with the pRGT81 (GFP expression vector) or mcp6+/pRGT81 (Mcp6-GFP expression vector) plasmid is indicated by `OP'.

View larger version (53K): [in a new window]   Fig. 4. Homologous pairing is reduced in mcp6 cells. (A) Time-lapse observation of the lys1 locus in a living cell, either mcp6 (ST197) or wild type (WT) (AY174-7B), during the horsetail stage. The lacO repeat sequence integrated into the lys1 loci was visualized by the LacI-NLS-GFP fusion protein. Images of a single cell were obtained at 5 minute intervals. The numbers at the bottom of each photograph represent the timing in minutes, with 0 minutes being when nuclear fusion (karyogamy) occurs. The rectangles under each photo indicate that the lys1 loci were paired (grey) or not paired (white). Bar, 5 µm. (B) Time course of homologous pairing frequency during the horsetail stage in mcp6 (red square) compared with that in the WT (blue circle). The average values were calculated from 20 independent cells. The lys1 locus in chromosome I is illustrated as an inset.

View larger version (31K): [in a new window]   Fig. 5. Homologous recombination is reduced in mcp6 cells but ectopic recombination is increased compared with wild-type (WT) cells. The chromosomal positions of the loci and centromeres are illustrated in the insets. (A) Intergenic recombination (crossing over) showing the intervals between leu1 and his2 (left), lys3 and cdc12 (middle) and the primary tetrad (right). Only those tetrads that generated four viable spores were used to calculate the genetic distances (cM). The strains examined for leu1-his2 crossing were WT (TT8-1 x NP32-2A) and mcp6 (TT398 x TT399), whereas the strains used for the lys3-cdc12 crossing were WT (TT8-1 x TT231-1) and mcp6 (TT399 x TT411). The data shown are the average values calculated from at least three independent assays (at least 40 tetrads were dissected per assay). (B) Intragenic recombination. The strains examined were WT (MS105-1B x MS111w1) and mcp6 (TT400 x TT401). The average values were calculated from at least three independent assays. (C) Ectopic intragenic recombination. The strains crossed were WT (MS105-1B x GP1123) and mcp6 (TT400 x TT1014). The average values were calculated from at least three independent assays. Standard deviations are indicated as error bars. (D) Spores of mcp6 cells are almost normal as judged by the frequency of abnormal ascospores. The haploid parental strains were mated and sporulated on ME plate. After overnight culture, the cells were fixed with 70% ethanol for staining with Hoechst 33342. At least 200 cells were counted. (E) Spore viability of WT (TP4-5A x TP4-1D) and mcp6 (TT397-5A x TT397-1D) cells. Random spore analysis was performed.

View larger version (49K): [in a new window]   Fig. 6. The localization of Mcp6-GFP is normal in taz1 and dhc1-d3 cells. (A) A typical immunofluorescence image of Mcp6-GFP at the horsetail phase in wild type (ST134) and taz1 (ST200) cells. (B) A typical image of Mcp6-GFP at the horsetail phase in wild-type (ST142) and dhc1-d3 (ST196) cells (living). Bar, 5 µm. (C) Frequency of cells in which the Mcp6-GFP signals localize with Sad1 to the leading edge of the horsetail nucleus in the wild type and taz1 cells. (D) Frequency of cells in which the Mcp6-GFP signals localize with Sad1-DsRed to the leading edge of the horsetail nucleus in the wild-type and dhc1-d3 cells.

View larger version (66K): [in a new window]   Fig. 7. The subcellular localization of GFP-tagged SPB components at the horsetail phase is normal in mcp6 cells. The h90 strains that express Sad1-DsRed (WT, CRL790; mcp6, ST148) (A), Spo15-GFP (WT, ST176; mcp6, ST171-1) (B) or Kms1-GFP (WT, ST191-1; mcp6, ST172-1) (C) fusion proteins were induced to enter meiosis by nitrogen starvation. After 6 hours, the cells were collected and observed under a fluorescence microscope. Typical images are shown. (D) The proportions of the cell population in which Sad1-DsRed localized to the leading edge of the nucleus with a single dot, as depicted in the inset. (E) The proportions of the cell population in which Spo15-GFP and Sad1-DsRed colocalized to the leading edge of the nucleus with a single dot, as depicted in the inset. (F) The proportions of the cell population in which Kms1-GFP and Sad1-DsRed colocalized to the leading edge of the nucleus, as depicted in the inset. Green, GFP; red, Sad1-DsRed; blue, Hoechst 33342. The dotted line depicts the contour of the cell. Bar, 5 µm.

View larger version (53K): [in a new window]   Fig. 8. GFP-tagged telomere components and -tubulin localize normally in mcp6 cells. The h90 strains that express Taz1-GFP (WT, ST178; mcp6, ST173) (A), Swi6-GFP (WT, ST179-1; mcp6, ST174) (B) or -tubulin-GFP (WT, YY105; mcp6, ST146) (C) were induced to enter meiosis by nitrogen starvation. After 6 hours, the cells were collected and observed under a fluorescence microscope. Images shown in (C) were obtained by immunofluorescence. Typical images are shown. (D) The proportions of the cell population in which Taz1-GFP and Sad1-DsRed colocalized to the leading edge of the nucleus with a single dot, as depicted in the inset. (E) The proportions of the cell population in which Swi6-GFP and Sad1-DsRed colocalized to the leading edge (L.E.) of the nucleus with extra dots of Swi6-GFP in the nucleus, as depicted in the insets. (F) The proportions of the cell population that display normal or abnormal (i) and (ii) tubulin positioning as depicted on the right. Green, GFP; red, Sad1-DsRed (A,B) or Sad1 (C); blue, Hoechst 33342. The dotted line indicates the contour of the cell. Bar, 5 µm.

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