spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 20 July 2004
doi: 10.1242/jcs.01248

Journal of Cell Science 117, 3875-3886 (2004)
Published by The Company of Biologists 2004
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamamoto, T. G.
Right arrow Articles by Hiraoka, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamamoto, T. G.
Right arrow Articles by Hiraoka, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Activation of the pheromone-responsive MAP kinase drives haploid cells to undergo ectopic meiosis with normal telomere clustering and sister chromatid segregation in fission yeast

Takaharu G. Yamamoto1,2, Yuji Chikashige1,2, Fumiyo Ozoe3, Makoto Kawamukai3 and Yasushi Hiraoka1,2,*

1 Cell Biology Group and CREST Research Project, Kansai Advanced Research Center, National Institute of Information and Communication Technology, 588-2 Iwaoka-cho, Iwaoka, Nishi-ku, Kobe 651-2492, Japan
2 Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, 560-0043, Japan
3 Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, 690-8504, Japan



View larger version (42K):

[in a new window]
  		Fig. 1. Progression of meiosis induced by the expression of byr1DD. (A) Amino acid sequences of H. sapiens MAPKK and S. pombe Byr1. MAPKKK phosphorylation sites of H. sapiens MAPKK and the corresponding sites of Byr1 are underlined. To make a constitutively active form of Byr1 (Byr1DD), these sites of Byr1 were replaced with aspartic acid (shown as `D'). (B) Mitotic growth defects in cells expressing byr1DD. h– wild-type (CRL239) cells were transformed with either pREP81 (vector), pREP81-byr1+ (byr1+) or pREP81-byr1DD (byr1DD). Each transformant was cultured to mid-log phase in liquid EMM2 with thiamine. After removing thiamine, fivefold serial dilutions of each transformant were spotted onto EMM2 plates with (right panel, `Repressed') or without thiamine (left panel, `Induced'). Plates were photographed after 4 days incubation at 30°C. (C) h– wild-type (CRL239) cells carrying pREP81-byr1DD were induced to undergo ectopic meiosis in liquid EMM2 at 30°C, and fixed at 30 hours after induction. Nuclei were stained with DAPI. Bright-field images of the same cells are also shown (Phase). Arrows indicate cells with three or four nuclei, or spores. Scale bar: 5 µm. (D) Progression of meiosis in cells expressing byr1DD. h– wild-type (CRL239) cells carrying pREP81-byr1DD were induced to undergo ectopic meiosis in liquid EMM2 at 30°C. The number of nuclei per cell was counted every 2 hours from 13 to 29 hours after induction. At least 300 cells were scored for each time point. During analysis, the cell concentration was kept between 1x106 and 2x107 cells/ml by diluting with EMM2. (E) DNA content, measured by flow cytometry. The same cell samples used in D were examined.

 


View larger version (36K):

[in a new window]
  		Fig. 2. Sister chromatid cohesion in cells expressing byr1DD. Segregation of sister chromatids in meiosis I (A,B) and in meiosis II (C,D). (A,C) h+/h– diploid (TG157) cells (left), h–/h– diploid (TG143) cells carrying pREP1-byr1DD (middle), and h– haploid (AY193-3A) cells carrying pREP81-byr1DD (right) were induced to undergo meiosis and observed by fluorescence microscopy without fixation. The lys1 locus near the centromere (green) was stained using the lacI-GFP/lacO recognition system. In diploid cells, one of two lys1 loci was stained. The nucleus (white) was stained with Hoechst 33342. Scale bars: 5 µm. (B,D) Patterns of segregation were classified into two categories in B or five categories in D based on the number of nuclei (open circles) and the number of lys1-GFP signals (dots in the circle). Numbers of the cells examined (N) are shown at the top of each graph.

 


View larger version (39K):

[in a new window]
  		Fig. 3. Mitotic growth defects in mutant cells expressing byr1DD. (A) Amino acid sequences of H. sapiens MAPK and S. pombe Spk1. MAPKK phosphorylation sites of H. sapiens MAPK and the corresponding sites of Spk1 are underlined. To make unphosphorylatable forms of Spk1, these sites in Spk1 were replaced with either or both alanine (A) and phenylalanine (F). These mutated Spk1 constructs are designated Spk1AY, Spk1TF, and Spk1AF as indicated. (B) h– spk1+ (TG7) cells, h– spk1{Delta} (TP106-3C) cells, h– spk1AF (TG43) cells, h– spk1AY (TG44) cells, h– spk1TF (TG45) cells, h– mei2{Delta} (CRL544) cells, and h– mei3{Delta} (TG30) cells were transformed with pREP81-byr1DD. Each transformant was cultured in liquid EMM2 with thiamine. After removing thiamine, fivefold serial dilutions of each transformant were spotted onto EMM2 plates with (right panel, `Repressed') or without thiamine (left panel, `Induced'). Plates were photographed after 4 days incubation at 30°C. (C) h– spk1{Delta} (TP106-3C) cells, h– mei2{Delta} (CRL544) cells, and h– mei3{Delta} (TG30) cells carrying pREP81-byr1DD were cultured in liquid EMM2 at 30°C to induce ectopic meiosis, and fixed at 30 hours after induction. The nuclei were stained with DAPI. Bright-field images of the same cells are also shown (Phase). Scale bar: 5 µm. (D) Progression of meiosis in mei3{Delta} cells expressing byr1DD. h– mei3{Delta} (TG30) cells carrying pREP81-byr1DD were induced to undergo meiosis in liquid EMM2 at 30°C. The number of nuclei per cell was counted every 2 hours from 13 to 29 hours after induction (open symbols). As a control, h– wild-type (CRL239) cells carrying pREP81-byr1DD were examined (filled symbols). At least 300 cells were scored for each time point. During analysis, the cell concentration was kept between 1x106 and 2x107 cells/ml by diluting with EMM2.

 


View larger version (25K):

[in a new window]
  		Fig. 7. A model for genetic regulation of meiosis and telomere clustering. Regulatory pathways in the normal process of meiosis (A), in meiosis induced by byr1DD expression (B), and in meiosis induced by Pat1 inactivation (C). Activation and inhibition are indicated by arrows and crossing bars, respectively. Direct biochemical interaction has been proved for the inhibition of Pat1 by Mei3, the inhibition of Mei2 by Pat1, and the activation of Spk1 by Byr1 (indicated by a single arrow or bar). Other regulatory pathways indicated by two successive arrows or bars are conceptual, and could be indirect.

 


View larger version (28K):

[in a new window]
  		Fig. 4. Nuclear localization of telomeres in cells expressing byr1DD. h– wild-type (TG309) cells (A) and h– spk1{Delta} (TG335) cells (B) carrying pREP81-byr1DD were cultured in liquid EMM2 for 27 hour at 30°C to induce ectopic meiosis, and observed by fluorescence microscopy without fixation. The SPB (red) was stained with CFP-Sad1, and telomeres (green) were stained with Taz1-GFP. Bright-field images of the same cells are also shown (Phase). Scale bars: 5 µm. (C) Time course of cell shape alteration (`altered shape') and telomere clustering (`tel clustering') after induction of byr1DD in h– wild-type (TG309) cells and h– spk1{Delta} (TG335) cells carrying pREP81-byr1DD. To calculate the percentage of cells with altered shape, at least 130 cells were scored for each time point. To calculate the percentage of cells with telomere clustering, at least 50 cells were scored for each time point. (D) h– wild-type (CRL239) cells carrying pREP81-byr1DD were induced to undergo ectopic meiosis in liquid EMM2 at 30°C, and fixed at 20 hours after induction. The SPB (red) was stained with anti-Sad1 antibodies; telomeres (green) and centromeres (blue) were stained by FISH using cos212 and pRS140 as DNA probes, respectively. The nucleus (white) was stained with DAPI. Scale bar: 5 µm.

 


View larger version (64K):

[in a new window]
  		Fig. 5. Nuclear localization of telomeres in cells treated with mating pheromone. h– sxa2{Delta} (TG208) cells (A,B), h– sxa2{Delta} mei2{Delta} (TG263) cells (C-E), and h– sxa2{Delta} spk1{Delta} (TG265) cells (F,G) were treated with the synthetic P-factor, and observed by fluorescence microscopy without fixation. (A,C,D,F) The SPB (red) and telomeres (green) were stained with Sad1-DsRed and Taz1-GFP, respectively. Bright-field images of the same cells are also shown (Phase). Scale bars: 5 µm. (B,E,G) Time course of cell shape alteration (`altered shape') and telomere clustering (`tel clustering') after P-factor treatment. To calculate the percentage of cells with altered shape, 200 cells (B,G) or 130 cells (E) were scored for each time point. To calculate the percentage of cells with telomere clustering, at least 50 cells were scored for each time point (B,E,G).

 


View larger version (48K):

[in a new window]
  		Fig. 6. Nuclear localization of telomeres in pat1-114 mutant cells. h– pat1-114 (TG292) cells (A-C), h– pat1-114 mei2{Delta} (TG202) cells (D-F), and h– pat1-114 spk1{Delta} (CRLc92) cells (G-I) were shifted to the restrictive temperature to induce ectopic meiosis, and observed by fluorescence microscopy without fixation. (A,D,G) Progression of meiosis after shifting to the restrictive temperature. The number of nuclei per cell was counted for up to 10 hours after induction of meiosis. At least 250 cells were scored for each time point. (B,E,H) The SPB (red) was stained with CFP-Sad1, and telomeres (green) were stained with Taz1-GFP. Bright-field images of the same cells are also shown (Phase). Scale bars: 5 µm. (C,F,I) Time course of telomere clustering after shifting to the restrictive temperature. To calculate the percentage of cells with telomere clustering, at least 50 cells were scored for each time point.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2004