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First published online 29 June 2004
doi: 10.1242/jcs.01231

Journal of Cell Science 117, 3547-3559 (2004)
Published by The Company of Biologists 2004
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Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-{alpha}-associated protein Ctf4 is essential for chromatid disjunction during meiosis II

Mark Petronczki1,*, Barbara Chwalla1,*, Maria F. Siomos1,*, Shihori Yokobayashi2, Wolfgang Helmhart1, Adam M. Deutschbauer3, Ronald W. Davis3, Yoshinori Watanabe2 and Kim Nasmyth1,{ddagger}

1 Research Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria
2 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan
3 Department of Genetics and Department of Biochemistry, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA



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  		Fig. 1. (A) CHL1, CTF8 and CTF4 are required for sister-chromatid cohesion during mitosis. Cycling cultures of wild-type (K10003), chl1{Delta} (K11652), ctf4{Delta} (K11692), ctf8{Delta} (K10576) and trf4{Delta} (K12661) strains expressing Pds1-Myc18 (securin) and carrying chromosome V marked by GFP at the URA3 locus 35 kbp away from the centromere were fixed and stained for DNA and with antibodies against tubulin and the Myc epitope. Fluorescence images of a wild-type (left) and chl1{Delta} (right) metaphase cell showing URA3-GFP (green), tubulin (red) and DNA (blue) in the large frame and securin (white) of the respective cell above in the small inserts. The proportions of securin-positive cells with a bipolar spindle containing one or two URA3-GFP signals in the different strains are given below the images. 100 metaphase cells were scored for each strain in this experiment. (B-E) Chl1 is required during S phase to support sister-chromatid cohesion. A haploid yeast strain (K12131) with chromosome V marked by GFP at the URA3 locus and expressing Pds1-Myc18 (securin) in which the endogenous CHL1 ORF was put under regulation of the Gal1-10 promoter and three HA tags were fused to the N-terminus was arrested with {alpha} factor and released in medium containing raffinose (B) with no addition of galactose, (C) with the addition of galactose at 0 minutes (min) or (D) with addition of galactose at 45 minutes. At the indicated time points, DNA replication was monitored by flow cytometry of DNA content and expression of HA3-Chl1 by immunoblotting with an anti-HA antibody and a loading control (Swi6). (E) Samples from all three cultures taken at 90 minutes were fixed and stained with antibodies against tubulin and the Myc tag of securin to asses the ratio of securin-positive cells with a bipolar spindle containing one or two URA3-GFP signals. 100 metaphase cells were scored for all three cultures in this experiment. (F) Chl1 localizes to the nucleus. Cycling cells of a strain expressing Chl1-Myc18 (K11770) (left) and of an untagged control strain (K8378) (right) were fixed and stained for DNA and with antibodies against tubulin and the Myc epitope. Fluorescence microscopy images of a G1 (left) and metaphase (right) cell showing tubulin (red) and DNA (blue) in the upper panel and anti-Myc staining of the respective cells above in the lower panel.

 


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  		Fig. 3. (A-D) CTF8 is required for sister-chromatid cohesion during meiosis. Diploid wild-type (K10003) (A,B) and ctf8{Delta} (K10576) (C,D) strains expressing Pds1-Myc18 (securin) and carrying one homolog of chromosome V marked by GFP (heterozygous URA3-GFP) were analysed in a meiotic time course experiment. Cells were fixed and stained for DNA and with antibodies against tubulin and the Myc epitope. Fluorescence microscopy images of various meiotic cell cycle stages (indicated below the images) of wild-type (A) and ctf8{Delta} (C) cells showing URA3-GFP (green), tubulin (red) and DNA (blue) are arranged in the upper panels as a simulated time course. The lower panels in (A,C) display the securin staining of the cells above them. Fluorescent-microscopy pictures of a sporulated wild-type (B) and ctf8{Delta} (D) tetrad stained for DNA (blue) and showing heterozygous URA3-GFP (green). (E,F) Deletion of CHL1 does not cause frequent meiosis I non-disjunction of homologues. Diploid wild-type (K8409) (E) and chl1{Delta} (K11603) (F) strains carrying both homologs of chromosome V marked by GFP (homozygous URA3-GFP) were analysed in meiotic time course experiment. Cells were fixed and stained for DNA and with antibodies against tubulin. Wild-type (E) and chl1{Delta} (F) anaphase I cells displaying URA3-GFP (green), tubulin (red) and DNA (blue) are shown. For each strain, 100 anaphase I cells were scored in this experiment.

 


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  		Fig. 2. The alternative RF-CCtf18/Dcc1/Ctf8, CHL1 and CTF4 are required for chromosome segregation during meiosis and for spore viability. Diploid wild-type (K8409), ctf18{Delta} (K10488), dcc1{Delta} (K10117), ctf8{Delta} (K10092), chl1{Delta} (K11603), ctf4{Delta} (K11681) and trf4{Delta} (K12384) strains in which both homologs of chromosome V were marked by GFP (homozygous URA3-GFP) were sporulated and chromosome segregation in tetrads was scored under the fluorescence microscope. Spore viability (n=100) was determined by dissection. Diploid wild-type (K10003), ctf18{Delta} (K10608), dcc1{Delta} (K10352), ctf8{Delta} (K10576), chl1{Delta} (K11652), ctf4{Delta} (K11692) and trf4{Delta} (K12661) strains in which only one copy of chromosome V was marked by GFP (heterozygous URA3-GFP) were sporulated and chromosome segregation in tetrads was scored under the fluorescence microscope. More than 200 tetrads of each strain were scored for URA3-GFP segregation. n.d., not determined.

 


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  		Fig. 6. Association of Rec8 with chromosomes and its retention at centromeres in meiosis II are not affected in the absence of CTF8. Diploid wild-type (K10003) (A,C) and ctf8{Delta} (K10572) (B,D) strains expressing Rec8-HA3 and Pds1-Myc18 (securin) were analysed in a meiotic time-course experiment by chromosome spreading (A,B) and in situ immunofluorescence (C,D). Chromosome spreads were stained for DNA and with antibodies against the HA tag. Pachytene-stage chromosome spreads of wild-type (A) and ctf8{Delta} (B) cells showing Rec8 (red) and DNA (blue) in the upper panels and Rec8 alone (white) in the lower panels. Fixed cells were stained for DNA and with antibodies against tubulin, the HA and Myc epitope, and analysed by fluorescence microscopy. Wild-type (C) and ctf8{Delta} (D) metaphase II cells showing tubulin (red) and DNA (blue) in the upper panel, Rec8 (white) in the middle panel, and securin (white) in the lower panel. 100 cells were scored for each strain in this experiment.

 


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  		Fig. 4. Mutations in the fission yeast ctf18 and dcc1 homologs cause meiosis II non-disjunction. Schizosaccharomyces pombe wild-type (PY796xJY333) (A), ctf18– (PX101xPX131) (B) and dcc1– (PX100xPX128) (C) strains carrying one homolog of chromosome 1 marked by GFP (heterozygous cen1-GFP) were sporulated and analysed under the fluorescence microscope. cen1-GFP is shown in white and the asci are highlighted using margins. (D) Diagram showing the percentages of cen1-GFP meiosis II non-disjunction in fission yeast wild type, ctf18– and dcc1– tetrads. More than 200 tetrads were scored for each strain.

 


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  		Fig. 5. Deletion of CTF8, CHL1 and CTF4 rescues the nuclear division block of mam1{Delta} cells in anaphase I. Diploid mam1{Delta} (K8923) (A,B) and mam1{Delta} ctf8{Delta} (K10572) (D,E) strains expressing Pds1-Myc18 (securin) and carrying one homolog of chromosome V marked by GFP (heterozygous URA3-GFP) were analysed in a meiotic time course. Cells were fixed and stained for DNA and with antibodies against tubulin and the Myc epitope. Fluorescent-microscopy images of securin-positive metaphase I cells with a bipolar spindle (A,D), a securin-negative `abortive' anaphase I (B) and a securin-negative elongated anaphase I (E) are shown. (A,B,D,E) The upper panels show URA3-GFP (green), tubulin (red) and DNA (blue); the lower panels show securin staining (white) of the cells above them. Schematic drawings illustrating an `abortive' anaphase of mam1{Delta} cells (C) and the rescued anaphase in mam1{Delta} ctf8{Delta} cells (F) showing meiotic cohesin in red and URA3-GFP in green. (G) The percentage of nuclear division and spindle elongation in wild-type (K10003), ctf8{Delta} (K10576), chl1{Delta} (K11652), ctf4{Delta} (K11692), mam1{Delta} (K8923), mam1{Delta} ctf8{Delta} (K10572), mam1{Delta} chl1{Delta} (K11703) and mam1{Delta} ctf4{Delta} (K11828) cells following the destruction of securin in anaphase I. 100 securin-negative cells with a bipolar metaphase I or anaphase I spindle were scored for each strain in this experiment.

 

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© The Company of Biologists Ltd 2004