doi:10.1016/j.ejcb.2008.05.0020171-9335/$ - se
The F658G substitution in Saccharomyces cerevisiae cohesin Irr1/Scc3 is
semi-dominant in the diploid and disturbs mitosis, meiosis and the cell cycle
Agata Cenaa, Ewa Kozowskab, Danuta Pochockaa, Marcin Grynberga, Takao Ishikawac, Jan Fronkc, Anna Kurlandzkaa,
aInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland bDepartment of Immunology, Institute of Zoology, Faculty of Biology, Warsaw University, Warsaw, Poland cInstitute of Biochemistry, Faculty of Biology, Warsaw University, Warsaw, Poland
Received 27 June 2007; received in revised form 29 April 2008; accepted 2 May 2008
Abstract
The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p and Smc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation (F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. The mutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able to interact with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects, irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, and experiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assembly checkpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to induce marked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability to interact with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation. r 2008 Elsevier GmbH. All rights reserved.
Keywords: Saccharomyces cerevisiae; Sister chromatid cohesion; Mitosis; Meiosis
Introduction
The maintenance of proper ploidy during cell division is crucial for the cell functioning. It requires both an accurate replication of chromosomes and their faithful segregation during mitosis and meiosis. In Saccharo-
myces cerevisiae the majority of cell cycle events are similar to the processes in other eukaryotes. The major
e front matter r 2008 Elsevier GmbH. All rights reserved.
cb.2008.05.002
4636.
ess: ania218@poczta.ibb.waw.pl (A. Kurlandzka).
differences are that in yeast, unlike in higher eukaryotes, DNA replication and spindle assembly are initiated simultaneously and the nuclear envelope remains intact throughout mitosis.
It is now generally accepted that the proper physical association of sister chromatids is maintained by the sister chromatid cohesion complex (SCC), and the unperturbed functioning of this complex is critical for genome stability (Hirano, 2000; Nasmyth, 2001, 2005; Uhlmann, 2004). The SCC, conserved in structure and function from yeast to mammals, is called cohesin in S.
cerevisiae. It is proposed that cohesins form a ring around sister chromatids (for a review, see Nasmyth and
ARTICLE IN PRESS A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844832
Haering, 2005). The cohesin complex is composed of a heterodimer of SMC (structural maintenance of chro- mosomes) proteins and of two non-SMC subunits: Mcd1/Scc1 (YDL003W) and Irr1/Scc3 (YIL026C). All genes encoding cohesins are essential – their deletion causes cell death. Mutations in genes encoding the proteins forming cohesin are either lethal or cause severe defects in chromosome segregation (Michaelis et al., 1997; Guacci et al., 1997; Klein et al., 1999; Kurlandzka et al., 1995; Uhlmann, 2003). The dissolution of the cohesion complex is precisely regulated by mechanisms that remain only partially known.
In mitosis, the cohesin complex holds sister chroma- tids together and, simultaneously, sister kinetochores are pulled to opposite cell poles by microtubules of the elongating mitotic spindle. This is probably the basis of the tension which signals proper bipolar attachment of kinetochores. This bipolar attachment is required for the proper assembly and functioning of the proteins engaged in the spindle assembly checkpoint (SAC). The correct functioning of this checkpoint is indispen- sable for mitosis exit (for reviews, see Lew and Burke, 2003; Pinsky and Biggins, 2005). Once all chromatids are aligned, the inhibitory signal from SAC that monitors bi-orientation is relieved. The anaphase- promoting complex (APC) then targets Pds1/securin for ubiquitin-mediated degradation. Pds1 proteolysis allows Esp1/separin to cleave the Mcd1/Scc1 cohesin subunit, triggering chromatid separation (Cohen-Fix et al., 1996; Ciosk et al., 1998).
When mitotically dividing yeast cells are induced to enter meiosis, cells exit the mitotic cell cycle at the G1 phase (Kupiec et al., 1997; Neiman, 2005). The switch from mitosis to meiosis has been thoroughly investi- gated at the transcriptional level (Kassir et al., 2003; Pnueli et al., 2004), and the mechanisms of chromosome segregation during meiosis are quite well known (Nasmyth, 2005). It has been established that accurate meiotic segregation requires replacement of the mitotic cohesin subunit Scc1 by the meiosis-specific kleisin Rec8. At the first meiotic division (meiosis I), Rec8 is cleaved along chromosome arms but is protected at the centromeres, where it is only cleaved during the second division (meiosis II) (Lee and Orr-Weaver, 2001; Watanabe, 2005).
The yeast Irr1/Scc3 protein is the least studied element of the cohesion complex, although homologues of this protein have been identified in other eukaryotes from fungi to plants and mammals (Wang et al., 2003; Valdeolmillos et al., 2004; Chelysheva et al., 2005; Hauf et al., 2005). In mammals, three homologues, called STAG1-3 or stromalins, were described, two of them being involved in mitotic cohesion and one being specific to meiosis (Carramolino et al., 1997; Prieto et al., 2001). The aminoterminal part of Irr1 (first 496 amino acids), conserved among eukaryotes, is 18–25% identical to
members of the stromal antigen protein family (Toth et al., 1999), whereas the C-terminal part is variable.
The proposed models of S. cerevisiae cohesin do not specify the position of Irr1p in the complex, although it is assumed that the primary role of Irr1p consists in closing the cohesin ring, and this protein is usually depicted as an element attached to the Mcd1/Scc1 kleisin subunit (Haering and Nasmyth, 2003; Nasmyth, 2005). Recent data obtained from proteome-wide purification of yeast protein complexes (Krogan et al., 2006) confirmed the previously identified interactions among Irr1p, Mcd1p and Smc3p, but also detected new Irr1p interactions with Hta2p (histone H2A subtype) and Yra1p (a nuclear protein required for export of mRNA from the nucleus).
The role of Irr1p/Scc3p in meiosis is poorly known. A meiosis-specific variant of Irr1p – STAG3 – has been described in mammalian cells (Pezzi et al., 2000). In Caenorhabditis elegans, the localization of a Rec8 homologue to chromosomes has been shown to depend on the presence of an Irr1p homologue – SCC-3 (Wang et al., 2003; Pasierbek et al., 2003). It has also been shown that Arabidopsis cohesins, AtREC8 (homologue of Rec8p) and AtSCC3 (homologue of Irr1p), are necessary for the monopolar orientation of kinetochores at meiosis I and for the maintenance of centromeric cohesion at anaphase I (Chelysheva et al., 2005).
Here we present data on an irr1-1 mutation (F658G substitution in the Irr1p/Scc3p cohesin) of S. cerevisiae
which is lethal in the haploid and semi-dominant in diploid yeast cells. In our recent paper (Cena et al., 2007), we described a rather unexpected influence of the presence of this mutated copy of IRR1 on the cell wall integrity. Here we show that the heterozygous diploid irr1-1/IRR1 exhibits significant irregularities in mitotic divisions: chromosome segregation errors, disturbances in segregation of nuclei and in cytokinesis. Moreover, this diploid is severely affected in meiosis. Our data suggest that these irregularities could result from malfunctioning of the spindle assembly checkpoint. These defects exhibit incomplete penetrance, in which they resemble phenotypes observed in pre-cancerous mammalian cells. Thus, our yeast irr1-1/IRR1 diploid may serve as a model to investigate the general aspects of genome integrity maintenance.
Materials and methods
Strains and media
Yeast strains used in the present study, isogenic with the strain W303, are listed in Table 1. Escherichia coli
XL1-Blue MRF0 (Stratagene, Saint Quentin en Yve- lines, France) was used for molecular manipulations.
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Strain Genotype Source
W303 MAT a ade2-1 trp1-1 leu2-3,112 his3-11 ura3-1 can1-100 Rothstein
collection
IRR1/IRR1
(DW303)
ura3-1 can1-100/can1-100
ura3-1 can1-100/can1-100 irr1DHkanMX4/IRR1
11 ura3-1/ura3-1 can1-100/can1-100 irr1DHkanMX4/IRR1
11/his3-11 ura3-1/ura3-1 can1-100/can1-100 irr1DHkanMX4 /IRR1-HA3HHIS3
This study
458 MAT a ade2-1 trp1-1 leu2-3,112 his3-11,15 ura3-1 can1-100 SCC3-HA3HHIS3 F. Uhlmann
615 MAT a ade2-1 trp1-1 leu2-3,112 his3-11,15 ura3 can1-100 SCC3-Myc18HTRP1 SCC1-
HA6HHIS3
F. Uhlmann
K7518 MAT a scc3-1 ade2-101 his3D200 leu2D1lys2-801 trp1-1 CAN+ ura3H3URA3 tet0112
leu2HLEU2tetR-GFP
ura3H3URA3tet0112 leu2HLEU2tetR-GFP/leu2HLEU2tetR-GFP
ura3H3URA3tet0112 leu2HLEU2tetR-GFP/leu2HLEU2tetR-GFP
11/his3-11 ura3-1/ura3-1 can1-100/can1-100 IRR1/IRR1 ura3H3URA3tet0112/ura3
This study
ura3H3URA3 tet0112/ura3H3URA3tet0112 leu2HLEU tetR-GFP/
leu2HLEU2tetR-GFP
(2004)
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 833
Yeast culture media were prepared as described (Rose et al., 1990). YPD contained 1% Bacto-yeast extract, 2% Bacto-peptone and 2% (all w/v) glucose. SD contained 0.67% yeast nitrogen base without amino acids (Difco, Detroit, MI, USA) and 2% glucose. For auxotrophic strains, the media contained appropriate supplements. Standard methods were used to genetically manipulate yeast cells (Rose et al., 1990). Routinely, whenever possible strains were backcrossed at least twice. For drop tests, cells were grown overnight in YPD or minimal media and adjusted to a density OD600 ¼ 1. Growth was analyzed by plating 5-ml drops of 10-fold serial dilutions of cell suspensions onto solid media. Reagents were tested in concentrations recommended by Rieger et al. (1999).
Plasmid construction, gene tagging, site-directed
mutagenesis
Plasmids used in this study are listed in Table 2. To construct the IRR1-Myc13 fusion (pAC1) the 30- terminal fragment of IRR1 was PCR-amplified using primers: F1: 50-GGAGAGTCTTTTCGCTAAGC-30
and R1: 50-ATCTTGTGTGATTTCGTCGC-30. The Myc13-TADH1 fragment was amplified using pFA6a-
13Myc-kanMX6 as a template (Longtine et al., 1998) and primers F2: 50-GCGACGAAATCACACAA- GATTTAATTAACGGTGAACAAAAGC-30 (the se- quence encoding a fragment of IRR1 is underlined) and R2: 50-GTCCCGGGATATTACCCTGTTATCC-30
complementary to TADH1 and introducing an XmaI restriction site (CCCGGG). Products of both PCRs were combined and served as a hybrid template from which a third PCR product was obtained using primers F1 and R2. This product was introduced into pAK11/3. Site-directed mutagenesis was carried out using the Altered Sites in vitro mutagenesis system (Promega). Details of the F658G replacement have been described before (Cena et al., 2007). The plasmid pAC1/2, bearing the irr1-1 allele 30-fused with 13 repetitions of Myc, was a derivative of pAC1.
Western blotting and co-immunoprecipitation
To visualize chimeric HA- or c-Myc-tagged proteins on Western blots, protein samples (100 mg/lane) were subjected to 8% SDS–PAGE. Electrophoresis was followed by blotting onto Hybond-C extra membrane and probing with an anti-HA monoclonal antibody (HA.11, clone 16B12) (BabCO) or anti-c-Myc 9E10
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Plasmid Relevant plasmid genotype Reference
pAK11/1 PIRR1-IRR1-TIRR1 URA3 CEN This study, based on pRS316
pAK11/3 PIRR1-IRR1-TIRR1 TRP1 CEN This study, based on pRS314
pAK11/3F-G PIRR1-irr1-1-TIRR1 TRP1 CEN This study, as above
pAK11/17 PIRR1-irr1-1-TIRR1 TRP1 INT This study, derivative of pRS305
pAC1 PIRR1-IRR1-Myc13-TADH1 TRP1 CEN This study
pAC1/2 PIRR1-irr1-1-Myc13-TADH1 TRP1 CEN This study
pAC1/3 PIRR1-irr1-1-Myc13-TADH1 TRP1 INT This study
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844834
(BabCO). For co-immunoprecipitation, cells expressing chimeric Irr1p-Myc and Scc1p-HA or Irr1-1p-Myc and Scc1p-HA proteins were harvested at an OD600 ¼ 1 and resuspended in 50mM sodium phosphate buffer (pH 7.5), 50mMNaCl, 2mMMgCl2, 0.2% Triton X-100, contain- ing protease inhibitor cocktail Complete (Roche) and 25U/ml Benzonase (Merck). Cells were homogenized with glass beads in a bead-beater, the homogenate was spun down in a microfuge. The whole procedure was carried out on ice or at 4 1C. Soluble protein extracts were incubated with Protein G-agarose (Sigma) covered with anti-HA (Covance, monoclonal antibody HA.11 clone 16B12) or anti-Myc (Covance, monoclonal antibody clone 9E10) overnight at 4 1C. The immunoprecipitate was analyzed by SDS–PAGE, blotted and probed with anti-HA and anti-Myc antibodies, as above.
Microscopy and chromosome spreads
To localize nuclei, a Nikon Eclipse E800 fluorescence microscope with a 63 objective was used. DAPI (40,60- diamino-2-phenylindole dichloride, Sigma) was used to stain DNA. To visualize c-Myc-tagged Irr1-1p, an anti- Myc antibody from Invitrogen was used. Chromosome segregation was monitored by following segregation of chromosome V carrying tandem repeats of the Tet operator integrated in the centromeric region which could be visualized by GFP because the cells expressed also the tet-GFP fusion, as described by Michaelis et al. (1997). For quantitative assays, cells were grown in YPD medium to log phase, stained with DAPI, and examined by fluorescence microscopy. Over 400 cells were counted for each strain. Experiments were done in duplicate, and the percentage of cells representing indicated morphology classes was determined. Chromo- some spreads were prepared as described (Klein et al., 1992; Michaelis et al., 1997), DNA was stained with DAPI.
Cell synchronization
For nocodazole (NZ, metaphase arrest) treatment experiments, diploid cells were grown in YPD complete
medium to OD600 ¼ 1 (2 107 cells/ml), corresponding to the exponential part of the growth curve (data not shown). Subsequently, cells were incubated in YPD medium with 15 mg/ml NZ at 30 1C for 3 or 5 h. After incubation, cells were washed with water twice and transferred into fresh YPD to allow the cell cycle to resume. Aliquots were withdrawn at 30-min intervals and transferred into meiosis-inducing medium (1% potassium acetate) or prepared for FACS analysis. For the 0-h time point the cells, after washing off the synchronizing agent, were directly suspended in meiosis- inducing medium or prepared for FACS. Meiosis was assayed after 3 days of incubation by observation in a light microscope.
FACS analysis
Flow cytometric DNA quantification was performed according to Mark Winey’s lab on-line protocols (http:// mcdb.colorado.edu/labs/winey/protocols.html). Cells were fixed in 70% ethanol and kept at 20 1C until use. For staining, the cells were incubated overnight in 50 mg/ml propidium iodide in the dark at 4 1C and sonicated for 15 s before the measurement.
Results
localization or ability to associate with chromosomes
Our main purpose was a systematic investigation of the role of various parts of the Irr1p/Scc3p cohesin, since functional domains of this protein (or its homo- logues) were not known. To achieve this we introduced amino acid substitutions in parts of the Irr1p molecule which were deemed, based on homology studies, to be important as potential functional or structural sites. As it was mentioned in our previous paper (Cena et al., 2007), the introduction of an allele encoding the F658G substitution, named irr1-1, into the irr1D/IRR1 diploid caused incorrect morphology in mitosis and meiosis, indicating a role of the mutated residue in Irr1p
synthesized and properly localized in the heterozygous irr1-1/
IRR1 diploid. Haploid strains expressing wild-type Scc3/Irr1-
HA3 or Scc3/Irr1-Myc18 protein were obtained from F.
Uhlmann. Myc13-tagged Irr1-1p was constructed as described
in Materials and methods. (A) Immunoblots of whole-cell
extracts were probed with anti-Myc and anti-HA antibodies.
Lane 1: Haploid strain expressing Scc3/Irr1-3HA, lane 2:
haploid strain expressing Scc3/Irr1-18Myc, lane 3: hetero-
zygous diploid expressing Irr1-1-Myc13 and Irr1-3HA. (B)
The mutated Irr1-1 protein is present in the nucleus. The
irr1D/IRR1 hemizygote was transformed with a plasmid
bearing the irr1-1-Myc13 gene, leading to its integration into
the TRP1 locus. Localization of the Myc13-tagged Irr1-1
protein was followed by indirect immunofluorescence; cells
were probed with anti-Myc primary antibodies and anti-mouse
Cy3-conjugated secondary antibodies. Nuclei were stained
with DAPI. (C) Irr1-1p localizes to chromosomes. Chromo-
some spreads from the heterozygous strain expressing Irr1-1-
Myc13p protein were analyzed by indirect immunofluores-
cence as detailed above.
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 835
functioning, and numerous phenotypes suggesting changes in the cell wall integrity. The irr1-1 mutation was lethal in the haploid state. To further assay the role of the F658G substitution and to facilitate further experiments, the irr1-1 gene was integrated into the TRP1 locus (chromosome IV, whereas IRR1 is on chromosome IX) of the irr1D/IRR1 hemizygote giving the strain irr1-1/IRR1 (Cena et al., 2007).
Since the phenotype of irr1-1/IRR1 was different from that of the irr1DHkanMX4/IRR1 hemizygote, which in fact did not differ from the wild type, except for spore germination (Cena et al., 2007), we expected that the mutated irr1-1 gene was expressed in the cell. To check whether Irr1-1p was indeed present, and also whether it affected the level of the original Irr1 protein, we constructed a diploid bearing the wild-type genomic copy of IRR1 30-terminally fused with a triple HA epitope- encoding sequence, and the irr1-1 allele 30-terminally fused with Myc13 (both under the native IRR1 promoter) and integrated into the TRP1 locus. Due to the various lengths of the epitopes, we could distinguish the wild-type and the mutated protein and we found that both proteins were indeed present in the irr1-1/IRR1 heterozygote and had the expected sizes (Fig. 1A). Importantly, the levels of both proteins were similar to the level of Irr1p in wild-type haploids (compare lane 3 with lanes 1 and 2). The Myc- tagged Irr1-1 mutant protein was also correctly localized to the nucleus (Fig. 1B). To check whether Irr1-1p retained its chromosome-binding properties, we examined chromo- some spreads of cells expressing the Irr1-1-Myc fusion. As unsynchronized mid-log cultures were used for the spreads, they must have contained cells in all phases of the cell cycle. In almost all cases the fluorescence signals coming from the Myc-fusion protein and from DAPI- stained DNA overlapped (Fig. 1C). This indicated that the Irr1-1 protein retained its ability to interact with chromo- somes. Further in this paper we show in more detail that Irr1-1p also retained its ability to interact with a specific component of the cohesin complex, Scc1p.
In conclusion, we found that the phenotypic effects of the irr1-1 allele in the heterozygous irr1-1/IRR1 diploid were not due to problems with Irr1-1p synthesis, stability, subcellular distribution or general properties.
The strain with both the wild-type and mutated copies of Irr1p tagged was constructed only to follow the fate of the proteins in the heterozygous diploid, but in all further studies the irr1-1/IRR1 strain with non-tagged versions of the genes was used to exclude possible side- effects of the tags.
The irr1-1 mutation causes irregularities in cell
divisions and affects meiosis
In the next step we characterized the defects caused by the presence of Irr1-1p. In Fig. 2 we show examples of
irr1-1/IRR1 cells with nuclear segregation defects, in which we observed two nuclei in one cell or which underwent irregular divisions. Here, binucleate cells constituted ca. 15–20% of all cells, whereas in wild-type control the percentage of aberrant cells did not exceed 3%. Although the presence of the F658G substitution in Irr1-1p caused aberrations in mitotic divisions of irr1-1/ IRR1, it did not affect the strain growth rate on solid media (Fig. 3).
Subsequently, we checked how the presence of the mutated Irr1-1p cohesin influenced meiosis. We found that meiosis of an asynchronous culture of irr1-1/IRR1
was initiated ca. 24 h after transferring to 1% potassium acetate (the same was observed in the wild-type diploid IRR1/IRR1), and after 48 h ca. 64% of cells sporulated (52% in the control). However, we observed that in irr1-
1/IRR1 asci were occasionally formed from cells that still had a bud attached, or asci containing more than four spores were present; the aberrant asci represented ca. 11% of all. Fig. 4 shows examples of such cells (see also Table 4). This aberration suggests that the irr1-1/ IRR1 cells were entering meiosis before having com- pleted mitotic cell division.
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Fig. 2. The presence of the irr1-1 mutation in the heterozygous
irr1-1/IRR1 diploid causes irregularities in mitotic divisions
and nucleus transmission to the progeny cell. Defects are
observed in ca. 15–20% of all cells. Cells were viewed under
fluorescence microscopy, nuclei were stained with DAPI.
Fig. 3. Strain irr1-1/IRR1 does not show growth defects on
YPD solid medium.
in cells which have not completed the mitotic cell cycle.
Examples of asci containing six or eight spores. DNA was
stained with DAPI.
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844836
In the population of sporulating cells we noticed asci containing four, six or eight spores. Eighteen asci were dissected and the viability of spores was low (28 germinated). None, one or two or three spores were viable in asci containing six or eight spores and none or one spore in those containing four spores. The ploidy of
the obtained spore clones was estimated by FACS analysis and, with one exception, the DNA content per cell was equivalent to haploid (not shown). The spore clones were able to mate exclusively with either a MATa or a MATa wild-type sex tester haploid strain; none mated with both. DAPI staining of nuclei showed that, with the exception of one clone, the cells had only one nucleus. The spores did not contain the irr1-1 allele as neither of them was Trp+. One spore gave progeny of cells with two nuclei which were Trp+ (but not G418R), thus they bore both irr1-1 (whose integration restored wild-type TRP1) and the wild-type copy of IRR1 (as it is absolutely required for cell viability).
To check whether the mutated Irr1-1p could be competed out by an excess of wild-type Irr1p we introduced a wild-type copy of IRR1 on the centromeric plasmid pAK11/3 to the irr1-1/IRR1 heterozygote. We found that the presence of the extra copy of IRR1
decreased the number of bi-nucleate mitotically dividing cells and the frequency of irregular tetrads to ca. 2–3%, i.e. to the wild-type level. This indicates that the additional wild-type copy of IRR1 phenotypically ‘‘cured’’ the majority of cells, most likely by decreasing the ratio of Irr1-1p to wild-type Irr1p.
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errors in mitotically dividing cells
Type of aberration Frequency (%)
IRR1/IRR1 irr1-1/IRR1
0.2 3.0
0 0.9
0 0.2
0 0.5
0 2.2
0 1.0
0 0.5
0 0.5
0 0.2
0 0.2
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 837
The F658G substitution in Irr1-1p causes a
chromosome segregation defect in mitosis and
meiosis
Since the main function ascribed to cohesins is sister chromatid cohesion, we checked whether the irr1-1 mutation caused defects in chromosome segregation. First we investigated mitotically dividing cells. To detect gross changes in cell ploidy we performed a FACS analysis of the DNA content of irr1-1/IRR1 cells from unsynchronized mid-log cultures. This analysis revealed mainly cells of 2C and 4C DNA content, similar to normal IRR1/IRR1 (Fig. 5). To verify the range of aneuploidies we introduced a GFP-labeled chromosome V into irr1-1/IRR1. To analyze mitosis, we used strain ACD5 with only one copy of chromosome V labeled and the other not, and to follow meiosis, strain ACD7 was used, with both chromosome V copies labeled. Tables 3 and 4 summarize the results of microscopic observations, and in Fig. 6 we show examples of chromosome missegregation. We found that the pre- sence of Irr1-1p increased chromosome segregation errors of mitotically dividing cells from 0.2% (control IRR1/IRR1) to 9.4%. However, the chromosomal segregation errors were not coupled to defects in nuclei segregation since they were frequently observed in cells exhibiting regular nuclear divisions (Table 3). The chromosome segregation errors were also elevated in meiosis, both in asci of correct and incorrect morphol- ogy (Table 4). Basing on the above observations we conclude that the presence of Irr1-1p decreases chromo- some segregation fidelity in mitosis and meiosis.
Chromosome V segregation (black dots) was followed in IRR1/IRR1
and irr1-1/IRR1 ACD5 strains bearing one copy of chromosome V
labeled with tetR-GFP close to the centromere. Irregular chromosome
segregation was observed in 0.2% of the IRR1/IRR1 cells and in 9.4%
of the irr1-1/IRR1 cells (415 cells were counted for each strain).
The irr1-1 mutation influences the response to
nocodazole and the induction of meiosis
Since meiosis was frequently observed in cells which had not completed mitotic cell division, an influence of the irr1-1 mutation on the progression of the cell cycle was implicated. To check whether this defect could be coupled to the malfunctioning of the spindle assembly
Fig. 5. The presence of the irr1-1 mutation in the heterozygous
irr1-1/IRR1 diploid does not cause significant changes in
ploidy. DNA content was estimated in non-synchronized
cultures by FACS.
checkpoint we subjected cells to nocodazole (NZ, a microtubule-destabilizing agent causing metaphase ar- rest following activation of the spindle assembly checkpoint). After 3 h of incubation of IRR1/IRR1
and irr1-1/IRR1 with NZ (see Materials and methods for details), the drug was washed off and the cultures were allowed to resume the cell cycle. To follow its course, samples were removed every 30min for 2.5 h. Two samples were withdrawn at each time point: one was subjected to FACS analysis and the other was transferred into meiosis-inducing medium. Fig. 7A shows the DNA content estimated by FACS, and meiosis data are presented in Table 5.
The treatment with NZ stopped IRR1/IRR1 at the 4C stage, as expected, whereas irr1-1/IRR1 was not arrested
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chaotic chromosome segregation
and irr1-1/IRR1 ACD7 strains bearing two copies of chromosome V
labeled with tetR-GFP close to the centromere. The efficiency of
sporulation was 52% for IRR1/IRR1 cells and 63.7% for irr1-1/IRR1
cells (424 cells were counted for each strain). For asci type analysis, the
number of all sporulating cells was set as 100%. Small ovals in column
1 represent spores, nuclei are not marked.
Fig. 6. The presence of the irr1-1 mutation in the heterozygous
irr1-1/IRR1 diploid causes chromosome missegregation. Ex-
amples of (A) mitotically dividing cells, one chromosome
labeled and (B) irregular asci, two chromosomes labeled. In the
control and mutant strains, the centromeric region of
chromosome V was visualized by GFP (tetR-GFP), DNA
was stained with DAPI.
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844838
by this agent since at time 0 cells of various DNA contents were present. After being released from the NZ treatment, a small fraction of IRR1/IRR1 cells were able to enter meiosis at 30min, and a significant number of cells after 60 and 90min. The irr1-1/IRR1 strain treated with NZ entered meiosis throughout the 150min course of the post-synchrony recovery, with a maximum efficiency after 30–90min. These results, summarized in Table 5, supported the observation that in the irr1-1/ IRR1 mutant the SAC could be compromised.
To exclude the possibility that the lack of the arrest of irr1-1/IRR1 was caused by the experimental conditions used, we prolonged the NZ treatment up to 5 h to ensure that all cells had the chance to reach the point in their
cycle where the NZ action would stop its progress. In the above conditions, the majority of the irr1-1/IRR1
cells subjected to FACS analysis contained more than 4C DNA (Fig. 7B). Cells were alive and under the microscope we did not observe a significant increase of the number of binucleate cells. Thus, we assume that during prolonged NZ treatment, the majority of the irr1-1/IRR cells did not arrest at 4C but resumed DNA replication thus producing polyploid cells. This observa- tion supported the claim of SAC malfunctioning.
To check whether the presence of Irr1-1p causes other defects associated with DNA metabolism we performed drop tests on solid medium checking the sensitivity to HU (hydroxyurea), MMS (methyl methane sulfonate) and UV light, since they are well-known agents influencing cells bearing mutations involved in DNA metabolism. As expected, the irr1D/IRR1 strain did not exhibit an altered sensitivity to these agents compared to IRR1/IRR1. However, although we did not find an altered sensitivity of irr1-1/IRR1 to UV, this hetero- zygote exhibited a moderately increased sensitivity to
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Fig. 7. The presence of the irr1-1 mutation in the heterozygous irr1-1/IRR1 diploid causes alterations of the cell cycle course. Cells
were treated with 1 mg/ml nocodazole (NZ) for 3 h (A) or 5 h (B) and released into YPD medium. Samples were assessed for DNA
content by FACS at the indicated time points.
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 839
HU and to MMS (Fig. 8). These phenotypes suggest that irr1-1 may also affect DNA replication/repair systems.
The F658G substitution is likely to change the
structure of Irr1p/Scc3p, but it does not abrogate its
ability to interact with Scc1p
Since the F658G substitution causes strong pheno- typic effects, we attempted to predict whether it might
introduce a significant change in the Irr1p/Scc3p structure. The structure of this protein is not known, although the presence of HEAT repeats has been claimed (Neuwald and Hirano, 2000). We analyzed the sequence of the Irr1/Scc3 protein for possible structural homologs using three servers: MetaServer (Ginalski et al., 2003), PHYRE (http://www.sbg.bio.ic.ac.uk/phyre) and HHpred (Jones, 1999; Soding, 2005). Each of the applied tools indicated the following proteins as Irr1p/ Scc3p structural ‘‘neighbors’’: clathrin adapters AP2 (1GW5:A, 1GW5:B) and AP1 (1W63), karyopherin
nocodazole treatment
IRR1/IRR1 irr1-1/IRR1
120 12 34
150 3–5 20–30
Yeast culture was treated with 15 mg/ml NZ for 3 h. At indicated
intervals after release from the synchronizing agent cells were
transferred into liquid meiosis-inducing medium, and sporulation
was assayed after 36 h. Numbers represent the estimated percentage of
cells forming asci in meiosis-inducing medium. The experiment was
performed three times, for each sample ca. 200 cells were scored.
Fig. 8. The heterozygous diploid irr1-1/IRR1 exhibits in-
creased sensitivity to HU and MMS. Cells were grown in YPD
medium and diluted to identical concentrations. Serial 10-fold
dilutions were spotted in 5-ml portions onto YPD plates
supplemented with HU (6mg/ml) or MMS (0.01%).
Fig. 9. Ribbon diagram of structural models of a fragment of
Irr1p/Scc3p (light blue) and Irr1-1p (salmon). To show
changes in the general shape of the protein bearing the
F658G substitution, the N-parts of the protein structures were
superimposed. F658 is shown as sticks (green). Hinge
fragments are highlighted in dark blue and red for the wild-
type and mutant proteins, respectively.
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844840
beta-2 (1QBK:B), and importin beta (1QGR:A) (Protein Data Bank (Berman et al., 2000) entries are shown in brackets). All three structure prediction algorithms used concurred that Irr1p/Scc3p belongs to the Armadillo (ARM) repeat superfamily in the all-alpha class of proteins.
The ARM repeat is implicated in mediating protein– protein interactions and has a common origin with the HEAT repeat (Riggleman et al., 1989; Coates, 2003; Andrade et al., 2001), the presence of which in Irr1p/ Scc3p was postulated earlier (Neuwald and Hirano, 2000). The approximately 40-amino-acid ARM repeat, first identified in the Drosophila segment polarity gene product Armadillo, has since been identified in over 240 different proteins of diverse cellular functions, from protists to mammals. ARM repeat proteins contain several helices which are connected by variable-length
loops. The mutual orientation of the helices changes, so the proteins assume a curved structure.
The whole amino acid sequence of Irr1p/Scc3p was submitted to the PHYRE server which generated a putative model only for a fragment of Irr1p comprising amino acids 514–860, which, fortunately, included F658. To check the effect of the F658G substitution, the model structures of the wild-type and mutant protein were subjected to energy minimization in vacuo using consistent valence force field as implemented in DIS- COVER ver.98 (Accelrys, San Diego, CA).
F658 is located in the central part of the Irr1/Scc3 protein and the 514–860 fragment of Irr1p/Scc3p which could be modeled comprises several amino acid repeats surrounding F658. These repeats form two arms connected by a loop in which F658 is located. The model shows that the side chain of F658 is directed into the cavity formed by P642, L653, S675, I679 and the hydrophobic part of the side chain of K673. In consequence, F658 is inaccessible from the outside. A comparison of the wild-type and F658G mutant structures (Fig. 9) shows a substantial change of the relative orientation of the N- and C-parts of the mutant protein compared to that in the wild type. The shape of the individual repeats seems not to change, except for the fragment 650-665 containing F658.
An overall root-mean-square (RMS) difference be- tween the Irr1-1p and Irr1p structures is about 7 (A, while when measured individually for the N- and C- parts of the two proteins included in the model these differences are only about 2 (A. This suggests that the
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with Scc1p. Proteins extracted from diploid yeast strains
expressing Scc1-HA6 fusion protein and Scc3/Irr1-Myc18 or
Irr1-1-Myc13 fusion proteins were precipitated with anti-HA
or anti-Myc antibodies. (A) Soluble protein extracts (I) and
anti-Myc (M) or anti-HA (H) precipitates were analyzed for
the presence of Irr1-Myc or Irr1-1-Myc and Scc1-HA fusion
proteins. (B) Control experiment showing the specificity of
binding of the fusion proteins to anti-Myc- or anti-HA-
covered beads.
A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 841
655–675 fragment plays a role of a ‘‘hinge’’. Particularly, the arms of the F658G mutant protein fragment are closer to each other than those of the wild-type Irr1p/ Scc3p. Thus, replacing F658 by G changes the interac- tions with surrounding amino acids, which in turn results in changes of the overall protein geometry.
Since it had been suggested that Scc3p (Irr1p) physically interacts with Scc1p and associates with the cohesin complex in an Scc1-dependent manner (Haering et al., 2002; Ho et al., 2002; Krogan et al., 2006; Mc Intyre et al., 2007), we assumed that the predicted change in Irr1p/Scc3p geometry might hamper its interaction with Scc1p. To check this, we performed co-immunoprecipitation of Scc1p and Irr1-1p. As shown in Fig. 10, despite the predicted structure changes, Irr1- 1p retains its ability to interact with Scc1p. Thus, the introduced substitution, although lethal in the haploid and causing strong effects in the heterozygote, does not exert its effects through abolishing the Scc1p-Irr1p/ Scc3p interaction.
Discussion
The participation of the Irr1p/Scc3p cohesin in mitotic chromosome segregation has been documented since 1999 (Toth et al., 1999) but details of its role in this and other processes are not available yet. Here we observed that a single substitution in Irr1p not only disturbed chromosome segregation in mitosis and
meiosis, but also caused errors in segregation of nuclei, the course of meiosis and the cell cycle.
Since the hemizygote irr1D/IRR1 does not show significant mitotic aberrations, but the irr1-1/IRR1
heterozygote does, one is inclined to think that indeed the phenotypes of irr1-1/IRR1 result from the presence of the mutated Irr1-1p and not from an insufficient amount of wild-type Irr1p. Since the irr1-1/IRR1 strain synthesizes two forms of Irr1p, the wild-type and the mutated one, three types of cohesin complexes may potentially be formed: those containing the wild-type protein only, those containing the mutated protein only, and mixed ones (assuming that two or more Irr1p molecules are present per one cohesin complex). It is not known whether both proteins have the same affinity for other elements of the complex, but since the mutant exhibits sporadic chromosome segregation errors it seems likely that cohesion complexes containing both versions of Irr1p/Scc3 (wild-type and Irr1-1) are formed in a stochastic manner. It is highly unlikely that Irr1-1p displaces all wild-type Irr1p/Scc3 molecules from the complexes, as without the wild-type Irr1p/Scc3 the cell cannot function (the irr1-1 haploid is lethal). Besides, since introduction of an additional copy of wild-type IRR1 to irr1-1/IRR1 almost fully reversed the pheno- types of the mutant strain, it probably did so by increasing the ratio of Irr1p to Irr1-1p. This also suggests that the mutated Irr1-1 protein accesses the cohesin complex by competing with non-mutated Irr1p/ Scc3.
Although the heterozygous irr1-1/IRR1 diploid dis- played numerous defects associated with mitotic and meiotic divisions, the errors in chromosome segregation in mitosis seemed not to be coupled with defects in segregation of nuclei. Although there are data showing that interfering with proper cohesion of chromatids may somehow influence nuclear migration, mostly in meiosis (Pasierbek et al., 2003; Salah and Nasmyth, 2000), in yeast the mechanisms linking errors of chromosome segregation with the aberrant transmission of the nucleus to the bud are far from being understood.
The observed irregularities in meiosis could be due to SAC malfunctioning. One should bear in mind that induction of meiosis in mitotically dividing cells requires exit from the mitotic cell cycle at the G1 phase, when chromosome segregation is completed (Kupiec et al., 1997). The irr1-1/IRR1 strain enters meiosis without completing cell division, which is manifested by progeny cells forming asci while still attached to the mother. Such a phenotype could result from the mutant failing to recognize an un-completed mitotic division before entering meiosis. The experiment, in which we induced meiosis in cells treated with NZ in attempt to synchronize them at metaphase, indicated that in fact NZ failed to cause the G2/M arrest of the mutant. Also the FACS analysis after 5 h of NZ treatment showing
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substantial population of cells with more than 4C DNA, indicated partial failure of the synchronization attempt. These data suggest malfunctioning of the mitotic spindle control. It is well established that the cohesin complexes concentrate in the pericentromeric domain and resist the tendency of the microtubules to pull chromatids apart during their bi-orientation (Glynn et al., 2004). It is believed that in the absence of proper bipolar attach- ment or sufficient tension, the SAC is triggered to inhibit the onset of anaphase. In case of spindle malfunction- ing, the SAC inhibits subsequent mitotic events, thus helping to maintain co-ordination of the cell cycle (reviewed in Lew and Burke, 2003; Gillett et al., 2004). Since properly assembled cohesin complexes are a prerequisite for subsequent correct assembling of kinetochores, we assume that the presence of Irr1-1p in a fraction of cohesin complexes could perturb these structures. As a result, the malformed kinetochores could not sufficiently support the correct assembling and functioning of the SAC. In consequence, it is likely that irr1-1/IRR1 cannot be arrested in mitotic divisions by NZ treatment because of SAC malfunctioning. Unable to halt the progress of the cell cycle until a properly organized mitotic spindle is in place, the cells chaotically enter meiosis in response to a meiosis-inducing signal or aberrantly continue the mitotic cell cycle by synthesizing DNA without having completed the chromosome segregation phase.
The role of cohesin in kinetochore assembly and functioning is poorly known. One report indicates an enhancement of cohesin association in pericentromeric regions by kinetochores (Weber et al., 2004). Another study points to a role of cohesin in the kinetochore- microtubule interactions exemplified by prolonged Bub1p binding to centromeres that could result from errors in these interactions (Toyoda et al., 2002). Several observations concern the role of Scc1 and its orthologs in centromere and kinetochore organization and func- tioning (Sonoda et al., 2001; Hoque and Ishikawa, 2002; Vass et al., 2003; Vagnarelli et al., 2004). Thus, since cohesin constitutes the structural basis of the kineto- chore, on which elements of the SAC are assembled, we assume that the phenotypes of irr1-1/IRR1 discussed above could likely be caused by SAC malfunctioning.
The increased sensitivity of the irr1-1/IRR1 mutant to HU and MMS implicates a defect in DNA replication/ repair. It is known that DNA replication and chromatid cohesion are coupled, although the mechanisms which link them are far from being understood (Petronczki et al., 2004; Wysocka et al., 2004; Skibbens, 2005; Laha et al., 2006). Our observations suggest a possible role of Irr1p/Scc3p in these processes.
To sum up, our results show that the presence of the mutated Irr1-1 protein in the heterozygous diploid irr1-
1/IRR1 triggers numerous effects. Besides errors in chromosome segregation we observed aberrations of the
cell cycle which could cause meiotic defects. The described diploid yeast cell mimics to some extent a mammalian cell bearing a semi-dominant mutation in the genome-segregation machinery. Cells bearing such a defect are not eliminated and may lead to the develop- ment of tumors. The incomplete penetrance of the defects could be due to the stochastic nature of Irr1-1p incorporation into the cohesion complexes, or to a significant proportion of errors caused by the pre- sence of Irr1-1p being compensated by an unknown mechanism.
Acknowledgments
This work was supported by the Ministry of Science and Higher Education, Grant 2 P04C01130. We thank F. Uhlmann (London Research Institute, London, UK) for yeast strains, and Teresa Zoladek for critical comments.
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Introduction
Western blotting and co-immunoprecipitation
Microscopy and chromosome spreads
Results
The F658G substitution does not affect Irr1p level, localization or ability to associate with chromosomes
The irr1-1 mutation causes irregularities in cell divisions and affects meiosis
The F658G substitution in Irr1-1p causes a chromosome segregation defect in mitosis and meiosis
The irr1-1 mutation influences the response to nocodazole and the induction of meiosis
The F658G substitution is likely to change the structure of Irr1p/Scc3p, but it does not abrogate its ability to interact with Scc1p
Discussion
Acknowledgments
References