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First published online 3 June 2008
doi: 10.1242/jcs.031799

Journal of Cell Science 121, 2148-2158 (2008)
Published by The Company of Biologists 2008
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DNA double-strand breaks, but not crossovers, are required for the reorganization of meiotic nuclei in Tetrahymena

Kazufumi Mochizuki1, Maria Novatchkova2 and Josef Loidl3,*

1 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), A-1030 Vienna, Austria
2 Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria
3 Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr Gasse 1, A-1030 Vienna, Austria


Figure 1
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  		Fig. 1. Expression patterns and multiple sequence alignment of Tetrahymena meiotic genes and proteins. (A) Expression of genes, demonstrated by RT-PCR. SPO11 is expressed only in meiosis. Of the two Tetrahymena HOP2 homologs, HOP2A is meiosis-specific and HOP2B is ubiquitously expressed. Likewise, two MND1-related genes show meiotic and ubiquitous expression, respectively. E, exponentially growing cells; S, cells growing under starvation conditions; numbers indicate the time after mixing of the cultures. RPL21 is a ubiquitously expressed control gene. (B) Multiple sequence alignment of the N-terminal–central-region of Hop2-group proteins that have highest sequence conservation within the family and show similarity to the `winged helix' fold. Protein-sequence identifiers are composed of two-letter species codes (Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Mm, Mus musculus; At, Arabidopsis thaliana; Tt, Tetrahymena thermophila) and common protein symbols. Accession numbers of the sequences used: NP_011482 (Sc), NP_001018191 (Sp), NP_032975 (Mm), NP_172791 (At), XP_001020981 (Tt Hop2A) and XP_001013509 (Tt Hop2B). Visualization was performed with Jalview with BLOSUM62-score/conservation shading (Clamp et al., 2004Go). Identical (*), strongly conserved (:) and weakly conserved (.) amino acids as well as predicted {alpha}-helices (white bars) and β-strands (arrows) are indicated below the alignment. Tetrahymena TTHERM_01190440 protein (Hop2Bp) shows closer sequence similarity to members from other species than does TTHERM_00794620 protein (Hop2Ap). With a predicted length of 422 and 314 amino acids (aa), respectively, Hop2Bp and Hop2Ap would be notably longer than the other members of the Hop2 orthology group, which have around 220 aa.

 

Figure 2
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  		Fig. 2. Meiotic MIC reorganization in wild type and in mutants. (A) Micrographs with each panel showing the MICs and MACs of a pair of conjugating cells (DAPI staining). (a) During wild-type prophase, the MIC progressively elongates and reaches its maximal extension at stage IV. From then on, it shortens again, and chromatin condenses and reaches maximal condensation at stage VI, which corresponds with diakinesis–metaphase-I. This is followed by a first and a second meiotic division. (b) In the hop2A{Delta} mutant, MIC development is normal up to stage V. At stage VI, condensed chromatin entities, which are more numerous than in the wild type, appear. (c) In the spo11{Delta} mutant, stage II is normal but the shape of stage-III MICs is irregular and their ends do not taper. Stage IV is missing. Instead, MICs progress from an aberrant stage III directly to stage V. (d) Cisplatin treatment restores full elongation of the MICs. Scale bar: 10 µm. (B) Meiotic time-courses of wild-type, hop2A{Delta} and spo11{Delta} meiosis. Whereas in the hop2A{Delta} mutant, meiosis progresses similarly to wild type, there are no stage IV crescents formed in the spo11 deletion mutant. Aberrant stage-III MICs (dark shading) directly progress to stage V. The heights of the columns represent the percentages of cells in each stage for each time point. At least 200 MICs were evaluated for each time point.

 

Figure 3
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  		Fig. 3. Meiotic divisions in the wild type (A-C), and in hop2A{Delta} (D-F) and spo11{Delta} (G-I) mutants. (A) In wild type, five bivalents can be discriminated in diakinesis or metaphase I. In anaphase I (B) and anaphase II (C), equal amounts of chromatin are segregated. In mutant meioses, chromosomes appear to be unpaired (D,G) and anaphase I is often clearly asymmetric (E,H) with a large (large arrow) and a small (small arrow) number of chromosomes. (F,I) Correspondingly, asymmetric anaphase II configurations with large numbers of chromatids (large arrowheads) and small numbers of chromatids (small arrowheads) occur in the mutants. Notably, chromosome fragments are absent in hop2A{Delta} meioses. Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. Immunostaining of wild-type, hop2A{Delta} and spo11{Delta} meiotic MICs. (A-C) Cna1 staining of centromeres demonstrates that, starting from a clustered arrangement and passing through a stage of dispersal, centromeres occupy a small area in the tip of the elongated MIC in wild type and in the hop2A{Delta} mutant. In the spo11{Delta} mutant they remain dispersed. The DSB marker {gamma}-H2A.X delineates MICs in wild type (D) and in the hop2A{Delta} mutant (E), and in the spo11{Delta} mutant only after DSB induction by cisplatin (F). The recombination marker Rad51p forms foci in the elongating MICs of wild type (G) and the hop2A{Delta} mutant (H), and displays a uniform nuclear distribution at later stages. It does not form foci in the spo11{Delta} mutant (I). Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. Scoring meiotic pairing and bouquet arrangement by FISH. (A-J) Two loci, one close (region B) and one at a larger distance (region A) from the telomeric end of the MIC (arrowheads), which is tapered and DAPI-pale (see Loidl and Scherthan, 2004Go), were delineated by FISH. Separate FISH signals (A-C,F-H) are scored as indicating the absence of pairing. Single FISH signals (D,E,J) and double dots (I) reflect pairing of the corresponding homologous loci. Examples are shown from wild type (A-F), the hop2A{Delta} mutant (G,I) and the spo11{Delta} mutant (H,J). (K-N) FISH with a compound telomere probe (orange) suggests that (some) telomeres are assembled near one end of stage-II MICs of the wild type (K) and the spo11{Delta} mutant (M). In the wild type, the telomere signal becomes very strong upon further elongation of the MIC (L), whereas in the spo11{Delta} mutant, only weak scattered signals remain visible, presumably indicating dispersal of the telomeres in stage-III-like MICs (N). The elongated MICs shown are relatively straight because the Carnoy fixation procedure releases nuclei from the cells, which improves FISH. Scale bar: 5 µm.

 

Figure 6
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  		Fig. 6. Homologous pairing of a chromosomal locus (region B, see Fig. 5) as determined by FISH. If a FISH probe to a chromosomal region produces two separate dots within a MIC, the chromosomes are considered not to be paired in this region. Pairing is indicated by two closely associated (double dot) FISH signals or by the fusion of FISH signals (single dot). In wild type, the frequency of nuclei with pairing increases throughout meiotic prophase. In the hop2A{Delta} mutant, the maximal level of pairing achieved in stage IV is somewhat lower. In the spo11{Delta} mutant, the maximal pairing (at the stage resembling stage III) achieves a level above the ground level (presumably owing to random contacts) at the beginning of meiosis. A total of 100 nuclei per stage and genotype were evaluated. Pairing levels at region A were similar (supplementary material Table S2).

 

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