Fig 1.
Meiotic centromere behaviors in budding yeast.
A. In meiosis of budding yeast, Zip1 (orange) mediates centromere coupling (green arrowheads) between non-homologous partner chromosomes (light blue and purple). As the cell proceeds through later stages of meiosis, homologs pair and the mature synaptonemal complex (SC) structure zips the chromosomes together. After pachytene, the SC disassembles, except at the centromeres (blue arrowhead). B. The Zip1 protein is predicted to have globular domains at its ends spanning a longer coiled-coil and forms parallel dimers with N-termini in the center of the SC (denoted by N) and the C-termini along the axial elements (denoted by C). C. We evaluated the same nine ZIP1 deletion mutants previously described by Tung and colleagues (Tung & Roeder, 1998). The mutations are named for their relative position along the genetic sequence–N for N-terminus, M for middle region, and C for C-terminus. The approximate SC structure formed in each mutant as described by Tung and Roeder (1998) is shown. D. The areas shaded in blue correspond to deletions (above) that significantly disrupted centromere coupling. Those shaded in green correlate with deletions that significantly disrupted achiasmate segregation. Note that not all deletions were screened for achiasmate segregation defects because some caused a meiotic arrest.
Fig 2.
Centromere coupling requires parts of the N and C-termini of Zip1.
A. Centromere coupling values were obtained by scoring the number of Mtw1-GFP foci in meiotic chromosome spreads. CEN1 loci were visualized by virtue of lacI-GFP localized to a lac operator array next to the centromere. B. Coupling data. Mutants are listed according to the severity of their coupling phenotype. The thin blue and red lines indicate average Mtw1 foci values for wild-type and zip1Δ, respectively. The mutants were split into three groups–like wild-type (light blue), intermediate (green), and like zip1Δ (orange). The “like wild-type” group had values indistinguishable from wild-type but were significantly different from zip1Δ (p<0.05); whereas the “like zip1Δ” group had values indistinguishable from zip1Δ but significantly different from wild-type (p<0.05). The zip1-M2 mutant had an intermediate phenotype that was significantly different from both wild-type and zip1Δ. A complete list of averages and statistical values are presented in S2 Table.
Fig 3.
Centromere plasmid disjunction requires the N-terminus of Zip1.
A. Representative binucleate cells with disjoined (a ZIP1 cell) and non-disjoined (a zip1-N1 cell) centromere plasmids. The segregation of CEN plasmids in anaphase I was assessed by monitoring the tetR-tdTomato and lacI-GFP foci localized to tet and lac operator repeats, respectively, inserted into a plasmid that contains 5.1 kb of CEN3 sequence. B. Non-disjunction frequencies for CEN plasmids in each strain. n values: ZIP1, 250; zip1-N1, 190; zip1- NM1, 200; zip1-M1, 143; zip1-M2, 54; zip1-MC1, 69; zip1-MC2, 55. Statistical comparisons were performed with Fisher’s exact test to compare all genotypes to WT. Bonferroni’s correction was utilized to adjust for the number of comparisons. *p ≤0.05.; ***p ≤0.00625. Scale bars equal 2 μm.
Fig 4.
Centromere plasmid pairing requires the N-terminus of Zip1.
Pairing of plasmid centromeres in prophase chromosome spreads was assessed by monitoring the pairing of tetR-tdTomato and lacI-GFP foci localized to tet operator and lac operator arrays on plasmids bearing a 5.1 kb region of chromosome III encompassing CEN3. A. An example of a spread with unpaired plasmid centromeres. B. Distances between the centers of the tdTomato and GFP foci in each spread (average and standard deviation). *** P = 0.0002. The grey cross-hatched region indicates separation of less-than 0.6 μm between the centers of the foci, a distance used to infer pairing of the centromeres. C. The percent of spreads scored as “paired” in the ZIP1 (58%, n = 50) and zip1-N1 (22%, n = 63) strains. ****p<0.0001. Scale bar equals 2 μm.
Fig 5.
The Zip1 N-terminal domain is required for efficient co-localization to centromeres.
Chromosome spreads were prepared from prophase ZIP1 and zip1-N1 cells expressing Mtw1-GFP as a kinetochore marker. Indirect fluorescence structured illumination microscopy was used to visualize Mtw1-GFP and Zip1 foci (Zip1 antibodies were raised against carboxy-terminal amino acids shared by both the wildtype and Zip1-N1 proteins). A and B. The overlap of Mtw1 foci with Zip1 foci (green circles) and Zip1 foci with Mtw1 foci (blue circles) was measured in each spread and the statistical significance of the difference between the observed Mtw1 co-localization with Zip1 from random simulations was evaluated with Costes’ P-value (gray triangles; greater than 95% is considered significant). Representative images from the two strains are shown. Zip1 (red), Mtw1-GFP (green), overlapping foci (white arrowhead), scale bars equal 2 μm. C. The average co-localization of Mtw1 foci with Zip1 across all the chromosome spreads was determined. * p<0.05. D. Centromere pairing was evaluated by counting the number of Mtw1-GFP foci in the chromosome spreads. ZIP1 (n = 27), zip1-N1 (n = 22), zip1Δ (n = 22). **P<0.01, ****P<0.0001.