Limits...
Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis.

Voelkel-Meiman K, Johnston C, Thappeta Y, Subramanian VV, Hochwagen A, MacQueen AJ - PLoS Genet. (2015)

Bottom Line: While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery.This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast.Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut, United States of America.

ABSTRACT
Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

No MeSH data available.


Relative distribution of Zip3-MYC and K. l. Zip1 on meiotic chromosomes in K. l. ZIP1-expressing cells.S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and expressing K. l. ZIP1 (K375) were surface-spread at 2 hour intervals during sporulation, beginning at 12 hours after entry into sporulation medium and ending at 24 hours. These strains are homozygous for an ndt80  allele, and thus will not progress beyond the pachytene stage of meiotic prophase. Immunofluorescence was used to label K. l. Zip1 (green) and Zip3-MYC (red) on meiotic chromosomes (labeled with DAPI, white in first column and blue in second and third columns). Note in the top row images, the polycomplex aggregate of K. l. Zip1 overlaps an aggregate of Zip3-MYC. Arrows point to a subset of apparent co-localization or adjacency events between K. l. Zip1 foci and Zip3-MYC. Scale, 1 micron.
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pgen.1005335.g004: Relative distribution of Zip3-MYC and K. l. Zip1 on meiotic chromosomes in K. l. ZIP1-expressing cells.S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and expressing K. l. ZIP1 (K375) were surface-spread at 2 hour intervals during sporulation, beginning at 12 hours after entry into sporulation medium and ending at 24 hours. These strains are homozygous for an ndt80 allele, and thus will not progress beyond the pachytene stage of meiotic prophase. Immunofluorescence was used to label K. l. Zip1 (green) and Zip3-MYC (red) on meiotic chromosomes (labeled with DAPI, white in first column and blue in second and third columns). Note in the top row images, the polycomplex aggregate of K. l. Zip1 overlaps an aggregate of Zip3-MYC. Arrows point to a subset of apparent co-localization or adjacency events between K. l. Zip1 foci and Zip3-MYC. Scale, 1 micron.

Mentions: At the 22 and 24 hour time points, a majority (> 80%) of chromosome spreads from ndt80Δ mutants appeared to be at the pachytene stage where homologous chromosomes are aligned and exhibit nearly full synapsis (Figs 2 and 3A). The DAPI-stained DNA morphology of surface-spread pachytene chromosomes from wild-type S. cerevisiae reveals distinct, individualized chromosome pairs with Zip1, Ecm11-MYC, and SUMO coinciding as linear structures at the interface of each chromosome pair (Figs 2 and 3A, [11–13]). In contrast, while the DAPI-stained DNA morphology of our K.l. ZIP1 meiotic time course nuclei suggested normal progression into the pachytene stage, none of the meiotic chromosome spreads from cells expressing K. l. ZIP1 at any of the seven time points (n = 560) exhibited full-length linear structures of Zip1, SUMO, or Ecm11-MYC. Across time points, the vast majority of nuclei displayed either no detectable K. l. Zip1 or a handful of K. l. Zip1 foci dispersed along meiotic chromosomes (Figs 2 and 3), accompanied by a punctate distribution of SUMO and Ecm11 on chromosomes. The number of K. l. Zip1 chromosome-associated foci exhibited by these nuclei ranged from 1–36, with an average of 11 K. l. Zip1 foci per nucleus. The most prominent K.l. Zip1 structure found associated with S. cerevisiae meiotic nuclei was an aggregate of K. l. Zip1, Ecm11-MYC and SUMO proteins (examples in Figs 2 and 3A and S1). K. l. Zip1 polycomplexes also contained the SIC protein, Zip3 (Fig 4). Such “polycomplex” aggregates of synapsis proteins are a characteristic feature of meiotic nuclei in S. cerevisiae mutants that fail to assemble SC [12,15,18,20]. K. l. Zip1 polycomplexes were exhibited by over half (358/560) of the surface-spread nuclei, and were observed at both early and later meiotic time points, regardless of whether they displayed detectable chromosomal K. l. Zip1 foci.


Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis.

Voelkel-Meiman K, Johnston C, Thappeta Y, Subramanian VV, Hochwagen A, MacQueen AJ - PLoS Genet. (2015)

Relative distribution of Zip3-MYC and K. l. Zip1 on meiotic chromosomes in K. l. ZIP1-expressing cells.S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and expressing K. l. ZIP1 (K375) were surface-spread at 2 hour intervals during sporulation, beginning at 12 hours after entry into sporulation medium and ending at 24 hours. These strains are homozygous for an ndt80  allele, and thus will not progress beyond the pachytene stage of meiotic prophase. Immunofluorescence was used to label K. l. Zip1 (green) and Zip3-MYC (red) on meiotic chromosomes (labeled with DAPI, white in first column and blue in second and third columns). Note in the top row images, the polycomplex aggregate of K. l. Zip1 overlaps an aggregate of Zip3-MYC. Arrows point to a subset of apparent co-localization or adjacency events between K. l. Zip1 foci and Zip3-MYC. Scale, 1 micron.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482702&req=5

pgen.1005335.g004: Relative distribution of Zip3-MYC and K. l. Zip1 on meiotic chromosomes in K. l. ZIP1-expressing cells.S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and expressing K. l. ZIP1 (K375) were surface-spread at 2 hour intervals during sporulation, beginning at 12 hours after entry into sporulation medium and ending at 24 hours. These strains are homozygous for an ndt80 allele, and thus will not progress beyond the pachytene stage of meiotic prophase. Immunofluorescence was used to label K. l. Zip1 (green) and Zip3-MYC (red) on meiotic chromosomes (labeled with DAPI, white in first column and blue in second and third columns). Note in the top row images, the polycomplex aggregate of K. l. Zip1 overlaps an aggregate of Zip3-MYC. Arrows point to a subset of apparent co-localization or adjacency events between K. l. Zip1 foci and Zip3-MYC. Scale, 1 micron.
Mentions: At the 22 and 24 hour time points, a majority (> 80%) of chromosome spreads from ndt80Δ mutants appeared to be at the pachytene stage where homologous chromosomes are aligned and exhibit nearly full synapsis (Figs 2 and 3A). The DAPI-stained DNA morphology of surface-spread pachytene chromosomes from wild-type S. cerevisiae reveals distinct, individualized chromosome pairs with Zip1, Ecm11-MYC, and SUMO coinciding as linear structures at the interface of each chromosome pair (Figs 2 and 3A, [11–13]). In contrast, while the DAPI-stained DNA morphology of our K.l. ZIP1 meiotic time course nuclei suggested normal progression into the pachytene stage, none of the meiotic chromosome spreads from cells expressing K. l. ZIP1 at any of the seven time points (n = 560) exhibited full-length linear structures of Zip1, SUMO, or Ecm11-MYC. Across time points, the vast majority of nuclei displayed either no detectable K. l. Zip1 or a handful of K. l. Zip1 foci dispersed along meiotic chromosomes (Figs 2 and 3), accompanied by a punctate distribution of SUMO and Ecm11 on chromosomes. The number of K. l. Zip1 chromosome-associated foci exhibited by these nuclei ranged from 1–36, with an average of 11 K. l. Zip1 foci per nucleus. The most prominent K.l. Zip1 structure found associated with S. cerevisiae meiotic nuclei was an aggregate of K. l. Zip1, Ecm11-MYC and SUMO proteins (examples in Figs 2 and 3A and S1). K. l. Zip1 polycomplexes also contained the SIC protein, Zip3 (Fig 4). Such “polycomplex” aggregates of synapsis proteins are a characteristic feature of meiotic nuclei in S. cerevisiae mutants that fail to assemble SC [12,15,18,20]. K. l. Zip1 polycomplexes were exhibited by over half (358/560) of the surface-spread nuclei, and were observed at both early and later meiotic time points, regardless of whether they displayed detectable chromosomal K. l. Zip1 foci.

Bottom Line: While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery.This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast.Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut, United States of America.

ABSTRACT
Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

No MeSH data available.