Our lab is interested in the process of chromosome segregation and how defects in this process can affect the development of a multicellular organism. Over the past few years we have focused on the meiotic divisions that produce haploid gametes. We have been studying a class of temperature-sensitive (ts) embryonic lethal mutants from C. elegans that arrest in metaphase of meiosis I. In wildtype animals, oocytes in prophase of meiosis I are fertilized by sperm. Following fertilization, the oocyte chromosomes undergo two meiotic divisions, discarding the extra chromosomes in the polar bodies. These first meiotic divisions are important as any errors in chromosome segregation at this stage can lead to embryos with an abnormal number of chromosomes, which would likely lead to lethality. In our mutants, the oocyte chromosomes arrest in metaphase of meiosis I and never separate their chromosome homologs and never extrude polar bodies. Our meiotic mutants define five genes;they encode subunits of the Anaphase Promoting Complex or Cyclosome (APC/C). This complex serves as an E3 ubiquitin ligase that targets proteins for destruction (by the 26S proteasome) during the metaphase to anaphase transition of the cell cycle. We have named these mutants mat for their defects in the metaphase to anaphase transition during meiosis I. To identify extragenic regulators or substrates of these APC/C subunits, we have carried out a genetic suppression screen using a mat-3 mutant. The majority of our 27 suppressor mutations are dominant. These suppressors define at least 9 complementation groups. A large number of alleles represent mutations in three spindle assembly checkpoint components, mdf-1, mdf-2, and mdf-3. Our results suggest that this checkpoint operates during meiosis. We believe that our mat mutants are not triggering the checkpoint, but rather that the checkpoint normally operates during meiosis as a negative regulator of the APC/C. Perhaps the checkpoint functions to regulate the proper timing of the meiotic divisions. We also identified three dominant suppressors that were mutations in the Cdc20/Fzy ortholog, a positive regulator of the APC/C. In the past year, we have characterized another suppressor allele that harbors a mutation in an APC subunit, such-1. We had previously tested this gene for a role in the meiotic divisions (using RNAi) yet failed to find an early embryonic phenotype. A temperature-sensitive reduction-of-function allele, h1960, does exist but does not display the same early arrest as our other APC alleles. RNAi of the such-1 gene in the suppressed strain reverts the strain back to the meiotic 1-cell arrest phenotype. This finding strongly suggests that our suppressor allele is a gain-of-function allele in such-1. Sequencing of the such-1 gene in this mutant background confirmed that such-1 harbored a mutation in its coding sequence. Our suppressor screen was instrumental in identifying this rare gain-of-function allele that revealed to us that this APC subunit could function during the meiotic divisions. The such-1 gene encodes an APC-5 ortholog and interestingly, there are two apc-5-like genes in C. elegans. We have recently shown that the other apc-5 gene, gfi-3, is not essential based on RNAi treatment. There are no existing mutations in gfi-3. RNAi of gfi-3 does not enhance other APC mutants, while RNAi of such-1 does. The such-1(h1960) allele mentioned above also does enhance other APC loss-of-function phenotypes. Interestingly, depletion of gfi-3 and such-1 from such-1(h1960) animals does result in 1-cell meiotic arrest. These results suggest that such-1 and gfi-3 are redundantly required for the meiotic divisions and that they can both function as meiotic APC-5 subunits. Using GFP transgenes, we have shown that they are both expressed in the hermaphrodite and male germline, and in early embryos. Their post-embryonic expression patterns vary and thus their somatic roles may differ later in development. Why only nematodes harbor two APC5 paralogs remains a mystery. We also have been pursuing the molecular identification of emb-1, a gene which we believe is a novel subunit or regulator of the APC. Temperature-sensitive alleles of emb-1 behave very much like our previously characterized APC mutants;they arrest as 1-cell embryos, are enhanced when combined with other APC alleles, and are suppressed weakly by our previously characterized APC suppressors. Three factor mapping, RNAi phenocopy, and transgenic rescue revealed that emb-1 encodes a small 81 amino acid protein. This protein is likely the APC16 subunit of the APC that was recently identified in human cells. Further support for our genetic conclusion comes from the findings of our colleagues who showed that EMB-1 co-purifies with numerous APC subunits. C. elegans is the only organism to date in which alleles of this new APC subunit exist. The function of this subunit with the larger complex remains to be determined.
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