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 have been mapped using single nucleotide polymorphism (SNP) technology and define at least 9 complementation groups. One allele is a second site mutation within the mat-3 gene itself. A large number of alleles represent mutations in three spindle checkpoint components. These are the C. elegans orthologs of MAD1, MAD2, and MAD3. The spindle checkpoint prevents the metaphase to anaphase transition when chromosomes are not properly attached to the mitotic spindle. Our results suggest that this checkpoint also operates during meiosis. We identified one allele in the mdf-1 (the C. elegans Mad1 ortholog), two alleles in the mdf-3 gene (the Mad3 ortholog), and 12 alleles in the mdf-2 gene (the Mad2 ortholog). 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 a positive regulator of the APC/C. This gene is called fzy-1 and is the Cdc20/Fzy ortholog. These three mutations cluster in a small region of the protein thought to be important for its interaction with MDF-2. These mutations presumably disrupt the interaction with MDF-2 and thus prevent MDF-2 inhibition of the APC/C. In the past year, we have mapped many of the remaining suppressors. One of them is a mutation in the C. elegans Zeste White 10 ortholog, named czw-1. This gene is thought to also function as a spindle checkpoint gene. RNAi depletion of czw-1 causes embryonic lethality and sterility. The weak allele from our suppressor screen does not have these phenotypes on its own, yet does suppress the embryonic lethality of our mat-3 mutant. Another suppressor is a gain-of-function allele of 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. 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 are currently testing whether these two apc-5 genes function redundantly, thus explaining why we never observed a meiotic function earlier when only depleting one of them.
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