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. In order to molecularly identify the genes required for the first meiotic division, we have mapped our mutants and sequenced candidate genes. Five of the six genes have now been identified and 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 proteosome) during the metaphase to anaphase transition of the cell cycle. We have named our mutants ?mat? for their defects in the metaphase to anaphase transition during meiosis I. These ts mutants also display defects in spermatocyte meiosis; primary spermatocytes arrest in metaphase of meiosis I with a normal meiotic spindle, yet fail to separate chromosome homologs. Thus, these mutants disrupt meiosis in both oocytes and spermatocytes. To address the role of the mat genes in mitosis, we have performed shift-up experiments during embryogenesis and larval development. Temperature shift-up experiments during embryogenesis do not result in embryonic phenotypes, however, somatic defects in the gonad, vulva, and male tail are apparent in adults. This observation suggests that mitotic divisions in the soma are affected by the mat mutants. For many of the alleles, these shift-up experiments also result in sterility, suggesting mitotic defects in germline proliferation. To identify extragenic regulators or substrates of these APC/C subunits, we have carried out a genetic suppression screen. The majority of our 29 suppressor mutations are dominant. These suppressors have been mapped using single nucleotide polymorphism (SNP) technology and define at least 6 complementation groups. A few alleles are linked to mat-3; sequencing of mat-3 has revealed that only one of these represents a second site mutations within the mat-3 gene. Three unlinked alleles have been well-mapped and sequenced and represent mutations in two spindle checkpoint components. These are the C. elegans orthologs of 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 currently do not know if our mat mutants are triggering the checkpoint, or if the checkpoint normally operates during meiosis as a negative regulator of the APC/C. We are currently mapping the remaining suppressors and anticipate finding novel molecules that shed light on how the APC/C is regulated during meiosis. These suppressors may also reveal how the APC/C functions in different tissues and at different times during the development of a multicellular organism. So far, none of our suppressor mutations have a phenotype on their own. By RNAi, we are currently testing if depletion of spindle checkpoint proteins can suppress other mat-3 alleles or other mat genes. We have taken a similar genetic approach to identify regulators and substrates of an indirect downstreaam component of the APC/C pathway. This target is a protease called separase, which is released when APC/C targets securin for destruction. Securin normally sequesters separase so that it cannot cleave the cohesin molecules that hold sister chromatids together. With securin destroyed, separase is free to cleave cohesin and sister chromatid separation occurs. We have four ts alleles of sep-1 and have carried out a suppression screen with one of these alleles. To date, we have identified three suppressors that restore viability to sep-1 mutants at the non-permissive temperature. One of these mutants is an intragenic suppressor; the other two are extragenic. We are currently mapping these two suppressors so that we can molecularly identify them. We also want to examine the composition of the APC/C in C. elegans. For these studies, we are generating transgenic lines expressing epitope-tagged APC/C subunits so that we can then purify the complex with epitope-specific antibodies. These proteins that we purify will be subjected to mass spectrometry to identify the components of the APC/C. We will then examine whether this complex varies during development. In a separate study, we are examining the function of the C. elegans Myt1 ortholog. Myt1 belongs to the Wee1 family of kinases and is thought to down regulate Cdk1 during the cell cycle. RNAi studies with the Myt1 ortholog, wee-1.3, result in sterility. Mothers injected with dsRNA quickly become sterile; the oocyte chromosomes are no longer paused in diakinesis of meiosis I. These chromosomes have many hallmarks of being mitotic; they stain with a number of mitotic marker antibodies. Oocyte maturation also appears to be precocious. We propose that WEE-1.3 normally functions to keep maternal CDK-1 inactive during oogenesis, and that upon fertilization, CDK-1 becomes activated to allow for the meiotic and mitotic divisions of the embryo. In the absence of WEE-1.3, CDK-1 becomes precociously active and drives oocyte maturation and chromosome maturation in immature oocytes that are not fully differentiated. These oocytes fail to be fertilized presumably because they have not synthesized all the proper oocyte/embryo products they need for further development. We are further characterizing this phenotype and plan to use RNAi screens to identify other components of this pathway.
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