Cell Cycle Regulation In C. elegans

线虫的细胞周期调控

基本信息

批准号：
7593438
负责人：
ANDY GOLDEN
金额：
$ 32.84万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

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. We are currently mapping the remaining suppressors and anticipate finding novel molecules that shed light on how the APC/C is regulated during meiosis. To address whether our suppressor mutations have phenotypes on their own, we have crossed them away from the original mat-3 mutation. For some of our mutants, there is a significant reduction in brood size. However, we have not observed any obvious embryonic lethal phenotypes for these genes, suggesting that our screen uncovered viable alleles of essential spindle checkpoint genes. We are currently characterizing the meiotic defects associated with the strict paternal-effect lethal mutant spe-11. SPE-11 is a sperm-specific factor that is contributed to the oocyte upon fertilization. Homozygous spe-11 mutant males produce dead embryos when mated with wild-type hermaphrodites or females. These embryos fail to extrude their polar bodies, secrete an eggshell, and initiate proper mitosis and cytokinesis. As a result, the embryos die as 1-cell embryos with eggshell and cytokinesis defects. We are further characterizing the defects associated with the mutant spe-11 alleles in order to address what function spe-11 plays in influencing the meiotic divisions of the oocyte. We intend to screen for other genes that phenocopy the spe-11 phenotype using classical genetic screens and RNAi screens in order to identify other factors that operate in the SPE-11 pathway.

我们的实验室对染色体分离的过程以及该过程中的缺陷如何影响多细胞生物的发育感兴趣。在过去的几年里，我们一直关注产生单倍体配子的减数分裂。我们一直在研究一类来自秀丽隐杆线虫的温度敏感（ts）胚胎致死突变体，它们在减数分裂 I 中期停滞。在野生型动物中，减数分裂 I 前期的卵母细胞由精子受精。受精后，卵母细胞染色体经历两次减数分裂，丢弃极体中多余的染色体。这些第一次减数分裂很重要，因为此阶段染色体分离的任何错误都可能导致胚胎染色体数量异常，这可能会导致死亡。在我们的突变体中，卵母细胞染色体停滞在减数分裂 I 的中期，永远不会分离它们的染色体同源物，也永远不会挤出极体。我们的减数分裂突变体定义了五个基因；它们编码后期促进复合体或环体 (APC/C) 的亚基。该复合物充当 E3 泛素连接酶，在细胞周期的中期到后期转变期间靶向破坏蛋白质（通过 26S 蛋白酶体）。我们将这些突变体命名为 Mat，因为它们在减数分裂 I 期间的中期到后期转变中存在缺陷。为了鉴定这些 APC/C 亚基的外源调节因子或底物，我们使用 mat-3 突变体进行了遗传抑制筛选。我们的 27 个抑制突变中的大多数都是显性突变。这些抑制因子已使用单核苷酸多态性 (SNP) 技术进行定位，并定义了至少 9 个互补组。其中一个等位基因是 mat-3 基因本身的第二个位点突变。大量等位基因代表三个纺锤体检查点成分的突变。这些是 MAD1、MAD2 和 MAD3 的线虫直系同源物。当染色体未正确附着在有丝分裂纺锤体上时，纺锤体检查点可防止中期到后期的转变。我们的结果表明该检查点也在减数分裂期间发挥作用。我们在 mdf-1（线虫 Mad1 直向同源物）中鉴定出 1 个等位基因，在 mdf-3 基因（Mad3 直向同源物）中鉴定出 2 个等位基因，在 mdf-2 基因（Mad2 直向同源物）中鉴定出 12 个等位基因。我们相信我们的 mat 突变体不会触发检查点，而是检查点通常在减数分裂期间作为 APC/C 的负调节因子发挥作用。也许检查点的功能是调节减数分裂的正确时间。我们还鉴定了三个显性抑制因子，它们是 APC/C 正调节因子的突变。该基因称为 fzy-1，是 Cdc20/Fzy 直向同源基因。这三个突变聚集在蛋白质的一个小区域，被认为对其与 MDF-2 的相互作用很重要。这些突变可能会破坏与 MDF-2 的相互作用，从而阻止 MDF-2 对 APC/C 的抑制。我们目前正在绘制其余抑制因子的图谱，并预计会发现新的分子，以阐明 APC/C 在减数分裂过程中的调节方式。为了解决我们的抑制突变是否有自己的表型，我们将它们与原始的 mat-3 突变进行了交叉。对于我们的一些突变体来说，巢的大小显着减少。然而，我们没有观察到这些基因的任何明显的胚胎致死表型，这表明我们的筛选发现了必需纺锤体检查点基因的可行等位基因。我们目前正在表征与严格父本效应致死突变体 spe-11 相关的减数分裂缺陷。 SPE-11 是一种精子特异性因子，在受精后形成卵母细胞。纯合 spe-11 突变雄性与野生型雌雄同体或雌性交配时会产生死亡胚胎。这些胚胎无法挤出极体、分泌蛋壳并启动适当的有丝分裂和胞质分裂。结果，胚胎作为具有蛋壳和胞质分裂缺陷的 1 细胞胚胎死亡。我们正在进一步表征与突变 spe-11 等位基因相关的缺陷，以便解决 spe-11 在影响卵母细胞减数分裂中发挥的功能。我们打算使用经典遗传筛选和 RNAi 筛选来筛选与 spe-11 表型相似的其他基因，以确定在 SPE-11 途径中起作用的其他因素。