Cell Cycle Regulation In C. elegans

线虫的细胞周期调控

• 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
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593438
• 关键词: 26S proteasome; Address; Affect; Alleles; Anaphase; Animals; Caenorhabditis elegans; Cell Cycle; Cell Cycle Regulation; Chromosome Segregation; Chromosomes; Class; Complex; Cytokinesis; Defect; Deposition; Development; Embryo; Essential Genes; Female; Fertilization; Genes; Genetic; Genetic Screening; Genetic Suppression; Germ Cells; Goals; Haploidy; Homologous Gene; I Kappa B-Alpha; Lead; Light; MXI1 gene; Maps; Meiosis; Metaphase; Mitosis; Mitotic spindle; Molecular; Mutation; Names; Numbers; Oocytes; Organism; Orthologous Gene; Partner in relationship; Pathway interactions; Phenocopy; Phenotype; Play; Process; Prophase; Protein Region; Proteins; RNA Interference; Single Nucleotide Polymorphism; Site; Staging; Suppressor Mutations; Technology; Temperature; Thinking; Time; anaphase-promoting complex; embryo cell; interest; male; mutant; novel; prevent; size; sperm cell; ubiquitin-protein ligase

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此过程中的缺陷如何影响多细胞生物的发展感兴趣。 在过去的几年中，我们专注于产生单倍配子的减数分裂分裂。 我们一直在研究来自秀丽隐杆线虫的一类温度敏感（TS）胚胎致死突变体，这些突变体在减数分裂的中期中停滞。在野生型动物中，减数分裂的预言中的卵母细胞被精子施肥。 受精后，卵母细胞染色体经历了两种减数分裂师，从而丢弃了极性体内的额外染色体。 这些第一个减数分裂分裂很重要，因为此阶段染色体隔离的任何错误都可能导致胚胎异常数量的染色体导致胚胎，这可能会导致致死性。 在我们的突变体中，卵母细胞染色体在减数分裂I的中期中停滞，从不将它们的染色体同源物分开，从不挤压极性体。 我们的减数分裂突变体定义了五个基因；它们编码促进复合物或循环体（APC/C）的后期亚基。 该复合物用作E3泛素连接酶，该连接酶靶向蛋白质在中期期间（由26S蛋白酶体）靶向细胞周期的后期转变。 我们命名了这些突变体MAT，因为它们在减数分裂过程中的中期为后期过渡中的缺陷。 为了鉴定这些APC/C亚基的基因外调节剂或底物，我们使用MAT-3突变体进行了遗传抑制筛查。我们27个抑制突变中的大多数是主导的。 这些抑制剂已使用单核苷酸多态性（SNP）技术进行了映射，并定义了至少9个互补组。 一个等位基因是MAT-3基因本身中的第二个位点突变。 许多等位基因代表三个主轴检查点组件中的突变。 这些是MAD1，MAD2和MAD3的秀丽隐杆线虫直系同源物。 当染色体未正确连接到有丝分裂纺锤体时，主轴检查点可防止中期向后期转变。 我们的结果表明，该检查点在减数分裂过程中也工作。 我们确定了MDF-1（秀丽隐杆线虫MAD1直系同源物）中的一个等位基因，MDF-3基因中的两个等位基因（MAD3直系同源物）和MDF-2基因中的12个等位基因（MAD2 Ortholog）。 我们认为，我们的垫子突变体并没有触发检查站，而是检查点通常在减数分裂过程中作为APC/C的负调节剂进行操作。 也许检查站的功能可以调节减数分裂分裂的适当时机。 我们还确定了三个主要的抑制剂，它们是APC/c的正调节剂中突变。 该基因称为fzy-1，是cdc20/fzy直系同源物。 这三个突变聚集在蛋白质的一小部分中，认为对其与MDF-2的相互作用很重要。 这些突变大概破坏了与MDF-2的相互作用，从而防止了APC/C的MDF-2抑制。 我们目前正在绘制剩余的抑制剂，并预计找到新的分子，这些分子阐明了在减数分裂过程中如何调节APC/C的方式。 为了解决我们的抑制突变是否单独具有表型，我们将它们脱离了原始的MAT-3突变。 对于我们的某些突变体，育雏尺寸显着降低。 但是，我们尚未观察到这些基因的任何明显的胚胎致死表型，这表明我们的屏幕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的父亲效应致死突变体SPE-11相关的减数分裂缺陷。 SPE-11是一个精子特异性因素，在受精时会导致卵母细胞。 与野生型雌雄同体或雌性交配时，纯合SPE-11突变雄性会产生死胚。 这些胚胎无法挤出其极性，分泌蛋壳，并启动有丝分裂和细胞因子。 结果，胚胎死于带蛋壳和细胞因子缺陷的1细胞胚胎。 我们正在进一步表征与突变体SPE-11等位基因相关的缺陷，以解决SPE-11在影响卵母细胞的减数分裂分裂时发挥的功能。 我们打算使用经典的遗传筛选和RNAi筛选来筛选表观复制SPE-11表型的基因，以识别SPE-11途径中运行的其他因素。