Cell Cycle Regulation In C. elegans

线虫的细胞周期调控

• 批准号: 7967187
• 负责人: Andy Golden
• 金额: $ 35.89万
• 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/7967187
• 关键词: 26S proteasome; Affect; Alleles; Anaphase; Animals; Back; Caenorhabditis elegans; Cell Cycle; Cell Cycle Regulation; Cells; Chromosome Segregation; Chromosomes; Code; Complex; Data; Defect; Deposition; Development; Embryo; Essential Genes; Fertilization; Frequencies; Genes; Genetic; Genetic Screening; Genetic Suppression; Germ Cells; Goals; Haploidy; Homologous Gene; I Kappa B-Alpha; Lead; MXI1 gene; Maps; Meiosis; Metaphase; Mitotic spindle; Molecular; Mutation; Names; Oocytes; Organism; Orthologous Gene; Phenotype; Process; Prophase; Protein Region; Proteins; RNA Interference; Reporter; Role; Single Nucleotide Polymorphism; Staging; Suppressor Mutations; Technology; Temperature; Testing; Time; Tissues; Work; anaphase-promoting complex; base; embryo cell; gain of function; gene function; interest; loss of function; mutant; prevent; 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. 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 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 does suggest that this gene functions during meiosis; meiotic 1-cell embryos are observed at the non-permissive temperature, but at a low frequency. 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 other than a transposon insertion allele. We are working to determine if this allele has an associated phenotype. RNAi of gfi-3 does not enhance other APC mutants, while RNAi of such-1 does. The such-1 reduction-of-function allele mentioned above also does enhance other APC loss-of-function phenotypes. These results suggest that such-1 is a meiotic APC-5 subunit. Depletion of this gene enhances weak APC mutants while a gain-of-function allele suppresses weak APC mutants. This suppression data suggests that a mutant gain-of-function version of the SUCH-1 protein might maintain the function of the multisubunit APC. Our hypothesis for the GFI-3 subunit is that this APC-5 subunit acts in other tissues or at other times during development. We are trying to prove this prediction using GFP reporter constructs.

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。 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。 在过去的一年中，我们描述了另一个抑制等位基因，该等位基因在APC亚基中含有突变，例如1。 我们以前曾测试过该基因在减数分裂师（使用RNAi）中的作用，但未能找到早期的胚胎表型。 对温度敏感的功能等位基因的降低确实表明该基因在减数分裂过程中起作用。减数分裂1细胞的胚胎在非抗药性温度下观察到，但频率低。 抑制菌株中这种1基因的RNAi将菌株恢复为减数分裂1细胞阻止表型。 这一发现强烈表明，我们的抑制等位基因是这种1中的功能奖励等位基因。 在这种突变体背景中，这种1基因的测序证实，这种1在其编码顺序中具有突变。 我们的抑制剂屏幕有助于识别这个罕见的功能奖励等位基因，该等位基因向我们揭示了该APC亚基在减数分裂划分期间可以起作用。 这种1基因编码APC-5直系同源物，有趣的是，秀丽隐杆线虫中有两个APC-5样基因。 我们最近表明，基于RNAi处理，其他APC-5基因GFI-3并不是必需的。 除了转座子插入等位基因外，GFI-3中没有现有突变。 我们正在努力确定该等位基因是否具有相关的表型。 GFI-3的RNAi不会增强其他APC突变体，而这种1的RNAi则不增强。 上面提到的1降低功能等位基因还可以增强其他APC功能丧失表型。 这些结果表明，这种1是减数分裂的APC-5亚基。 该基因的耗竭增强了弱APC突变体，而功能收益等位基因抑制了弱APC突变体。 该抑制数据表明，这种1-1蛋白的功能增益版本可能会维持多亚基APC的功能。 我们对GFI-3亚基的假设是，该APC-5亚基在其他组织中作用于其他组织。 我们正在尝试使用GFP记者构造来证明这一预测。