Cell Cycle Regulation In C. elegans

线虫的细胞周期调控

• 批准号: 8349664
• 负责人: Andy Golden
• 金额: $ 29.58万
• 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/8349664
• 关键词: 26S proteasome; Affect; Alleles; Amino Acids; Anaphase; Animals; Back; Caenorhabditis elegans; Cell Cycle; Cell Cycle Regulation; Cells; Chromosome Segregation; Chromosomes; Code; Complex; Defect; Deposition; Development; Embryo; Essential Genes; Fertilization; Genes; Genetic; Genetic Screening; Genetic Suppression; Germ Cells; Goals; Haploidy; Homologous Gene; Human; Lead; Maps; Meiosis; Metaphase; Molecular; Mutation; Names; Nematoda; Oocytes; Organism; Orthologous Gene; Pattern; Phenocopy; Phenotype; Process; Prophase; Proteins; RNA Interference; Role; Staging; Suppressor Mutations; Temperature; Testing; Time; Transgenes; Transgenic Organisms; anaphase-promoting complex; base; embryo cell; gain of function; interest; loss of function; male; mutant; novel; paralogous gene; 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 define at least 9 complementation groups. A large number of alleles represent mutations in three spindle assembly checkpoint components, mdf-1, mdf-2, and mdf-3. Our results suggest that this checkpoint operates during meiosis. 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 the Cdc20/Fzy ortholog, a positive regulator 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, h1960, does exist but does not display the same early arrest as our other APC alleles. 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. RNAi of gfi-3 does not enhance other APC mutants, while RNAi of such-1 does. The such-1(h1960) allele mentioned above also does enhance other APC loss-of-function phenotypes. Interestingly, depletion of gfi-3 and such-1 from such-1(h1960) animals does result in 1-cell meiotic arrest. These results suggest that such-1 and gfi-3 are redundantly required for the meiotic divisions and that they can both function as meiotic APC-5 subunits. Using GFP transgenes, we have shown that they are both expressed in the hermaphrodite and male germline, and in early embryos. Their post-embryonic expression patterns vary and thus their somatic roles may differ later in development. Why only nematodes harbor two APC5 paralogs remains a mystery. We also have been pursuing the molecular identification of emb-1, a gene which we believe is a novel subunit or regulator of the APC. Temperature-sensitive alleles of emb-1 behave very much like our previously characterized APC mutants; they arrest as 1-cell embryos, are enhanced when combined with other APC alleles, and are suppressed weakly by our previously characterized APC suppressors. Three factor mapping, RNAi phenocopy, and transgenic rescue revealed that emb-1 encodes a small 81 amino acid protein. This protein is likely the APC16 subunit of the APC that was recently identified in human cells. Further support for our genetic conclusion comes from the findings of our colleagues who showed that EMB-1 co-purifies with numerous APC subunits. C. elegans is the only organism to date in which alleles of this new APC subunit exist. The function of this subunit with the larger complex remains to be determined.

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。 这些抑制器定义至少9个互补组。 许多等位基因代表三个主轴组件检查点组件MDF-1，MDF-2和MDF-3中的突变。我们的结果表明，该检查点在减数分裂过程中起作用。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，这些抑制剂是Cdc20/fzy直系同源物中的突变，这是APC/C的阳性调节剂。 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h1960确实存在，但与其他APC等位基因相同的早期停滞。 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。 GFI-3中没有现有突变。 GFI-3的RNAi不会增强其他APC突变体，而这种1的RNAi则不增强。 上面提到的这样的1（H1960）等位基因也可以增强其他APC功能丧失表型。 有趣的是，这种1（H1960）动物的GFI-3和1-1的耗竭确实会导致1细胞减数分裂骤停。这些结果表明，这种1和GFI-3是减数分裂划分的必要条件，并且它们都可以用作减数分裂APC-5亚基。 使用GFP转基因，我们已经证明它们均以雌雄同体和雄性生殖线以及早期的胚胎表示。 他们的胚胎后表达模式有所不同，因此它们的躯体作用可能在后来的发展中有所不同。 为什么只有线虫藏有两个APC5旁系同源物仍然是个谜。 我们还一直在追求EMB-1的分子鉴定，EMB-1是一种我们认为是APC的新型亚基或调节剂。 EMB-1的温度敏感等位基因的表现与我们先前表征的APC突变体非常相似。它们作为1细胞胚胎被捕获，与其他APC等位基因结合使用，并被我们先前表征的APC抑制器薄弱地抑制。三因子映射，RNAi表观和转基因救援表明，EMB-1编码小的81个氨基酸蛋白。该蛋白可能是最近在人类细胞中发现的APC的APC16亚基。 对我们的遗传结论的进一步支持来自我们的同事的发现，他们表明EMB-1与许多APC亚基共同治疗。秀丽隐杆线虫是迄今为止唯一存在这种新APC亚基等位基因的生物。该亚基具有较大复合物的功能尚待确定。