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。 这些抑制因子定义了至少 9 个互补组。 大量等位基因代表三个纺锤体组装检查点组件 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期间发挥作用。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 Cdc20/Fzy 直向同源物（APC/C 的正调节因子）中的突变。 去年，我们鉴定了另一种抑制等位​​基因，该等位基因在 APC 亚基中存在突变，即 such-1。 我们之前测试过该基因在减数分裂中的作用（使用 RNAi），但未能找到早期胚胎表型。 温度敏感的功能降低等位基因 h1960 确实存在，但没有表现出与我们的其他 APC 等位基因相同的早期停滞。 对受抑制菌株中 such-1 基因进行 RNAi 可使菌株恢复至减数分裂 1 细胞停滞表型。 这一发现强烈表明我们的抑制等位基因是 such-1 中的功能获得等位基因。 对这种突变背景中的 such-1 基因进行测序证实，such-1 在其编码序列中存在突变。 我们的抑制子筛选有助于识别这种罕见的功能获得等位基因，该等位基因向我们揭示了该 APC 亚基可以在减数分裂期间发挥作用。 such-1 基因编码 APC-5 直向同源物，有趣的是，秀丽隐杆线虫中有两个类似 apc-5 的基因。 我们最近表明，基于 RNAi 治疗，另一个 apc-5 基因 gfi-3 并不是必需的。 gfi-3 中不存在突变。 gfi-3 的 RNAi 不会增强其他 APC 突变体，而 such-1 的 RNAi 则会增强。 上面提到的 such-1(h1960) 等位基因也确实增强了其他 APC 功能丧失表型。 有趣的是，从 such-1(h1960) 动物中去除 gfi-3 和 such-1 确实会导致 1 细胞减数分裂停滞。这些结果表明，such-1 和 gfi-3 对于减数分裂是多余的，并且它们都可以作为减数分裂 APC-5 亚基发挥作用。 使用 GFP 转基因，我们发现它们都在雌雄同体和雄性种系以及早期胚胎中表达。 它们的胚胎后表达模式各不相同，因此它们的体细胞作用在发育后期可能会有所不同。 为什么只有线虫拥有两个 APC5 旁系同源物仍然是个谜。 我们还一直在进行 emb-1 的分子鉴定，我们认为该基因是 APC 的一个新亚基或调节因子。 emb-1 的温度敏感等位基因的行为与我们之前表征的 APC 突变体非常相似；它们作为 1 细胞胚胎停滞，与其他 APC 等位基因结合时得到增强，并被我们之前表征的 APC 抑制子微弱抑制。三因子图谱、RNAi 表型和转基因拯救表明 emb-1 编码一个由 81 个氨基酸组成的小蛋白。这种蛋白质很可能是最近在人类细胞中发现的 APC 的 APC16 亚基。 我们同事的研究结果进一步支持了我们的遗传学结论，他们表明 EMB-1 与许多 APC 亚基共同纯化。线虫是迄今为止唯一存在这种新 APC 亚基等位基因的生物体。该亚基与较大复合体的功能仍有待确定。