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这一过程中的缺陷如何影响多细胞有机体的发育感兴趣。在过去的几年里，我们一直专注于产生单倍体配子的减数分裂。我们一直在研究一类来自线虫的温度敏感(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前期的卵母细胞通过精子受精。受精后，卵母细胞的染色体经历了两次减数分裂，丢弃了极体中的额外染色体。这些第一次减数分裂是重要的，因为在这个阶段染色体分离的任何错误都可能导致胚胎的染色体数量异常，这可能会导致致命性。在我们的突变体中，卵母细胞的染色体停滞在减数分裂I的中期，并且永远不会分离它们的染色体同源物，也不会伸出极体。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)的亚单位。这个复合体作为E3泛素连接酶，在细胞周期的中期到后期转变过程中，针对蛋白质进行破坏(由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的中期到后期过渡中存在缺陷。 为了确定这些APC/C亚基的基因外调节因子或底物，我们使用MAT-3突变体进行了遗传抑制筛选。我们的27个抑制子突变中的大多数是显性的。这些抑制子定义了至少9个互补基团。大量等位基因代表了三个主轴装配检查点组件MDF-1、MDF-2和MDF-3的突变。我们的结果表明，这个检查点在减数分裂过程中起作用。我们认为，我们的MAT突变体并没有触发检查点，而是检查点在减数分裂过程中作为APC/C的负调节因子正常工作。我们还鉴定了三个显性抑制基因，它们是APC/C的正调控因子CDc20/Fzy同源基因的突变。 在过去的一年里，我们鉴定了另一个含有APC亚单位突变的抑制等位基因，如-1。我们之前测试了该基因在减数分裂中的作用(使用RNAi)，但未能发现早期胚胎表型。温度敏感的功能降低等位基因h1960确实存在，但不像我们的其他APC等位基因那样表现出早期停滞。在被抑制的菌株中，这种基因的RNAi使该菌株恢复到减数分裂1细胞停滞表型。这一发现有力地表明，我们的抑制等位基因是这样-1中的功能获得等位基因。对该突变背景下的That-1基因进行测序证实，That-1的编码序列存在突变。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，它向我们揭示了这个APC亚单位可以在减数分裂过程中发挥作用。此类-1基因编码一个APC-5同源基因，有趣的是，线虫中有两个类似APC-5的基因。我们最近已经证明，另一个APC-5基因GFI-3在RNAi治疗的基础上并不是必需的。目前还没有GFI-3基因的突变。GFI-3的RNAi不增强其他APC突变体，而此类-1的RNAi则增强。上面提到的这样的-1(H1960)等位基因也增强了其他APC功能丧失的表型。有趣的是，从这类动物(H1960)中去除GFI-3和SOSE-1确实会导致1-细胞减数分裂停滞。这些结果表明，它们都是减数分裂所必需的，它们都可以作为减数分裂的APC-5亚基发挥作用。利用GFP转基因，我们已经证明它们都在两性和雄性生殖系中表达，并在早期胚胎中表达。它们在胚胎后的表达模式各不相同，因此它们在发育后期的躯体作用可能不同。为什么只有线虫有两个APC5并列基因，这仍然是个谜。 我们还一直在进行emb-1的分子鉴定，我们认为它是APC的一个新的亚单位或调节因子。Emb-1的温度敏感等位基因的行为与我们之前描述的APC突变体非常相似；它们作为1细胞胚胎停滞，当与其他APC等位基因结合时被增强，并被我们先前描述的APC抑制子微弱地抑制。三因子作图、RNAi表型分析和转基因救援结果表明，emb-1编码一个81个氨基酸的小蛋白。这种蛋白很可能是最近在人类细胞中发现的APC的APC16亚基。我们的同事的发现进一步支持了我们的遗传学结论，他们表明EMB-1与许多APC亚基共同净化。线虫是迄今为止唯一存在这种新的APC亚单位等位基因的生物体。这个具有较大复合体的亚基的功能还有待确定。