Cell Cycle Regulation In C. elegans

我们的实验室感兴趣的是染色体分离的过程，以及这个过程中的缺陷如何影响多细胞生物的发育。 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。 本实验研究了一组温度敏感型（ts）的胚胎致死突变体。在减数分裂中期停滞的线虫。 在野生型动物中，减数分裂I前期的卵母细胞被精子受精。 受精后，卵母细胞染色体经历两次减数分裂，将多余的染色体丢弃在极体中。 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染色体数量异常，这可能导致死亡。 在我们的突变体中，卵母细胞染色体在减数分裂I中期停滞，并且从不分离它们的染色体同源物，也从不挤出极体。 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物或细胞核小体（APC/C）的亚基。 该复合物作为E3泛素连接酶，在细胞周期的中期到后期过渡期间靶向蛋白质进行破坏（通过26 S蛋白酶体）。 我们将这些突变体命名为mat，是因为它们在减数分裂Ⅰ的中期向后期过渡过程中存在缺陷。 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选。我们的27个抑制突变中的大多数是显性的。 这些抑制子定义了至少9个互补组。 大量等位基因代表三个纺锤体组装检查点组分mdf-1、mdf-2和mdf-3中的突变。我们的研究结果表明，这个检查点在减数分裂过程中运作。我们相信我们的mat突变体没有触发检查点，而是检查点在减数分裂期间作为APC/C的负调节剂正常运作。 也许检查点的功能是调节减数分裂的适当时间。 我们还确定了三个显性抑制因子，它们是Cdc 20/Fzy直系同源物中的突变，Cdc 20/Fzy是APC/C的正调节因子。 在过去的一年中，我们已经确定了另一个抑制等位基因，它在APC亚基中含有突变，如-1。 我们之前已经测试了这个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。 一个温度敏感的功能降低等位基因，h1960，确实存在，但不显示相同的早期逮捕我们的其他APC等位基因。 抑制菌株中such-1基因的RNAi使菌株回复到减数分裂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一个功能获得等位基因在这样的-1。 在这种突变背景下对such-1基因进行测序，证实such-1在其编码序列中含有突变。 我们的抑制筛选有助于鉴定这种罕见的功能获得性等位基因，这向我们揭示了这种APC亚基可以在减数分裂期间发挥作用。 such-1基因编码APC-5同源基因，有趣的是，在C.优雅 我们最近已经证明，另一个apc-5基因gfi-3不是基于RNAi治疗所必需的。 目前在gfi-3中没有突变。 gfi-3的RNAi不增强其他APC突变体，而such-1的RNAi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确实导致1-细胞减数分裂停滞。这些结果表明，such-1和gfi-3是减数分裂所必需的，并且它们都可以作为减数分裂APC-5亚基起作用。 使用GFP转基因，我们已经表明，他们都表达在雌雄同体和男性生殖系，并在早期胚胎。 它们的胚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不同。 为什么只有线虫具有两个APC 5旁系同源物仍然是一个谜。 我们也一直在追求emb-1的分子鉴定，我们认为这是一个新的APC亚基或调节基因。温度敏感的等位基因emb-1的行为非常像我们以前的特点APC突变体，他们逮捕1细胞胚胎，增强与其他APC等位基因结合时，并被我们以前的特点APC抑制弱。三因子定位、RNAi表型复制和转基因拯救实验表明，emb-1编码一个81个氨基酸的小蛋白。这种蛋白质可能是最近在人类细胞中鉴定的APC的APC 16亚基。 对我们遗传学结论的进一步支持来自我们同事的发现，他们显示EMB-1与许多APC亚基共纯化。C.秀丽线虫是迄今为止唯一存在这种新APC亚基的等位基因的生物体。该亚基与较大复合物的功能仍有待确定。

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.