Cell Cycle Regulation In C. elegans

线虫的细胞周期调控

• 批准号: 6673374
• 负责人: ANDY GOLDEN
• 金额: --
• 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/6673374
• 关键词: Caenorhabditis elegans; antibody; cell cycle; developmental genetics; egg /ovum; embryogenesis; fertilization; gene expression; genetic screening; green fluorescent proteins; meiosis; sperm; stainings; temperature sensitive mutant; tissue /cell culture

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. In order to molecularly identify the genes required for the first meiotic division, we have mapped our mutants and sequenced candidate genes. Five of the six genes have now been identified and 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 proteosome) during the metaphase to anaphase transition of the cell cycle. We have named our mutants "mat" for their defects in the metaphase to anaphase transition during meiosis I. These ts mutants also display defects in spermatocyte meiosis; primary spermatocytes arrest in metaphase of meiosis I with a normal meiotic spindle, yet fail to separate chromosome homologs. Thus, these mutants disrupt meiosis in both oocytes and spermatocytes. To address the role of the mat genes in mitosis, we have performed shift-up experiments during embryogenesis and larval development. Temperature shift-up experiments during embryogenesis do not result in embryonic phenotypes, however, somatic defects in the gonad, vulva, and male tail are apparent in adults. This observation suggests that mitotic divisions in the soma are affected by the mat mutants. For many of the alleles, these shift-up experiments also result in sterility, suggesting mitotic defects in germline proliferation. We have constructed double mutants of many of these APC/C mutants and have found that many combinations are synthetic lethal at the permissive temperature. We are currently using some of the double mutants that are still viable at the permissive temperature to re-examine the role of APC/C during the mitotic cell cycles during embryogenesis and larval development. Specifically, we hope to observe more severe mitotic defects during the development of the germline and various tissues. To further understand the role of these mat genes during development, we are characterizing their expression patterns using antibody staining and GFP transgenic lines. We have also completed a genetic suppression screen to identify extragenic regulators or substrates of these APC/C subunits. The majority of our 29 suppressor mutations are dominant. We anticipate finding novel molecules that shed light on how APC/C functions and is regulated in different tissues and at different times during the development of a multicellular organism. We also want to examine the composition of the APC/C in C. elegans. For these studies, we will generate transgenic lines expressing epitope-tagged APC/C subunits and then purify the complex with epitope-specific antibodies. These proteins that we purify will be subjected to mass spectrometry to identify the components of the APC/C. We will then examine whether this complex varies during development. In a separate study, we are examining the function of the C. elegans Myt1 ortholog. Myt1 belongs to the Wee1 family of kinases and is thought to down regulate Cdk1 during the cell cycle. RNAi studies with the Myt1 ortholog, wee-1.3, result in sterility. Mothers injected with dsRNA quickly become sterile; the oocyte chromosomes are no longer paused in diakinesis of meiosis I. We propose that WEE-1.3 normally functions to keep maternal CDK-1 inactive during oogenesis, and that upon fertilization, CDK-1 becomes activated to allow for the meiotic and mitotic divisions of the embryo. We are further characterizing this phenotype and plan to use RNAi screens to identify other components of this pathway.

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。为了从分子上鉴定第一次减数分裂所需的基因，我们绘制了突变体图谱并对候选基因进行了测序。六个基因中的五个现已被鉴定，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）。我们将我们的突变体命名为“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。这些 ts 突变体还表现出精母细胞减数分裂的缺陷；初级精母细胞在减数分裂 I 中期停滞，具有正常的减数分裂纺锤体，但无法分离染色体同源物。因此，这些突变体破坏了卵母细胞和精母细胞的减数分裂。为了解决 mat 基因在有丝分裂中的作用，我们在胚胎发生和幼虫发育过程中进行了上移实验。胚胎发生过程中的温度升高实验不会产生胚胎表型，然而，性腺、外阴和雄性尾部的体细胞缺陷在成人中很明显。这一观察结果表明体细胞中的有丝分裂受到 mat 突变体的影响。对于许多等位基因来说，这些上移实验也会导致不育，表明种系增殖中存在有丝分裂缺陷。我们已经构建了许多这些 APC/C 突变体的双突变体，并发现许多组合在允许的温度下是合成致死的。我们目前正在使用一些在允许的温度下仍然存活的双突变体来重新检查 APC/C 在胚胎发生和幼虫发育期间有丝分裂细胞周期中的作用。具体来说，我们希望在种系和各种组织的发育过程中观察到更严重的有丝分裂缺陷。为了进一步了解这些 mat 基因在发育过程中的作用，我们使用抗体染色和 GFP 转基因系来表征它们的表达模式。我们还完成了基因抑制筛选，以鉴定这些 APC/C 亚基的外源调节因子或底物。我们的 29 个抑制突变中的大多数都是显性突变。我们期望找到新的分子，以阐明 APC/C 在多细胞生物体发育过程中不同组织和不同时间的功能和调节方式。我们还想检查线虫中 APC/C 的组成。对于这些研究，我们将生成表达表位标记的 APC/C 亚基的转基因系，然后用表位特异性抗体纯化复合物。我们纯化的这些蛋白质将进行质谱分析以鉴定 APC/C 的成分。然后我们将检查该复合体在发育过程中是否发生变化。 在另一项研究中，我们正在检查线虫 Myt1 直系同源物的功能。 Myt1 属于 Wee1 激酶家族，被认为在细胞周期中下调 Cdk1。使用 Myt1 直向同源物 wee-1.3 进行的 RNAi 研究导致不育。注射双链RNA的母亲很快就会变得不育；卵母细胞染色体在减数分裂 I 的终变过程中不再暂停。我们认为 WEE-1.3 通常在卵子发生过程中发挥保持母体 CDK-1 不活跃的作用，并且在受精时，CDK-1 被激活以允许卵子的减数分裂和有丝分裂。胚胎。我们正在进一步表征这种表型，并计划使用 RNAi 筛选来识别该途径的其他组成部分。