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。为了分子识别第一个减数分裂分裂所需的基因，我们绘制了突变体和测序的候选基因。现在已经鉴定出六个基因中的五个，它们编码了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，以破坏（由26S蛋白体）在中期为细胞周期的后期转变。我们将突变体命名为“ MAT”，因为它们在减数分裂过程中的中期向后期转变中的缺陷。原发性精子细胞在减数分裂I的比例中停滞，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，这些突变体在卵母细胞和精子细胞中都破坏了减数分裂。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转移实验。胚胎发生过程中的温度转移实验不会导致胚胎表型，但是，成年人显然是性腺，外阴和雄性尾巴的体细胞缺陷。该观察结果表明，躯体中的有丝分裂分裂受MAT突变体的影响。对于许多等位基因，这些转移实验也导致不育，表明种系增殖中有丝分裂缺陷。我们已经构建了许多此类APC/C突变体的双重突变体，并发现许多组合在允许温度下都是合成的致命。目前，我们正在使用一些在允许温度下仍然可行的双突变体，以重新检查APC/C在胚胎生成和幼虫发育过程中有丝分裂细胞周期期间的作用。具体而言，我们希望在种系和各种组织的发展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基因在发育中的作用，我们正在使用抗体染色和GFP转基因线来表征它们的表达模式。我们还完成了一个遗传抑制筛查，以识别这些APC/C亚基的基因外调节剂或底物。我们的29个抑制突变中的大多数是主导的。我们预计找到新的分子，这些分子阐明了APC/C在多细胞生物体开发过程中在不同组织和不同时间的调节。我们还想检查秀丽隐杆线虫中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亚基的转基因线，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法，以识别APC/C的成分。然后，我们将检查该复合物在开发过程中是否有所不同。 在另一项研究中，我们正在研究秀丽隐杆线虫MYT1直系同源物的功能。 MYT1属于激酶的WEE1家族，被认为可以在细胞周期中调节CDK1。 RNAi对MYT1直系同源物WEE-1.3进行研究导致不育。注射dsRNA的母亲迅速变得无菌。卵母细胞染色体不再在减数分裂I的垂体性中暂停。我们提出WEE-1.3通常在卵子发生过程中保持母体CDK-1的非活性，并且在受精后，CDK-1被激活以允许胚胎的减数分裂和有丝分裂的分裂。我们正在进一步表征这种表型，并计划使用RNAi屏幕来识别该途径的其他组件。