Cell Cycle Regulation In C. elegans

线虫的细胞周期调控

• 批准号: 6983651
• 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/6983651
• 关键词: Caenorhabditis elegans; antibody; cell cycle; cell growth regulation; cyclin dependent kinase; developmental genetics; egg /ovum; embryogenesis; fertilization; gene expression; mass spectrometry; meiosis; single nucleotide polymorphism; sperm; 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. To identify extragenic regulators or substrates of these APC/C subunits, we have carried out a genetic suppression screen. The majority of our 29 suppressor mutations are dominant. These suppressors have been mapped using single nucleotide polymorphism (SNP) technology and define at least 6 complementation groups. A few alleles are linked to mat-3; sequencing of mat-3 has revealed that only one of these represents a second site mutations within the mat-3 gene. Three unlinked alleles have been well-mapped and sequenced and represent mutations in two spindle checkpoint components. These are the C. elegans orthologs of MAD2 and MAD3. The spindle checkpoint prevents the metaphase to anaphase transition when chromosomes are not properly attached to the mitotic spindle. Our results suggest that this checkpoint also operates during meiosis. We currently do not know if our mat mutants are triggering the checkpoint, or if the checkpoint normally operates during meiosis as a negative regulator of the APC/C. We are currently mapping the remaining suppressors and anticipate finding novel molecules that shed light on how the APC/C is regulated during meiosis. These suppressors may also reveal how the APC/C functions in different tissues and at different times during the development of a multicellular organism. So far, none of our suppressor mutations have a phenotype on their own. By RNAi, we are currently testing if depletion of spindle checkpoint proteins can suppress other mat-3 alleles or other mat genes. We have taken a similar genetic approach to identify regulators and substrates of an indirect downstreaam component of the APC/C pathway. This target is a protease called separase, which is released when APC/C targets securin for destruction. Securin normally sequesters separase so that it cannot cleave the cohesin molecules that hold sister chromatids together. With securin destroyed, separase is free to cleave cohesin and sister chromatid separation occurs. We have four ts alleles of sep-1 and have carried out a suppression screen with one of these alleles. To date, we have identified three suppressors that restore viability to sep-1 mutants at the non-permissive temperature. One of these mutants is an intragenic suppressor; the other two are extragenic. We are currently mapping these two suppressors so that we can molecularly identify them. We also want to examine the composition of the APC/C in C. elegans. For these studies, we are generating transgenic lines expressing epitope-tagged APC/C subunits so that we can 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. These chromosomes have many hallmarks of being mitotic; they stain with a number of mitotic marker antibodies. Oocyte maturation also appears to be precocious. 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. In the absence of WEE-1.3, CDK-1 becomes precociously active and drives oocyte maturation and chromosome maturation in immature oocytes that are not fully differentiated. These oocytes fail to be fertilized presumably because they have not synthesized all the proper oocyte/embryo products they need for further development. 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。我们命名了我们的突变体？因为它们在减数分裂过程中的中期向后期转变中的缺陷。这些TS突变体还显示出精子细胞减数分裂的缺陷；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。 为了识别这些APC/C亚基的基因外调节剂或底物，我们进行了遗传抑制筛查。我们的29个抑制突变中的大多数是主导的。这些抑制剂已使用单核苷酸多态性（SNP）技术进行了映射，并定义了至少6个互补组。一些等位基因链接到MAT-3； MAT-3的测序表明，其中仅代表MAT-3基因内的第二个位点突变。三个未链接等位基因的映射和测序已得到很好的测序，并表示两个主轴检查点组件中的突变。这些是MAD2和MAD3的秀丽隐杆线虫直系同源物。当染色体未正确连接到有丝分裂纺锤体时，主轴检查点可防止中期向后期转变。我们的结果表明，该检查点在减数分裂过程中也工作。我们目前不知道我们的垫子突变体是否触发了检查点，或者检查点在减数分裂期间通常作为APC/c的负调节剂在减数分裂时运行。我们目前正在绘制剩余的抑制剂，并预计找到新的分子，这些分子阐明了在减数分裂过程中如何调节APC/C的方式。这些抑制剂还可以揭示APC/C在多细胞生物体开发过程中如何在不同组织和不同时间的功能。到目前为止，我们的抑制突变都没有单独具有表型。通过RNAi，我们目前正在测试主轴检查点蛋白的耗竭是否可以抑制其他MAT-3等位基因或其他MAT基因。 我们采用了类似的遗传方法来识别APC/C途径间接下区分数的调节剂和底物。该靶标是一种称为分离酶的蛋白酶，当APC/C靶向Securin进行破坏时，该靶酶将会释放。 Securin通常是隔离分离酶，因此它不能裂解将姐妹染色质固定在一起的粘蛋白分子。随着证人林的破坏，分离酶可以自由切割粘着蛋白，而姐妹染色单体分离发生。我们有四个sep-1等位基因，并用其中一个等位基因进行了抑制屏幕。迄今为止，我们已经确定了三个在非疗法温度下恢复对Sep-1突变体的生存力的抑制剂。这些突变体之一是基因内抑制剂。另外两个是外部的。我们目前正在绘制这两个抑制器，以便我们可以分子识别它们。 我们还想检查秀丽隐杆线虫中APC/C的组成。在这些研究中，我们正在生成表达表位标记的APC/C亚基的转基因线，以便我们可以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的炎性I。这些染色体具有许多有丝分裂的标志。它们用多种有丝分裂标记抗体染色。卵母细胞的成熟似乎也很早熟。我们建议WEE-1.3通常功能以使母体CDK-1在卵子发生过程中保持不活跃，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分裂。在没有WEE-1.3的情况下，CDK-1的活跃性就变得活跃，并在未完全区分的未成熟卵母细胞中驱动卵母细胞成熟和染色体成熟。这些卵母细胞可能没有受精，因为它们没有合成他们进一步开发所需的所有适当的卵母细胞/胚胎产品。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。