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）。我们已经将我们的突变体命名为“mat”。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。 为了鉴定这些 APC/C 亚基的外源调节因子或底物，我们进行了基因抑制筛选。我们的 29 个抑制突变中的大多数都是显性突变。这些抑制因子已使用单核苷酸多态性 (SNP) 技术进行定位，并定义了至少 6 个互补组。一些等位基因与 mat-3 相关； mat-3 的测序表明，其中只有一个代表 mat-3 基因内的第二个位点突变。三个未连锁的等位基因已被很好地定位和测序，代表两个纺锤体检查点成分的突变。这些是 MAD2 和 MAD3 的线虫直系同源物。当染色体未正确附着在有丝分裂纺锤体上时，纺锤体检查点可防止中期到后期的转变。我们的结果表明该检查点也在减数分裂期间发挥作用。我们目前不知道我们的 mat 突变体是否触发了检查点，或者检查点是否在减数分裂过程中正常运行作为 APC/C 的负调节因子。我们目前正在绘制其余抑制因子的图谱，并预计会发现新的分子，以阐明 APC/C 在减数分裂过程中的调节方式。这些抑制因子还可能揭示 APC/C 在多细胞生物发育过程中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自己的表型。通过 RNAi，我们目前正在测试纺锤体检查点蛋白的消耗是否可以抑制其他 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鉴定 APC/C 途径间接下游成分的调节因子和底物。该目标是一种称为分离酶的蛋白酶，当 APC/C 以 securin 为目标进行破坏时，会释放该酶。 Securin 通常会隔离分离酶，使其无法裂解将姐妹染色单体固定在一起的粘连蛋白分子。随着 securin 的破坏，分离酶可以自由地裂解粘连蛋白，并且发生姐妹染色单体分离。我们有 sep-1 的四个 ts 等位基因，并用这些等位基因之一进行了抑制筛选。迄今为止，我们已经鉴定出三种抑制因子，可以在不允许的温度下恢复 sep-1 突变体的活力。这些突变体之一是基因内抑制基因；另外两个是外源性的。我们目前正在绘制这两种抑制因子的图谱，以便我们能够从分子水平上识别它们。 我们还想检查线虫中 APC/C 的组成。对于这些研究，我们正在生成表达表位标记的 APC/C 亚基的转基因系，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；卵母细胞染色体不再暂停在减数分裂 I 的终变过程中。这些染色体具有许多有丝分裂的特征；它们用许多有丝分裂标记抗体染色。卵母细胞成熟似乎也早熟。我们认为，WEE-1.3 通常的作用是在卵子发生过程中保持母体 CDK-1 不活跃，并且在受精时，CDK-1 被激活以允许胚胎的减数分裂和有丝分裂。在缺乏 WEE-1.3 的情况下，CDK-1 变得早熟活跃，并驱动未完全分化的未成熟卵母细胞的卵母细胞成熟和染色体成熟。这些卵母细胞未能受精，可能是因为它们没有合成进一步发育所需的所有适当的卵母细胞/胚胎产物。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。