Centrosome Maturation and Duplication in the C. elegans Embryo

线虫胚胎中的中心体成熟和复制

• 批准号: 7733978
• 负责人: Kevin F O'Connell
• 金额: $ 25.77万
• 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/7733978
• 关键词: Address; Affect; Anaphase; Antibodies; Biochemical; Biological Models; Caenorhabditis elegans; Cell Cycle; Cell Cycle Progression; Cell Nucleus; Cell Polarity; Cell physiology; Cells; Centrioles; Centrosome; Chromosome Segregation; Cytokinesis; Cytoplasmic Granules; Data; Defect; Embryo; Ensure; Event; Genes; Genetic; Goals; Laboratories; Localized; Maps; Microtomy; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic spindle; Molecular; Morphology; Mutation; Nematoda; Nuclear; Numbers; Organelles; Phase; Phenocopy; Phenotype; Phosphoric Monoester Hydrolases; Play; Positioning Attribute; Process; Protein Overexpression; Protein phosphatase; Proteins; Proteomics; RNA Binding; RNA Interference; RNA-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; Role; Staining method; Stains; Structure; Testing; Time; Translations; Transmission Electron Microscopy; Zinc Fingers; cell type; gamma Tubulin; mutant; phosphatase-1 kinase; size; trafficking

In most cell types, microtubules are organized by the centrosome, an organelle composed of an orthogonal pair of centrioles surrounded by a matrix of pericentriolar material (PCM). During the cell cycle, the centrosome duplicates precisely once. This event is of critical importance to mitotic spindle assembly as it ensures that two centrosomes are available to form the poles of the bipolar spindle. Duplication involves splitting of the existing centriole pair followed by the synthesis of a new centriole next to each old centriole. As the cell progresses toward mitosis, the centrosome "matures" or gradually acquires increased levels of microtubule nucleating capacity and PCM. Despite the importance of centrosome duplication and maturation, little is known of how these processes are regulated at a molecular level. In my laboratory, we are using the nematode Caenorhabditis elegans to study centrosome duplication and maturation. Specifically, our goals are to identify the factors that regulate these processes and to understand how they function on a molecular level. During 2008, we continued to study two factors that regulate centrosome duplication and have initiated studies on two others. All were identified in a screen for genes that genetically interact with zyg-1, a conserved upstream regulator of centrosome duplication. SZY-20 is a negative regulator of ZYG-1 and localizes to centrosomes. We have demonstrated that SZY-20 limits centrosome size and that in its absence centrosomes are enlarged. These centrosomes possess elevated levels of ZYG-1 and PCM components such as SPD-2, SPD-5, and gamma-tubulin. Further, we have found that these enlarged centrosomes nucleate more microtubules than their wild-type counterparts and that the enlarged centrosome phenotype is associated with defects in microtubule-dependent processes. Of particular significance we find that two centrosome duplication factors, ZYG-1 and SAS-6 are involved in defining centrosome size. In particular, inhibition of ZYG-1 in the szy-20 mutant restores normal centrosome size and rescues the microtubule-related defects. Importantly, thin section transmission electron microscopy has revealed that szy-20 mutants possess centrioles of normal size. This and other data argue that the role of ZYG-1 in controlling centrosome size is separable from its role in centriole replication. Last year we cloned the szy-20 gene and found that it encodes a widely conserved, yet unstudied protein. At the time, our analysis indicated that SZY-20 was likely to be an RNA-binding protein. To address this we performed overexpression studies and have found that overexpressing SZY-20 reduces centrosome size in wild-type embryos yet no effect is seen when an RNA-binding defective mutant is overexpressed. Our data indicates that SZY-20 regulates the level of ZYG-1 at the centrosome and may act by controlling the translation, stability, or localization of centrosome-associated mRNAs. We are also continuing our studies on SZY-5, another negative regulator of ZYG-1. SZY-5 encodes a zinc-finger protein but does not seem to regulate centrosome-levels of ZYG-1, indicating that it interacts with ZYG-1 in a manner distinctly different from that of SZY-20. We have postulated that SZY-5 might control the expression of one or more centrosome duplication factors and have used RT-PCR and quantitative immunostaining to address this issue. So far, SZY-5 does not appear to affect the expression or localization of any of the known centrosome duplication factors. To further define the function of SZY-5 we have sought to determine its subcellular distribution and to characterize defects that arise in its absence. Toward this end, we have raised an antibody to SZY-5 and find that it stains nuclei and cytoplasmic granules. We have also found that embryos lacking SZY-5 display defects in osmotic integrity, cell cycle progression, polar body extrusion, cytokinesis, and anaphase spindle positioning. As some of the processes affected by the szy-5 mutation involve centrosome function, we are examining centrosome structure and function in the mutant. Finally, we are initiating a proteomics approach to study SZY-5, as the identification of interacting factors should help us understand the role of SZY-5 in centrosome duplication. This past year we have also initiated analysis of two other factors that interact with ZYG-1. Mutation of the szy-1 gene suppresses the centrosome duplication defect of zyg-1 mutants and disrupts normal centrosome morphology. We have mapped szy-3 to a small physical interval and hope to identify the gene using RNAi to phenocopy szy-1 mutant defects. The szy-2 gene is also another suppressor of ZYG-1. We have cloned szy-2 and find that it encodes a regulatory subunit of protein phosphatase 1 (PP1) called I-2. We find that I-2 opposes the activity of ZYG-1 and are currently testing to see if I-2 negatively or positively regulates PP1. Since ZYG-1 is a kinase, PP1 may interact directly with ZYG-1 substrates. In addition to its interaction with ZYG-1, we find that I-2 is required for proper chromosome segregation.

在大多数细胞类型中，微管是由中心体组织的，中心体是由一个正交的中心元组组成的细胞器，该细胞体被周围的中心三醇材料（PCM）组成。 在细胞周期中，中心体精确地重复了一次。 该事件对于有丝分裂纺锤体组件至关重要，因为它可以确保两个中心体可用于形成双极纺锤体的极。 重复涉及分裂现有的中心对，然后在每个旧的中心旁旁边合成一个新的中心。 随着细胞朝着有丝分裂的发展，中心体“成熟”或逐渐获得了微管成核能力和PCM的水平。 尽管中心体重复和成熟很重要，但对这些过程在分子水平的调节方式鲜为人知。 在我的实验室中，我们正在使用线虫秀丽隐杆线虫来研究中心体重复和成熟。 具体而言，我们的目标是确定调节这些过程的因素，并了解它们在分子水平上的功能。 在2008年期间，我们继续研究两个调节中心体重复的因素，并对另外两个因素进行了研究。 所有这些都在与Zyg-1相互作用的基因中鉴定出来，该基因是centrosome重复的保守上游调节剂Zyg-1。 SZY-20是ZYG-1的负调节剂，并定位于中心体。 我们已经证明，SZY-20限制了中心体的大小，并且在缺席的情况下，中心体被扩大。 这些中心体具有较高水平的ZYG-1和PCM成分，例如SPD-2，SPD-5​​和γ-微管蛋白。 此外，我们发现这些扩大的中心体比其野生型对应物更大的微管成核，并且增大的中心体表型与微管依赖性过程中的缺陷有关。 特别重要的是，我们发现两个中心体重复因子ZYG-1和SAS-6参与定义中心体大小。 特别是，在SZY-20突变体中抑制Zyg-1可恢复正常的中心体大小并挽救与微管相关的缺陷。重要的是，薄截面的透射电子显微镜表明，SZY-20突变体具有正常大小的中心元素。 该数据和其他数据认为，Zyg-1在控制中心体大小中的作用与其在中心复制中的作用可分开。 去年，我们克隆了SZY-20基因，发现它编码了广泛保守但未研究的蛋白质。 当时，我们的分析表明SZY-20可能是一种RNA结合蛋白。 为了解决这一问题，我们进行了过表达的研究，发现过度表达SZY-20会减少野生型胚胎中的中心体大小，但是当RNA结合有缺陷的突变体过表达时，没有任何影响。 我们的数据表明，SZY-20调节中心体处的ZYG-1水平，并可以通过控制与中心体相关的mRNA的翻译，稳定性或定位来起作用。 我们还继续对ZYG-1的另一个负面调节剂SZY-5进行研究。 SZY-5编码锌指蛋白，但似乎没有调节Zyg-1的中心体级别，表明它与Zyg-1的相互作用与SZY-20的方式截然不同。 我们假设SZY-5可以控制一个或多个中心体重复因子的表达，并使用RT-PCR和定量免疫染色来解决此问题。到目前为止，SZY-5似乎没有影响任何已知的中心体重复因子的表达或定位。为了进一步定义SZY-5的功能，我们试图确定其亚细胞分布，并表征其缺乏的缺陷。 为此，我们提出了SZY-5的抗体，发现它染色了核和细胞质颗粒。我们还发现，缺乏SZY-5显示出渗透性完整性，细胞周期进程，极性体挤出，细胞因子和后期纺锤体定位的胚胎。由于受SZY-5突变影响的某些过程涉及中心体功能，我们正在研究突变体中的中心体结构和功能。最后，我们正在启动一种研究SZY-5的蛋白质​​组学方法，因为相互作用因子的识别应有助于我们了解SZY-5在中心体重复中的作用。 在过去的一年中，我们还开始了与Zyg-1相互作用的其他两个因素的分析。 SZY-1基因的突变抑制了Zyg-1突变体的中心体重复缺陷并破坏正常的中心体形态。我们已经将SZY-3映射到一个小的物理间隔，并希望使用RNAi鉴定该基因，以表szy-1突变缺陷。 SZY-2基因也是Zyg-1的另一个抑制剂。 我们已经克隆了SZY-2，发现它编码称为I-2的蛋白质磷酸酶1（PP1）的调节亚基。 我们发现I-2反对ZYG-1的活性，目前正在测试，以查看I-2是否对PP1进行负面调节。 由于ZYG-1是一种激酶，因此PP1可能直接与Zyg-1底物相互作用。 除了与ZYG-1的相互作用外，我们发现I-2是正确的染色体分离所必需的。