Mechanisms of Centrosome Reproduction in Animal Cells

动物细胞中心体复制机制

• 批准号: 7680083
• 负责人: EDWARD H HINCHCLIFFE
• 金额: $ 25.47万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680083
• 关键词: Animals; Antineoplastic Agents; Biochemical; Biological; Biological Models; Cancer Biology; Candidate Disease Gene; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Proliferation; Cells; Centrioles; Centrosome; Colcemid; Competence; Complement; Complex; Cyclin E; Disease; Ensure; Event; Fluorescence Recovery After Photobleaching; Genetic; Genome; Genomic Instability; Human; Image; Immunofluorescence Immunologic; Intrinsic factor; Investigation; Kinetics; Knowledge; Lead; Licensing; Licensing Factor; Life; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methods; Microscopy; Microtubule Triplet; Microtubule-Organizing Center; Microtubules; Mitosis; Molecular Genetics; Organelles; Phase; Play; Proteins; Rana; Regulation; Reproduction; Role; Signal Transduction; Somatic Cell; Source; Structural Protein; Structure; System; Techniques; Testing; Video Microscopy; Work; Xenopus; daughter cell; design; egg; knock-down; neoplastic cell; next generation; novel; prevent; reproductive; research study; tektin A

DESCRIPTION (provided by applicant): Mitosis must be carried out with high fidelity to ensure that each daughter cell receives a complete complement of the genome. Bipolar spindle assembly is directed by the duplication of the centrosome, which serves as the major microtubule-organizing center in mammalian cells. At the core of each centrosome is a pair of centrioles, composed of triplet microtubules: the duplication and assembly of the centrioles mirrors the duplication of the centrosome as a whole. Mistakes in the duplication of the centrosome itself can lead to the formation of multipolar spindles that do contribute to genomic instability. Indeed, many human tumor cells have abnormal numbers of centrosomes. To continue the investigation of the cell cycle regulation of centrosome duplication we will use live-cell video microscopy and biophysical/cell biological techniques to analyze centrosome reproduction in mammalian cells and frog egg extracts. These studies are intended to reveal important novel aspects of centrosome function that have proved difficult to study using molecular genetic or biochemical approaches. In Aim I, we will determine if Cdk2-cyclin E licenses centrosome duplication in Xenopus egg extracts, as suggested by our previous work. We will also test whether centrosome re-duplication in cells arrested in S-phase requires licensing. Finally, we test the hypothesis that centrosome duplication does not require the onset of S-phase, also suggested by our previous studies. In Aim II, we will investigate centriole assembly. Centriole microtubules are thought to be very stable, but our preliminary work on the centriole protein tektin suggests they undergo turnover throughout the cell cycle. We will use FRAP to measure the rate of GFPtektin/ centriole microtubule turnover in living cells. We will also test the hypothesis that tektin, a core centriole component, is synthesized coordinate with centrosome duplication, suggesting a mechanism for the cell cycle regulation of duplication itself. We will also test whether knocking-down the expression of tektin leads to inhibition of centriole duplication/elongation, and whether this induces cell cycle arrest. In Aim III, we examine early events in centrosome reproduction. We have developed methods to assemble centrosome precursors in mammalian cells. We will examine the kinetics of precursor formation using live-cell microscopy. We will isolate these precursor structures, analyze their protein composition by multi-LC Mass Spectrometry, and clone candidate genes. Using these, we will test whether the initial duplication event (precursor formation) is coordinated with centriole assembly or occurs earlier in the cell cycle. A detailed understanding of the regulation of centrosome reproduction will advance our knowledge of cancer biology - a disease characterized by unregulated cell proliferation. Our experiments should complement the genetic and biochemical work currently being done in a variety of model systems, and ultimately provide a fertile source of potential targets for the next generation of anti-cancer drugs.

描述（由申请人提供）：有丝分裂必须以高保真度进行，以确保每个子细胞获得完整的基因组互补。双极纺锤体的组装是由中心体的复制所指导的，中心体是哺乳动物细胞中主要的微管组织中心。在每个中心体的核心是一对中心粒，由三重微管组成：中心粒的复制和组装反映了中心体作为一个整体的复制。中心体自身复制的错误会导致多极纺锤体的形成，从而导致基因组的不稳定性。事实上，许多人类肿瘤细胞具有异常数量的中心体。 为了继续研究中心体复制的细胞周期调控，我们将使用活细胞视频显微镜和生物物理/细胞生物学技术来分析哺乳动物细胞和青蛙卵提取物中的中心体繁殖。这些研究旨在揭示中心体功能的重要新方面，这些方面已被证明难以使用分子遗传学或生化方法进行研究。 在目的我，我们将确定如果Cdk 2-细胞周期蛋白E许可中心体复制爪蟾卵提取物，我们以前的工作所建议的。我们还将测试在S期停滞的细胞中中心体重复是否需要许可。最后，我们测试的假设，中心体复制并不需要S期的发病，也建议我们以前的研究。在目标II中，我们将研究中心粒组装。中心粒微管被认为是非常稳定的，但我们对中心粒蛋白tektin的初步研究表明，它们在整个细胞周期中进行周转。我们将使用FRAP来测量活细胞中GFP tektin/中心粒微管更新的速率。我们还将测试的假设，tektin，核心中心粒组件，合成协调与中心体复制，表明复制本身的细胞周期调控机制。我们还将测试敲低tektin的表达是否会导致中心粒复制/延伸的抑制，以及这是否会诱导细胞周期停滞。在目的III中，我们研究中心体生殖的早期事件。我们已经开发了在哺乳动物细胞中组装中心体前体的方法。我们将使用活细胞显微镜检查前体形成的动力学。我们将分离这些前体结构，通过多重LC质谱分析其蛋白质组成，并克隆候选基因。使用这些，我们将测试是否最初的复制事件（前体形成）是协调与中心粒组装或发生在细胞周期的早期。对中心体复制调控的详细了解将推进我们对癌症生物学的认识-癌症是一种以不受调控的细胞增殖为特征的疾病。我们的实验应该补充目前在各种模型系统中进行的遗传和生物化学工作，并最终为下一代抗癌药物提供潜在靶点的肥沃来源。

项目成果

Toxoplasma gondii actively remodels the microtubule network in host cells.

• DOI: 10.1016/j.micinf.2008.08.014
• 发表时间: 2008-11
• 期刊: Microbes and infection
• 影响因子: 5.8
• 作者: Walker ME;Hjort EE;Smith SS;Tripathi A;Hornick JE;Hinchcliffe EH;Archer W;Hager KM
• 通讯作者: Hager KM

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis.

Tektin 2是中央纺锤体微管组织所必需的，并完成细胞因子。

• DOI: 10.1083/jcb.200711160
• 发表时间: 2008-05-19
• 期刊: JOURNAL OF CELL BIOLOGY
• 影响因子: 7.8
• 作者: Durcan, Thomas M.;Halpin, Elizabeth S.;Rao, Trisha;Collins, Nicholas S.;Tribble, Emily K.;Hornick, Jessica E.;Hinchcliffe, Edward H.
• 通讯作者: Hinchcliffe, Edward H.