Defining the true nature of the minimal cell cycle with quantitative proteomics

用定量蛋白质组学定义最小细胞周期的真实本质

• 批准号: 8136234
• 负责人: Hanno Steen
• 金额: $ 34.42万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136234
• 关键词: Affect; African; Animal Model; Animals; Automobile Driving; Biological; Biological Models; Cell Cycle; Cell Cycle Regulation; Cell division; Cells; Communities; Complex; Cyclin B; Data; Dependence; Development; Disease; Embryo; Event; Explosion; Feedback; Fertilization; Foundations; G1 Phase; G2 Phase; Genetic Transcription; Genome; Germ Cells; Global Change; Growth; Human; In Vitro; Laboratories; Malignant Neoplasms; Mediating; Mining; Mitosis; Mitotic; Modeling; Molecular; Morphologic artifacts; Nature; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; Rana; Refractory; Regulation; Research; Resolution; S Phase; Sampling; Series; Somatic Cell; Specimen; Staging; System; Techniques; Time; Translations; Tyrosine; Variant; Xenopus sp.; Zebrafish; base; blastomere structure; cell growth; egg; forgetting; improved; in vivo; insight; prevent; public health relevance; research study; segregation; stoichiometry; transcriptomics; virtual

DESCRIPTION (provided by applicant): The cleavage cycles of early metazoan embryos are limited to the bare essence of genome replication and segregation, lacking the growth, transcription and checkpoints which embellish the somatic cell cycle. These cleavage cycles are therefore the natural framework upon which to construct models of the much more complicated somatic cell cycles. Such models are the intellectual foundation for thousands of laboratories world-wide intent on understanding cell division and growth, and how to prevent, counteract and treat their misregulation. But the true nature of the most minimal cell cycle, the metazoan cleavage cycle, is far from fully understood. In particular, the regulatory modules that are widely thought to initiate mitosis are not important in the cleavage cycles. The unknown mechanism that truly controls entry into mitosis in these minimal cleavage cycles, and which by extension could be extant in all metazoan cell cycles, remains obscure. We propose that this unknown mechanism, and perhaps many other aspects of the minimal cell cycle, could be revealed by comprehensive analyses. The molecular landscape of the cleavage cycles can only be generated by MS- based proteomics, particularly because the virtual absence of zygotic transcription makes these cleavage cell cycles refractory to transcriptomics. In striving towards radically improved models of the cell cycle, we aim to define the minimal animal cell cycle by i) detecting which proteins and which phosphorylation sites oscillate in a cell cycle dependent manner, ii) quantifying the extent of variations/oscillations, iii) provide information about absolute phosphorylation stoichiometries, and iv) provide absolute quantities for key cell cycle regulators and their post-translational modifications (not limited to phosphorylation) that mediate the minimal cell cycle. This study will be the first large-scale, quantitative proteomic study of cleavage cycles and the first global analysis of the metazoan cell cycle that doesn't rely on drug-based synchronization techniques. The embryos of the frog X. laevis embryos provide a compelling context for these experiments due to their large size, holoblastic cleavage, and the capability for naturally synchronized cell cycles. The proposed experiments have the potential for revolutionary insights into the cell cycle, as well as generating a trove of data which can be mined and further extended upon by the vibrant cell cycle community. PUBLIC HEALTH RELEVANCE: Instead of using artificial in vitro cultured systems, it is our aim to study the in vivo "minimal" embryonic cell cycle in order to decipher the true nature of its regulation. Because we aim to describe hundreds of changes in protein abundance and protein modifications that underlie this cycle in vivo with an unprecedented temporal resolution, this project will challenge, refine, and enlarge current models of the cell cycle that are central to our fundamental understanding of proliferation, and to many human developmental diseases and cancers. Apart from furthering our fundamental understanding of the cell cycle, the development of preventatives, treatments and therapies for/of these diseases and malignancies will benefit greatly from this study.

描述（由申请人提供）：早期后生动物胚胎的分裂周期仅限于基因组复制和分离的本质，缺乏修饰体细胞周期的生长、转录和检查点。因此，这些裂解周期是构建更复杂的体细胞周期模型的自然框架。这些模型是全球数千个实验室的知识基础，这些实验室致力于了解细胞分裂和生长，以及如何预防、抵消和治疗其失调。但是，后生动物卵裂周期这一最小细胞周期的真正本质还远未完全了解。特别是，被广泛认为启动有丝分裂的调节模块在卵裂周期中并不重要。在这些最小的分裂周期中真正控制进入有丝分裂的未知机制，并且通过扩展可能存在于所有后生动物细胞周期中，仍然是模糊的。我们建议，这种未知的机制，以及最小细胞周期的许多其他方面，可以通过综合分析来揭示。裂解周期的分子景观只能通过基于 MS 的蛋白质组学来生成，特别是因为合子转录的实际缺失使得这些裂解细胞周期难以进行转录组学。在努力从根本上改进细胞周期模型的过程中，我们的目标是通过以下方式定义最小的动物细胞周期：i) 检测哪些蛋白质和哪些磷酸化位点以细胞周期依赖性方式振荡，ii) 量化变化/振荡的程度，iii)提供有关绝对磷酸化化学计量的信息，iv) 提供关键细胞周期调节剂及其介导最小细胞周期的翻译后修饰（不限于磷酸化）的绝对数量。这项研究将是第一个针对裂解周期的大规模定量蛋白质组学研究，也是第一个不依赖于基于药物的同步技术的后生动物细胞周期的全局分析。青蛙胚胎的胚胎由于其大尺寸、全胚细胞分裂以及自然同步细胞周期的能力，为这些实验提供了令人信服的背景。所提出的实验有可能对细胞周期产生革命性的见解，并产生大量数据，这些数据可以被充满活力的细胞周期社区挖掘和进一步扩展。 公共健康相关性：我们的目标不是使用人工体外培养系统，而是研究体内“最小”胚胎细胞周期，以破译其调节的真正本质。因为我们的目标是以前所未有的时间分辨率描述体内该循环背后的蛋白质丰度和蛋白质修饰的数百个变化，所以该项目将挑战、完善和扩大当前的细胞周期模型，这些模型对于我们对增殖的基本理解至关重要，以及许多人类发育疾病和癌症。除了进一步加深我们对细胞周期的基本了解外，这些疾病和恶性肿瘤的预防、治疗和疗法的开发也将从这项研究中受益匪浅。