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

描述（由申请人提供）：早期后生动物胚胎的卵裂周期仅限于基因组复制和分离的基本要素，缺乏修饰体细胞周期的生长、转录和检查点。因此，这些分裂周期是构建更为复杂的体细胞周期模型的天然框架。这些模型是全世界数千个实验室的智力基础，这些实验室致力于了解细胞分裂和生长，以及如何预防、抵消和治疗它们的失调。但是，最小的细胞周期，后生动物卵裂周期的真正性质，是远远没有完全理解。特别是，被广泛认为启动有丝分裂的调控模块在切割周期中并不重要。在这些最小的卵裂周期中，真正控制进入有丝分裂的未知机制仍然不清楚，并且通过扩展可能存在于所有后生动物细胞周期中。我们建议，这种未知的机制，也许还有许多其他方面的最小细胞周期，可以揭示全面的分析。切割周期的分子景观只能通过基于MS的蛋白质组学产生，特别是因为合子转录的实际缺乏使得这些切割细胞周期对转录组学来说是难处理的。在努力实现细胞周期的根本改进模型的过程中，我们的目标是通过以下方式定义最小动物细胞周期：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）提供关于绝对磷酸化化学计量的信息，和iv）提供介导最小细胞周期的关键细胞周期调节因子及其翻译后修饰（不限于磷酸化）的绝对量。这项研究将是第一个大规模的，定量蛋白质组学研究的分裂周期和第一个全球分析的后生动物细胞周期，不依赖于基于药物的同步技术。青蛙X.由于其大尺寸、全胚分裂和自然同步细胞周期的能力，非洲乳鼠胚胎为这些实验提供了令人信服的背景。拟议的实验有可能对细胞周期产生革命性的见解，并产生大量的数据，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。 公共卫生相关性：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数百种蛋白质丰度和蛋白质修饰的变化，这些变化是体内细胞周期的基础，因此该项目将挑战，改进和扩大目前的细胞周期模型，这些模型对我们对增殖的基本理解至关重要，对许多人类发育疾病和癌症也是如此。除了促进我们对细胞周期的基本理解外，这些疾病和恶性肿瘤的预防、治疗和疗法的发展将从这项研究中受益匪浅。

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.