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

描述(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，缺乏点缀体细胞周期的生长、转录和检查点。因此，这些分裂周期是构建复杂得多的体细胞周期模型的自然框架。这些模型是世界各地数以千计的实验室的智力基础，这些实验室打算了解细胞的分裂和生长，以及如何预防、抵消和治疗它们的不当调控。但是，最小的细胞周期--后生动物分裂周期--的真正本质还远未完全被理解。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，以及延伸到所有后生动物细胞周期中可能存在的机制，仍然不清楚。我们认为，这种未知的机制，也许还有最小细胞周期的许多其他方面，可以通过综合分析来揭示。分裂周期的分子图景只能由基于MS的蛋白质组学产生，特别是因为合子转录的虚拟缺失使得这些分裂细胞周期对转录组学来说难以实现。为了从根本上改进细胞周期模型，我们的目标是通过i)检测哪些蛋白质和哪些磷酸化位点以细胞周期依赖的方式振荡，ii)量化变异/振荡的程度，iii)提供关于绝对磷酸化化学计量学的信息，以及iv)提供调节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)的绝对量来定义最小细胞周期。这项研究将是第一次对卵裂周期进行大规模、定量的蛋白质组学研究，也是第一次对不依赖基于药物的同步技术的后生动物细胞周期进行全球分析。青蛙X.laevis胚胎的胚胎由于其大小、全胚层分裂和自然同步细胞周期的能力，为这些实验提供了一个令人信服的背景。拟议中的实验有可能对细胞周期进行革命性的洞察，并产生大量数据，这些数据可以被挖掘出来，并由充满活力的细胞周期群体进一步扩展。 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”的胚胎细胞周期，以破译其调节的真实性质。由于我们的目标是以前所未有的时间分辨率描述体内这一周期背后的数百种蛋白质丰度和蛋白质修饰的变化，因此该项目将挑战、改进和扩大当前的细胞周期模型，这些模型对于我们对增殖以及许多人类发育疾病和癌症的基本理解至关重要。除了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、治疗和治疗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.