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分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更为复杂的体细胞周期模型的自然框架。这些模型是全世界成千上万的实验室的知识基础，这些实验室致力于了解细胞分裂和生长，以及如何预防、抵消和治疗它们的失调。但是，最微小的细胞周期，即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控模块在卵裂周期中并不重要。在这些最小的分裂周期中，真正控制有丝分裂进入的未知机制，以及通过扩展可能存在于所有后生动物细胞周期中的机制，仍然不清楚。我们建议，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。裂解周期的分子景观只能由基于质谱的蛋白质组学生成，特别是因为实际上没有合子转录使得这些裂解细胞周期难以转录组学。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：1)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；2)量化变化/振荡的程度；3)提供绝对磷酸化化学计量学的信息。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）的绝对数量。这项研究将是第一个大规模的、定量的蛋白质组学研究，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球分析。青蛙X. laevis胚胎的胚胎为这些实验提供了一个令人信服的背景，因为它们的大尺寸，全细胞分裂，以及自然同步细胞周期的能力。拟议的实验有可能对细胞周期产生革命性的见解，并产生一个数据宝库，可以被充满活力的细胞周期社区挖掘和进一步扩展。

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.