Timing and regulation of meiotic commitment in S cerevisiae

描述（由申请人提供）： 项目摘要/摘要：在配子发生过程中，外源信号启动细胞进入减数分裂并分化为配子。未能维持减数分裂过程可导致不适当的细胞增殖、基因组损伤和发育缺陷。尽管维持减数分裂的重要性，但缺乏对外部信号如何影响细胞周期调控网络以防止细胞不适当地退出减数分裂的分子理解。这个建议的目的是确定细胞周期调控网络，受外部信号的影响，保持减数分裂或有丝分裂细胞的身份。由于多细胞真核生物减数分裂细胞的外部环境难以控制，本研究采用S。酿酒酵母作为模式生物。微流体工具已经被开发用于在时间上和精确地操纵细胞的外部信号，监测细胞内信号传导通路的输出，并识别单个细胞中的细胞周期结果。这些创新允许研究细胞如何整合信号以维持减数分裂，蛋白质如何在减数分裂网络中发挥作用，以及检查点机制如何影响细胞周期结果。拟议研究的基本原理是，所选择的问题集中在可能高度保守的过程上，从而使芽殖酵母中的发现能够揭示细胞周期控制的一般机制。强有力的初步数据指导了以下三个具体目标：1）确定控制减数分裂定型的调控机制; 2）确定减数分裂检查点途径在协调减数分裂定型和调节有丝分裂恢复中的作用; 3）识别和分析细胞周期调节因子，其控制有丝分裂恢复期间的关键G2/M检查点。在第一个目标中， 微流控工具将被用于检验反馈途径建立维持减数分裂的承诺的假设。此外，细胞周期调节剂将测试其在减数分裂维持中的作用。第二个目的是检验减数分裂检查点机制协调维持减数分裂承诺的时间调节的假设。第三个目标将采用生物化学技术和遗传筛选来确定高度保守的G2/M检查点如何协调退出减数分裂并返回有丝分裂的细胞中的细胞周期事件。使用微流体来操纵外部信号的应用和撤回，同时监测单细胞的创新方法允许测试关于响应外部信号的细胞周期调节的新假设。这项研究意义重大，因为其结果有望揭示细胞周期调控的一般原则，即通过维持减数分裂或有丝分裂细胞的身份来保护基因组的完整性。 最终，这些结果将进一步加深我们对这一过程中的错误如何促进肿瘤形成和发育异常的理解。

DESCRIPTION (provided by applicant): Project Summary/Abstract: During gametogenesis, an exogenous signal initiates cells to enter meiosis and differentiate into gametes. A failure to maintain the meiotic process can result in inappropriate cell proliferation, genome damage, and developmental defects. Despite the importance of maintaining meiosis, there is a lack of a molecular understanding of how external signals influence the cell-cycle regulatory network to prevent cells from inappropriately exiting meiosis. The objective of this proposal is to determine how cell-cycle regulatory networks, influenced by external signals, maintain the meiotic or mitotic cell identity. Due to the difficult in manipulating the external environment of meiotic cells in multi-cellular eukaryotic organisms, these studies employ S. cerevisiae as the model organism. Microfluidic tools have been developed to temporally and precisely manipulate the cells' external signals, monitor the output of the intracellular signaling pathways, and identify cell cycle outcomes in individual cells. Thes innovations allow the investigation of how cells integrate signals to maintain meiosis, how proteins function within meiotic networks, and how checkpoint mechanisms affect cell-cycle outcomes. The rationale for the proposed research is that the questions chosen are focused on processes that are likely to be highly conserved, allowing the findings in budding yeast to uncover general mechanisms of cell-cycle control. Strong preliminary data has guided the following three specific aims: 1) Determine the regulatory mechanisms that govern meiotic commitment; 2) Determine the role of meiotic checkpoint pathways in coordinating meiotic commitment and regulating the return to mitosis; and, 3) Identify and analyze cell-cycle regulators that control the crucial G2/M checkpoint during the return to mitosis. In the first aim, the microfluidic tools will be implemented to test the hypothesis that a feedback pathway establishes the commitment to maintain meiosis. Moreover, cell-cycle regulators will be tested for their role in meiotic maintenance. The second aim tests the hypothesis that meiotic checkpoint mechanisms coordinate the temporal regulation of the commitment to maintain meiosis. The third aim will employ biochemical techniques and a genetic screen to determine how the highly conserved G2/M checkpoint coordinates cell cycle events in cells that exit meiosis and return to mitosis. The innovative approach of using microfluidics to manipulate the application and withdrawal of an external signal while monitoring single cells allows the testing of novel hypotheses about cell-cycle regulation in response to external signals. The proposed research is significant because the results are expected to uncover general principles of cell-cycle regulation that protect genome integrity by maintaining the meiotic or mitotic cell identity. Ultimately, the results will further our understanding of how errors in this process facilitate neoplasia and developmental abnormalities.