Coordination of Mitotsis and Cytokinesis in Animal Cells

在早期胚胎的快速细胞分裂过程中，染色体倍性的维持需要染色体分离和细胞质分裂（细胞质分裂）的精确协调，以便在后期开始之前收缩环不会组装。最近在酵母和动物细胞中的研究已经发现了一个有丝分裂检查点，它监测着丝粒附着在有丝分裂纺锤体上，并调节姐妹染色单体分离和CDK1失活（有丝分裂退出）的开始。在分裂酵母中，有丝分裂检查点还调节一个称为分裂起始网络（SIN）的信号级联，该信号级联调节细胞质分裂的起始。动物细胞中细胞分裂的时间调控尚不清楚，但对海胆胚胎的研究表明，调节收缩环放置和组装的信号通路受有丝分裂检查点的影响，但独立于有丝分裂退出。为了了解高级真核生物有丝分裂检查点如何协调有丝分裂和细胞分裂，在海胆胚胎中鉴定并表征了SIN途径的一个终端组分（Mob1）。本应用程序中描述的实验线试图将分子，生化和活细胞分析结合起来，以评估分裂起始网络在调节细胞分裂终端事件中的作用。本研究的第一个具体目标是鉴定Sid2激酶的海胆同源物，研究其与Mob1的关联，并表征其在细胞周期中的活性。特异性目标2将在体内和体外研究Mob1和Sid2与纺锤杆的关联动态。特异性目的3将评估Mob1和Sid2在体内通过表达指导细胞周期终末事件中的作用

Maintenance of chromosomal ploidy during the rapid cell divisions of the early embryo requires a precise coordination of chromosome segregation and cytoplasmic partitioning (cytokinesis) such that the contractile ring does not assemble before the onset of anaphase. Recent studies in both yeast and animal cells have identified a mitotic checkpoint that monitors kinetochore attachment to the mitotic spindle and regulates the onset of sister chromatid segregation and CDK1 inactivation (mitotic exit). In fission yeast, the mitotic checkpoint also regulates a signaling cascade termed the Septation Initiation Network (SIN) that regulates the initiation of cytokinesis. The temporal regulation of cytokinesis in animal cells is not well characterized, but studies in sea urchin embryos suggest that the signaling pathways regulating contractile ring placement and assembly are subject to the mitotic checkpoint but independent of mitotic exit. In an effort to understand how the mitotic checkpoint coordinates mitosis and cytokinesis in higher eukaryotes, a terminal component of the SIN pathway (Mob1) has been identified and characterized in sea urchin embryos. The lines of experimentation described in this application seek to combine molecular, biochemical and live cell analyses to assess the role of the Septation Initiation Network in regulating the terminal events of cell division. The first Specific Aim of this proposal seeks to identify the urchin homolog of Sid2 kinase, examine its association with Mob1 and characterize its activity during the cell cycle. Specific Aim 2 will study the dynamics of Mob1 and Sid2 association with the spindle poles in vitro and in vivo. Specific Aim 3 will assess the roles of Mob1 and Sid2 in directing the terminal events of the cell cycle in vivo by expressing dominant-negative forms of Sid2 and Mob1, and studying their effects on mitotic exit and cytokinesis using live cell fluorescence imaging. Lastly, constitutively activated Sid2 will be introduced into living cells to test whether uncoupling SIN from the mitotic checkpoint can result in unregulated cytokinesis. These efforts should lead to a clearer understanding of how mitosis and cytokinesis is coordinated, and confirm whether the strategies employed by unicellular fungi to regulate septation are conserved in higher eukaryotes.