Coordination of Mitotsis and Cytokinesis in Animal Cells

在早期胚胎快速细胞分裂期间维持染色体倍性需要精确协调染色体分离和细胞质分裂(胞质分裂)，使收缩环在后期开始之前不会组装。最近在酵母和动物细胞中的研究已经确定了一个有丝分裂检查点，它监测着动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被称为分离启动网络(SIN)的信号级联，该信号级联调节胞质分裂的启动。动物细胞胞质分裂的时间调控尚未得到很好的描述，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制于有丝分裂检查点，但与有丝分裂退出无关。为了了解有丝分裂检查点如何协调高等真核生物的有丝分裂和胞质分裂，已在海胆胚胎中鉴定并鉴定了SIN途径的末端成分(Mob1)。本申请中描述的实验路线试图结合分子、生化和活细胞分析来评估分离起始网络在调节细胞分裂的末端事件中的作用。这项建议的第一个具体目标是鉴定Sid2激酶的海胆同源物，检查其与Mob1的关系，并表征其在细胞周期中的活性。特定目的2将在体外和体内研究Mob1和Sid2与纺锤体极点的关联动力学。具体目标3将评估Mob1和Sid2在体内引导细胞周期终末事件中的作用 显性-阴性形式的Sid2和Mob1，并利用活细胞荧光成像研究它们对有丝分裂退出和胞质分裂的影响。最后，结构性激活的Sid2将被引入活细胞中，以测试从有丝分裂检查点解偶联SIN是否会导致无调控的胞质分裂。这些努力应该会导致对有丝分裂和胞质分裂是如何协调的更清楚的理解，并证实单细胞真菌用来调节隔膜的策略在高等真核生物中是否保守。

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.