Coordination of Mitotsis and Cytokinesis in Animal Cells

在早期胚胎的快速细胞分裂期间维持染色体倍性需要染色体分离和细胞质分配（胞质分裂）的精确协调，使得收缩环在后期开始之前不组装。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，该检查点监测动粒附着到有丝分裂纺锤体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称为分隔起始网络（SIN）的信号级联，其调节胞质分裂的起始。在动物细胞中胞质分裂的时间调节还没有得到很好的表征，但在海胆胚胎中的研究表明，调节收缩环放置和组装的信号转导途径受到有丝分裂检查点，但独立于有丝分裂出口。为了理解有丝分裂检查点如何协调高等真核生物中的有丝分裂和胞质分裂，在海胆胚胎中鉴定并表征了SIN途径的末端组分（Mob1）。本申请中描述的实验系列试图将联合收割机分子、生物化学和活细胞分析结合起来，以评估分隔起始网络在调节细胞分裂的终末事件中的作用。该提案的第一个具体目标旨在鉴定Sid2激酶的海胆同源物，检查其与Mob1的关联并表征其在细胞周期中的活性。具体目标2将在体外和体内研究Mob1和Sid2与纺锤体极的结合动力学。具体目标3将评估Mob1和Sid2在体内指导细胞周期终末事件中的作用， 显性阴性形式的Sid2和Mob1，并研究其对有丝分裂出口和胞质分裂使用活细胞荧光成像的影响。最后，组成型激活的Sid2将被引入活细胞中，以测试SIN与有丝分裂检查点解偶联是否会导致不受调节的胞质分裂。这些努力应导致更清楚地了解有丝分裂和胞质分裂是如何协调的，并确认是否单细胞真菌所采用的策略，以调节分隔在高等真核生物中是保守的。

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.