Mechanisms of Cytokinesis

项目总结 在染色体被基于微管的有丝分裂纺锤体分离后，胞质分裂完成了细胞 分裂，将母细胞的内容分成两个子细胞。胞质分裂是通过以下方式完成的 在围绕细胞的赤道带中，在皮质上形成的肌球蛋白收缩环的收缩 赤道。胞质分裂失败会产生四倍体细胞，而四倍体细胞是癌症发生的常见中间细胞。 胞质分裂调节因子的上调和突变也与不同的癌症和抑制物有关 靶向胞质分裂作为潜在的化疗药物是有兴趣的，促使人们努力了解 细胞质分裂过程中形成皮质收缩能力的机制。以确保每个子单元格收到 相当于基因组的补充，收缩环的位置由后期纺锤体指定。这个 纺锤体向皮质发送两个叠加的信号：(1)促进收缩环组装的积极信号 这是由一组被称为中心纺锤体的捆绑微管产生的，这些微管形成于分离的 染色体，以及(2)由微管产生的抑制细胞收缩的负信号。 非赤道皮质。拟议的工作集中在这两个信号的分子基础上， 集体直接收缩环组件。正信号促进收缩环的中心成分 组装是中心纺锤体，是由激动素-6和细胞周期蛋白4的二聚体形成的四聚体复合体。 Centralspindlin被有丝分裂激酶PLK1磷酸化，PLK1集中在中央纺锤体上；它 随后扩散到邻近的质膜，在那里，通过在很大程度上保持 尚不清楚的是，其CYK4亚基与ECT2鸟嘌呤核苷酸交换因子(Global)结合并激活。 有源ECT2进而为收缩环组件的主调节器产生丰富的赤道区， RhoA-GTP。在目标1中，我们解决了与阳性胞质分裂信号有关的主要未决问题： 中枢纺锤体激活ECT2的机制。为了了解微管是如何抑制 非赤道皮质的收缩，这与来自中枢的积极信号同时发生 纺锤体，我们建立了一种监测收缩环蛋白从C。 优雅的胚胎。利用这一检测方法，我们确定Aurora A激酶是基于Aster的一个重要的介体。 收缩抑制。在目标2中，我们在这项工作的基础上确定了调节收缩的Aurora A靶标 抑制这一机制，并将其扩展到人类细胞。目标3解决了以下方面的一个重要差距 了解支持收缩环组装的细胞周期状态是如何产生的，并评估 胞质分裂的持续时间，如有丝分裂的持续时间，是否由基于p53的有丝分裂秒表监测 从种群中消除潜在问题细胞的机制。

PROJECT SUMMARY After the chromosomes are segregated by the microtubule-based mitotic spindle, cytokinesis completes cell division, partitioning the contents of the mother cell to the two daughter cells. Cytokinesis is accomplished by constriction of an acto-myosin contractile ring that forms on the cortex in an equatorial band encircling the cell equator. Cytokinesis failure generates tetraploid cells that are a common intermediate in the genesis of cancers. Upregulation and mutation of cytokinesis regulators has also been implicated in different cancers, and inhibitors targeting cytokinesis are of interest as potential chemotherapeutic agents, motivating efforts to understand the mechanisms that pattern cortical contractility during cytokinesis. To ensure that each daughter cell receives an equivalent genomic complement, the position of the contractile ring is specified by the anaphase spindle. The spindle sends two superimposed signals to the cortex: (1) a positive signal promoting contractile ring assembly that is generated by set of bundled microtubules, called the central spindle, that forms between the separating chromosomes, and (2) a negative signal generated by the microtubule asters that suppresses the contractility of the non-equatorial cortex. The proposed work focuses on the molecular basis for these two signals that collectively direct contractile ring assembly. A central component of the positive signal promoting contractile ring assembly is centralspindlin, a tetrameric complex formed by a dimer of kinesin-6 and a dimer of CYK4. Centralspindlin is phosphorylated by the mitotic kinase PLK1, which concentrates on the central spindle; it subsequently diffuses to the adjacent plasma membrane where, through mechanisms that remain largely unclear, its CYK4 subunit engages with and activates the ECT2 guanine nucleotide exchange factor (GEF). Active ECT2 in turn generates an equatorial zone enriched for the master regulator of contractile ring assembly, RhoA-GTP. In Aim 1, we address the major open question with respect to positive cytokinesis signaling: the mechanism of ECT2 activation by centralspindlin. To understand how the microtubule asters suppress contractility on the non-equatorial cortex, which occurs at the same time as positive signaling from the central spindle, we developed an assay monitoring clearing of contractile ring proteins from the cell poles in the C. elegans embryo. Using this assay, we identified Aurora A kinase as an essential mediator of aster-based contractility suppression. In Aim 2, we build on this work to identify the Aurora A targets that mediate contractility suppression and extend analysis of this mechanism to human cells. Aim 3 addresses an important gap in understanding how the cell cycle state that supports contractile ring assembly is generated, and assesses whether the duration of cytokinesis, like that of mitosis, is monitored by a p53-based mitotic stopwatch mechanism that eliminates potentially problematic cells from the population.