REGULATION OF CYTOKINESIS IN ANIMAL CELLS

收缩环是一种短暂的、以肌动球蛋白为基础的结构，负责有丝分裂结束时两个子细胞的物理分离。为了保证姐妹染色单体传递到每个子细胞的保真度，收缩环的形成必须与纺锤体组装和染色单体分离过程在时间和空间上协调调节。然而，实现这一目标的分子机制仍然难以捉摸，以及这些途径的修饰如何在发育过程中引起细胞不对称分裂。为了了解胚胎发生过程中收缩环形成的调控，研究人员开发了一种清洁剂提取的海胆受精卵细胞骨架模型，该模型在体外保留了重新激活收缩环收缩的能力，具有细胞周期调节激酶，能够在体外和体内磷酸化特定的皮质细胞骨架底物，并且可用于结构、生化和生物物理分析。以这些研究为基础，本提案寻求对细胞周期激酶（及其底物）在收缩环形成的空间和时间调节中所起作用的详细分析的支持。海胆胚胎具有高度的细胞周期同行性，并且易于显微注射和显微操作，这使其成为细胞分裂生化分析的一个有吸引力的模型系统。鉴于细胞周期调节机制在系统发育系中的高度保守性，本细胞质分裂研究的结果很可能具有普遍适用性。关于细胞分裂调节的三个突出问题形成了本提案的具体目的。本提案中描述的实验线将寻求：1)定义细胞周期激酶对收缩环形成的空间和时间调节的机制；2)明确细胞分裂过程中肌球蛋白活化的机制；3)确定确定卵裂沟位置的分子决定因素。收缩环调节的这三个方面的机制不太可能是相互排斥的，预计从每条实验线得出的结果将为其他两个目标中解决的机制提供洞察力和方向。这些努力将使我们更清楚地了解有丝分裂装置、细胞周期的调节机制和细胞信号通路如何协调作用，在空间和时间上调节细胞分裂。

The contractile ring is the transient, actomyosin-based structure responsible for the physical separation of the two daughter cells at the end of mitosis. To ensure the fidelity of sister chromatid transmission into each daughter cell, the formation of the contractile ring must be coordinately regulated both temporally and spatially with the processes of spindle assembly and chromatid separation. However, the molecular mechanisms by which this is accomplished remain elusive, as well as how modifications of these pathways give rise to assymetric cell divisions during development. In an effort to understand the regulation of contractile ring formation during embryogenesis, a detergent-extracted model of the sea urchin zygote cytoskeleton was developed that retains the capacity to reactivate contraction of the contractile ring in vitro, possesses cell cycle regulated kinases able to phosphorylate specific cortical cytoskeletal substrates in vitro and in vivo, and is amenable to structural, biochemical and biophysical analysis. Using these studies as a foundation, this proposal seeks support for a detailed analysis of the role(s) that cell cycle kinases (and their substrates) play in the spatial and temporal regulation of contractile ring formation. The high degree of cell cycle synchrony, as well as the amenability to microinjection and micromanipulation make the sea urchin embryo an attractive model system for the biochemical analysis of cytokinesis. Given the high degree of conservation of cell cycle regulatory mechanisms across phylogenetic lines, it is likely that the results obtained in this study of cytokinesis will be generally applicable. Three outstanding issues regarding the regulation of cytokinesis form the Specific Aims of this proposal. The lines of experimentation described in this proposal will seek to: 1) Define the mechanisms by which cell cycle kinases contribute to the spatial and temporal regulation of contractile ring formation; 2) Characterize the mechanism of myosin activation during cytokinesis; and 3) Identify the molecular determinants that specify the position of the cleavage furrow. It is unlikely that the mechanisms underlying these three aspects of contractile ring regulation are mutually exclusive, and it is expected that the results derived from each line of experimentation will lend insight and direction into the mechanisms addressed in the other two aims. These efforts should lead to a clearer understanding of how the mitotic apparatus, the regulatory machinery of the cell cycle, and cell signaling pathways coordinately act to spatially and temporally regulate cytokinesis.