REGULATION OF CYTOKINESIS IN ANIMAL CELLS

收缩环是一种瞬时的、基于肌动球蛋白的结构，负责有丝分裂结束时两个子细胞的物理分离。为了确保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进行协调调节。然而，完成这一过程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不对称的细胞分裂。为了了解胚胎发育过程中收缩环形成的规律，建立了一种洗涤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的能力，在体外和体内具有细胞周期调节蛋白能磷酸化特定的皮质细胞骨架底物，并适合于结构、生化和生物物理分析。以这些研究为基础，这项建议寻求支持对细胞周期蛋白激酶(及其底物)在收缩环形成的时空调节中所起的作用的详细分析(S)。海胆胚胎细胞周期的高度同步性，以及对显微注射和显微操作的适应性，使其成为细胞质分裂的生物化学分析的一个有吸引力的模型系统。鉴于细胞周期调控机制在系统发育系中的高度保守性，在细胞质分裂的研究中获得的结果很可能是普遍适用的。关于胞质分裂调控的三个悬而未决的问题构成了这项提案的具体目标。这项建议中描述的实验路线将寻求：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)表征细胞质分裂过程中肌球蛋白激活的机制；3)确定指定分裂沟位置的分子决定因素。收缩环调节的这三个方面的基础机制不太可能是相互排斥的，预计来自每一条线的实验结果将为其他两个目标中涉及的机制提供洞察力和方向。这些努力应该会导致对有丝分裂装置，细胞周期的调节机制，以及细胞信号通路如何协调作用，在空间和时间上调节细胞质分裂的更清晰的理解。

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.