REGULATION OF CYTOKINESIS IN ANIMAL CELLS

收缩环是瞬时的，基于肌动球蛋白的结构，负责在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的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不对称细胞分裂。 在努力了解在胚胎发生过程中的收缩环形成的调节，洗涤剂提取模型的海胆受精卵细胞骨架的开发，保留在体外重新激活收缩环的收缩的能力，具有细胞周期调节激酶能够磷酸化特定的皮质细胞骨架底物在体外和体内，并适合于结构，生物化学和生物物理分析。 以这些研究为基础，该提案寻求支持的作用（S），细胞周期激酶（及其底物）在收缩环形成的空间和时间调节的详细分析。 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，使其成为胞质分裂生化分析的一个有吸引力的模型系统。 鉴于细胞周期调控机制在系统发生系中的高度保守性，本研究中获得的胞质分裂结果可能具有普遍适用性。 关于胞质分裂调控的三个悬而未决的问题形成了本提案的具体目标。 本提案中描述的实验路线将寻求：1）确定细胞周期激酶对收缩环形成的空间和时间调节的机制; 2）描述胞质分裂期间肌球蛋白激活的机制; 3）确定指定卵裂沟位置的分子决定因素。 这是不可能的，这三个方面的收缩环调节的机制是相互排斥的，它是预期的结果，从每一个实验线将提供洞察力和方向到其他两个目标中解决的机制。 这些努力应该导致更清楚地了解有丝分裂装置，细胞周期的调节机制，和细胞信号转导途径协调行动，空间和时间调节胞质分裂。

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.