MOLECULAR ANALYSIS OF CYTOKINESIS IN DICTYOSTELIUM

在动物细胞分裂过程中，肌动球蛋白细胞骨架 经过剧烈的重组形成收缩环 的 这种“细胞器”的形成受到时间和空间的严格控制。 (at有丝分裂结束）和空间（在中期平面上）方式。 到 迄今为止，还没有明确的分子机制， 控制胞质分裂。 本提案的主要目的是了解 胞质分裂过程中细胞骨架重组的分子基础。 我们将使用盘基网柄藻作为模型系统， 生物体为这里讨论的研究提供了许多优势。 在 除了经历胞质分裂非常像动物细胞（不像 酵母细胞），网骨藻在生物化学和生物化学方面都具有很大的可塑性， 遗传水平及其细胞骨架已得到很好的表征。 这项建议旨在回答下列问题： 1.肌球蛋白是如何定位在收缩环中的？ 的机制 将阐明肌球蛋白在收缩环中的定位。 的 肌球蛋白分子的一部分需要定位在 将确定收缩环。 肌球蛋白的磷酸化状态 将分析细胞周期中的重链和轻链。 2.什么是收缩环？ 的分子组成， 将识别收缩环并分析其功能。 收缩环将被纯化， 表征了 收缩环蛋白将被克隆， 测序 这些蛋白质的功能将通过基因评估- 敲除和过表达实验。 3.还有哪些基因参与胞质分裂？ 一种遗传学方法， 胞质分裂将开始。 网囊藻突变体， 将分离胞质分裂。 胞质分裂突变体 表征了 肌动球蛋白细胞骨架的组织将是 在孤立的突变体中确定。 受影响的基因将被克隆 通过DNA介导的互补作用。

During cell division in animal cells the actomyosin cytoskeleton undergoes a dramatic reorganization to form the contractile ring. The formation of this "organelle" is tightly controlled both in a temporal (at the end of mitosis) and spatial (on the metaphase plane) manner. To date there is no clear understanding of the molecular mechanisms that control cytokinesis. The major goal of this proposal is to understand the molecular basis of cytoskeletal reorganization during cytokinesis. We will use Dictyostelium discoideum as a model system because this organism offers many advantages for the studies discussed here. In addition to undergoing cytokinesis very much like animal cells (unlike yeast cells), Dictyostelium is very malleable both at the biochemical and genetic level and its cytoskeleton has been very well characterized. This proposal is aimed at answering the following questions: 1. How is myosin localized in the contractile ring? The mechanisms of myosin localization in the contractile ring will be elucidated. The portion of the myosin molecule required for localization in the contractile ring will be determined. The phosphorylation state of myosin heavy and light chains during the cell cycle will be analyzed. 2. What constitutes a contractile ring? The molecular components of the contractile ring will be identified and their functions analyzed. Contractile rings will be purified and their protein components characterized. The contractile-ring proteins will be cloned and sequenced. The function of these proteins will be assessed by gene- knockout and overexpression experiments. 3. What other genes are involved in cytokinesis? A genetic approach to cytokinesis will be initiated. Dictyostelium mutants defective in cytokinesis will be isolated. The cytokinesis mutants will be characterized. The organization of the actomyosin cytoskeleton will be determined in the isolated mutants. The affected genes will be cloned by DNA-mediated complementation.