MOLECULAR ANALYSIS OF CYTOKINESIS IN DICTYOSTELIUM

盘基网柄菌细胞分裂的分子分析

• 批准号: 3469027
• 负责人: ARTURO DE LOZANNE
• 金额: $ 9.2万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3469027
• 关键词: Dictyostelium; cell cycle; cytoskeletal proteins; density gradient ultracentrifugation; fluorescence microscopy; gene complementation; gene expression; immunoprecipitation; laboratory rabbit; molecular cloning; myosin light chain kinase; myosins; phosphorylation; polymerase chain reaction; protein biosynthesis; protein purification; protein structure function; southern blotting; video recording system; western blottings

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.

在动物细胞分裂过程中，肌动球蛋白的细胞骨架 经历了戏剧性的重组，形成了收缩环。这个 这种“细胞器”的形成是在时间上受到严格控制的 (有丝分裂末期)和空间(中期面上)方式。至 目前还没有明确的分子机制 控制胞质分裂。这项建议的主要目标是理解 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菌作为模型系统，因为这 有机体为这里讨论的研究提供了许多优势。在……里面 除了进行胞质分裂外，还非常像动物细胞(不同于 酵母细胞)，网柄菌在生物化学和生物化学方面都具有很强的延展性。 基因水平及其细胞骨架已经得到了很好的表征。 这项建议旨在回答以下问题： 1.肌球蛋白在收缩环中是如何定位的？它的作用机制 肌球蛋白在收缩环中的定位将被阐明。这个 肌球蛋白分子中定位所需的部分 收缩环将被确定。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.收缩环由什么构成？它的分子组成 将识别收缩环并分析其功能。 收缩环将被提纯，其蛋白质组分 特色化的。收缩环蛋白将被克隆并 已排序。这些蛋白质的功能将通过基因- 基因敲除和过度表达实验。 3.参与胞质分裂的基因还有哪些？一种遗传方法来研究 胞质分裂将被启动。网柄网柄菌突变体缺陷于 胞质分裂将被分离。胞质分裂突变体将是 特色化的。肌动球蛋白细胞骨架的组织将是 在分离的突变体中确定的。受影响的基因将被克隆 通过DNA介导的互补作用。