Myosin-II Dynamics and Cytokinesis

肌球蛋白-II 动力学和细胞分裂

基本信息

批准号：
6916509
负责人：
James Spudich
金额：
$ 39.25万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 2007-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Myosin-II bipolar thick filament formation is highly regulated in cells and is required for cytokinesis as a key component of the contractile ring. The long-term objective of this application is to understand in molecular terms the regulation of myosin-II bipolar thick filament assembly, and to elucidate the spatial and temporal control of contractile ring formation, maintenance, and dissolution during cell division. D. discoideum has a number of advantages for the study of cytokinesis, and will be used as the model system. The research plan is designed to answer the following questions. How does myosin-II heavy chain phosphorylation control the assembly of myosin-II bipolar thick filaments at the molecular level? How are the three known myosin-II heavy chain kinases organized during the cell cycle, and how does that organization relate to the dynamics of the myosin-IIcontaining contractile ring? What are the other essential proteins involved in the establishment, maintenance and dissolution of the myosin-II-containing contractile ring, and what are their cellular organizations and dynamics during cell division? Molecular genetic approaches will be used to create directed mutations in the myosin-II tail domain, to test specific hypotheses of the mechanism of regulation of thick filament assembly. These mutant myosin-IIs will be transformed into Dictyostelium myosin-II null cells to test for rescue of cytokinesis in suspension. Fragments of the myosin-II tail will be analyzed in vitro by biochemical and biophysical methods to examine phosphorylation-dependent changes in conformational states, and to characterize the kinetics and thermodynamics of the assembly process. Several approaches will be used to identify proteins that are crucial players in myosin-II-dependent cytokinesis. cDNA complementation will define suppressors of myosin-II -impaired mutant strains of Dictyostelium. Proteins that bind directly to the myosin-II tail domain will be pursued by affinity column chromatography. Finally, Dictyostelium genes homologous to cytokinesis-related genes in other organisms will be searched for. All identified new proteins will be characterized in vitro and in vivo. The spatial and temporal organization of proteins of the contractile ring and of proteins involved in its formation will be followed in live cells by fluorescent tagging ans visualization in vivo using computer-linked, low-light-level imaging. Total internal reflection fluorescence microscopy allows visualization of single myosin-II bipolar thick filaments in the cell cortex just beneath the cell membrane. This method will allow dual visualization of myosin-II and other fluorescent-labeled cytokinesis-related proteins.

描述（由申请人提供）：肌球蛋白 - II双极厚细丝形成在细胞中受到高度调节，是细胞因子作为收缩环的关键组成部分所必需的。该应用的长期目标是用分子术语理解肌球蛋白-II双极厚细丝组件的调节，并阐明收缩环形成，维护和细胞分裂期间溶解的空间和时间控制。 D. Discoideum在研究细胞因子方面具有许多优势，并将用作模型系统。该研究计划旨在回答以下问题。肌球蛋白-II重链磷酸化如何控制分子水平的肌球蛋白-II双极厚丝的组装？在细胞周期中组织的三个已知的肌球蛋白 - II重链激酶如何与肌球蛋白iicontaining收缩环的动力学有何关系？在含有肌球蛋白-II的收缩环的建立，维持和溶解中涉及的其他必要蛋白质是什么？在细胞分裂期间，其细胞组织和动态是什么？分子遗传方法将用于在肌球蛋白-II尾部结构域中产生定向突变，以测试厚丝组装调节机理的特定假设。这些突变的肌球蛋白IIS将转化为肌球蛋白-II无效细胞，以测试悬浮液中的细胞因子。肌球蛋白-II尾巴的片段将通过生化和生物物理方法在体外进行分析，以检查构象状态的磷酸化依赖性变化，并表征组装过程的动力学和热力学。几种方法将用于鉴定蛋白质是肌球蛋白-II依赖性细胞因子的关键参与者。 cDNA互补将定义肌球蛋白-II损伤突变菌株的抑制。亲和色谱柱色谱法将追求直接与肌球蛋白-II尾域结合的蛋白质。最后，将搜索与其他生物体中与细胞因子相关基因同源的柱状基因。所有鉴定出的新蛋白质都将在体外和体内表征。在活细胞中，通过使用计算机连接的低光级成像在体内可视化收缩环和参与其形成的蛋白质的蛋白质的空间和时间组织。总内反射荧光显微镜可以可视化细胞膜下方细胞皮质中的单个肌球蛋白 - II双极厚细丝。该方法将允许双重可视化肌球蛋白II和其他荧光标记的细胞因子相关蛋白。